iGEM 2018 — Team Hamburg

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# Team:Hamburg/Notebook

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# Notebook

Use the tabs on the left to navigate through ten months of research. The notebook contains over 20,000 HTML lines, so it may take a while to load.

## Preparation of Ca/glycerol buffer and LB media

Ca-/glycerol buffer:

* 8.8233 g CaCl2 \* H2O (60 MM)
* 3.0255 g PIPES (10 mM)
* 189.6 g glycerol

The pH 7 adjusted with NAOH, filled up with water to 1 L and the buffer was sterilized by filter sterilization.

LB medium (Luria/Miller): 25 g for 1 L, pH 7

LB agar (Luria/Miller): 40 g for 1 L, pH 7

Both media were autoclaved.

## Preparation of agar plates

1 L agar mixed with 1 mL Camp and plated.

## Overnight culture of DH5a

We made one 5 mL DH5a overnight culture and incubated at 37°C and 22 rpm.

## Preparation of competent cells

We made competent cells with the protocol by Zhang Gong. 2x 500 mL grew at 37°C and 220 rpm. Afterwards we measured the OD600.

|  |  |
| --- | --- |
| Time [min] | OD |
| 55 | 0.068 |
| 120 | 0.21 |
| 138 | 0.362 |

## Test transformation

The protocol is by Zhang Gong. We put 0.4 µL pSB1C3 on the cells and incubated at 4°C on ice for 1 h. After that we heat shocked at 42°C for 45 s and incubated at 4°C on ice for 2 min. The cultures grew LB with Camp overnight at 37°C.

## Resuspension of required plasmids

|  |  |  |
| --- | --- | --- |
| Plate | Well | Insert |
| 2017 Kit Plate 3 | 3F | pSB1C3/BBa\_B0015 |
| 2017 Kit Plate 4 | 13L | pSB1A2/BBa\_E0040 |
| 2017 Kit Plate 2 | 24D | pSB1C3/BBa\_E0840 |
| 2017 Kit Plate 3 | 17C | pSB1C3/BBa\_I13504 |
| 2017 Kit Plate 3 | 8P | pSB1C3/BBa\_I20270 |
| 2017 Kit Plate 3 | 17L | pSB1C3/BBa\_I11032 |

We pierced through foil with a sterile pipette tip and added 10 µL sterile H2O. Then we incubated for 10 min at room temperature and transferred 10 µL of the samples into microtubes. Afterward the samples stored at -20°C.

|  |  |
| --- | --- |
| Lotnumber | Part |
| 180202NK01 | pSB1C3/BBa\_B0015 |
| 180202NK02 | pSB1A2/BBa\_E0040 |
| 180202NK03 | pSB1C3/BBa\_E0840 |
| 180202NK04 | pSB1C3/BBa\_I13504 |
| 180202NK05 | pSB1C3/BBa\_I20270 |
| 180202NK06 | pSB1C3/BBa\_I11032 |

## Transformation

The protocol was by Zhang Gong. We made the transformation off pSB1C3\_BBa\_B0015, pSB1A2\_BBa\_E0040, pSB1C3\_BBa\_E0840, pSB1C3\_BBa\_I13504, pSB1C3\_BBa\_I20270 into DH5a cells. The competent cells defrosted on ice for 1 h at 4°C. Then were added 1 µL of each plasmid to one of the alquots and incubated on ice for 35 min at 4°C. After that we did a heatshock for 1 min at 42°C and incubated again on ice for 3 min. Afterwards there were 850 µL prewarmed LB-Medium added to each tube and incubated 1 h at 37°C and 900 rpm. 200 µL of psB1C3\_BBa\_I13504 and 100 µL of every other aliquot were plated out onto cAmp/Amp agar plates and incubated at 28°C over the weekend.

## Supplement to the transformation

Only pSB1A2\_BBa\_E0040 and pSB1C3\_BBa\_I20270 showed colonies. pSB1C3\_BBa\_I20270 showed less colonies. Repetition of the transformation of pSB1C3\_BBa\_B0015, pSB1C3\_BBa\_E0840, pSB1C3\_BBa\_I13504, pSB1C3\_BBa\_I20270.

## Transformation

Protocol by Zhang Gong. Transformation of pSB1C3\_BBa\_B0015, pSB1C3\_BBa\_E0840, pSB1C3\_BBa\_I13504, pSB1C3\_BBa\_I20270. The competent cells defrosted on ice for 1 h at 4°C. Then were added 1 µL of each plasmid to one of the alquots and incubated on ice for 35 min at 4°C. After that we did a heatshock for 1 min at 42°C and incubated again on ice for 3 min. Afterwards there were 850 µL prewarmed LB-Medium added to each tube and incubated 1 h at 37°C and 900 rpm. After that we centrifuged for 1 min at 4.500 rpm and removed the supernatant until 100 µL and resuspended the pellet in remained medium. Then we plated the samples out onto cAmp/Amp agar plates and incubated overnight at 37°C.

## Examining the transformation

|  |  |
| --- | --- |
| Plate | Colonies |
| pSB1C3\_BBa\_B0015 | 0 |
| pSB1A2\_BBa\_E0040 | ~14 |
| pSB1C3\_BBa\_E0840 | 4 |
| pSB1C3\_BBa\_I13504 | 2 |
| pSB1C3\_BBa\_I20270 | 12 |

Set up overnight cultures of pSB1C3\_BBa\_E0840, pSB1A2\_BBa\_E0040, pSB1C3\_BBa\_I13504, pSB1C3\_BBa\_I20270 and incubated them overnight at 37°C and 220 rpm.

## Transformation

Protocol by Zhang Gong. Transformed pSB1C3\_BBa\_B0015 in DH5a. As result we had no colonies.

## Minipreparation of overnight cultures from 180206

The miniprep was made by GeneJET Plasmid Miniprep Kit by Thermo Scientific. Used the kit for pSB1A2\_BBa\_E0040, pSB1C3\_BBa\_E0840, pSB1C3\_BBa\_I13504, pSB1C3\_BBa\_I20270. The purified DNA was stored at -20°C.

## Preparation of the overnight cultures for growing competent cells

We prepared two overnight cultures (DH5a) in 3 mL LB-medium and incubated overnight at 37°C.

## Cryostocks of the overnight cultures from 180206

We mixed 1.35 µL of each overnight cultures with 450 µL 50% glycerol and stored at -80°C.

New lotnumbers:

|  |  |
| --- | --- |
| Lotnumber | Part |
| 180208AK01 | pSB1A2-BBa\_E0040 |
| 180208AK02 | pSB1C3-BBa\_E0840 |
| 180208AK03 | pSB1C3-BBa\_I13504 |
| 180208AK04 | pSB1C3-BBa\_I20270 |

## Preparation of competent cells

Protocol by Zhang Gong. We put 180209NK01 into 500 mL medium.

## Transformation

Transformation of pSB1C3\_BBa\_B0015 and pSB1C3\_BBa\_K206000 in DH5a. The samples was plated out onto cAmp agar plates and incubated over the weekend at room temperature. As result we had no colonies on both plates.

|  |  |
| --- | --- |
| Lot number | Part |
| 180202NK01 | pSB1C3\_BBa\_B0015 |
| 180202NK02 | pSB1C3\_BBa\_K206000 |

## Test transformation with cells from 180209 and by team Ignatova

The protocol was by Zhang Gong. The cells defrosted on ice for 20 min at 4°C. We got plasmid PRU (cAmp resistance) of team Ignatova (110 ng/µL). Added the 2.5 µL DNA and incubating on ice for 30 min and then we did a heat shock for 45 s at 42°C. After that we incubated on ice for 5 min and then incubated in preheated LB medium for 1 h at 37°C and 900 rpm. Afterwards the samples was plated out onto cAmp agar plates.

## Transformation with BBa\_K206000 and BBa\_B0015

Protocol was by Zhang Gong. The cells defrosted on ice for 30 min. Added 1.48 µL BB\_B0015 and 3 µL BBa\_K20600 each two tubes with 60 µL DH5a. After that we incubated on ice for 52 min at 4°C and then we did a heat shock for 60 s at 42°C. Afterwards the cells chilled on ice for 5 min at 4°C and added 850 µL preheated LB medium to each tube. After that we incubated for 1 h at 37°C and 900 rpm. Afterwards we centrifuged for 2 min at 4500 rpm. Next the samples plated out onto cAmp agar plates and incubated at 37°C.

## Preparation of two overnight cultures (ONCs)

Preparation of two ONCs with DH5a BBa\_B0015 and DH5a BBa\_K20600 . Then mixed the colony each into 8 mL LB medium and added 8 µL cAmp (10-3 g/L). Afterwards the samples were incubated overnight at 37°C and 900 rpm.

## PCR for amplification of parts by E.coli

Mastermix for 30 samples:

* 300 µL 5 x Phusion HF Buffer
* 30 µL dNTPs
* 30 µL Template DNA (E. coli cells)
* 15 µL Phusion DNA polymerase
* 1065 µL H2Odest

The new primers were dissolved in sterile H2O (100 µM). The primer prepared 1:10 dilution (10 µM) and added 1 µL primer to each 48 µL mastermix.

|  |  |  |  |
| --- | --- | --- | --- |
| Tube | Part | Fw Primer | Rv Primer |
| 1 | MlcRE | Pre-MlcRE-Fw | MlcRE-Suf-Rv |
| 1 | MlcRE | MlcRE-Fw | MlcRE-Rv |
| 3 | IdhA | Pre-IdhA-Fw | IdhA-Suf |
| 4 | IdhA | IdhA-Fw | IdhA-Rv |
| 5 | sulA | Pre-sulA-Fw | sulA-Suf-Rv |
| 6 | sulA | sulA-Fw | sulA-Rv |
| 7 | BBa\_B0031\_IdhA | 31-IdhA-Fw | IdhA-Suf-Rv |
| 8 | BBa\_B0032\_IdhA | 32-IdhA-Fw | IdhA-Suf-Rv |
| 9 | BBa\_B0034\_IdhA | 34-IdhA-Fw | IdhA-Suf-Rv |
| 10 | BBa\_B0031\_IdhA | 31-sulA-Fw | sulA-Suf-Rv |
| 11 | BBa\_B0032\_IdhA | 32-sulA-Fw | sulA-Suf-Rv |
| 12 | BBa\_B0034\_IdhA | 31-sulA-Fw | sulA-Suf-Rv |
| 13 | oHypB Promotor | Pre-HypB-Fw | HypB-Suf-Rv |
| 14 | oHypB Promotor | HypB-Fw | HypB-Rv |

Annealing:

|  |  |  |  |
| --- | --- | --- | --- |
| Tube | Part | Fw Primer | Rv Primer |
| 15 | BBa\_1718018 | dapA-Fw | dapA-Rv |

PCR program:

|  |  |
| --- | --- |
| 98 °C | 3 min |
| 98 °C | 10 s |
| 60 °C | 30 s |
| 72 °C | 40 s |
| Go to step 2 30x | |
| 72 °C | 5 min |

Annealing program, afterwards chilled on ice:

|  |  |
| --- | --- |
| 98 °C | 30 s |
| 50 °C | 30 s |
| 72 °C | 5 min |

## Minipreparation of BBa\_K206000 and BBa\_B0015

The miniprep was made with a GeneJET Plasmid Miniprep Kit by Thermo Scientific.

## Nanodrop measurement

Measurement for λ = 260 nm.

|  |  |  |
| --- | --- | --- |
| Lotnumber | Part | DNA-concentration [ng/µL] |
| 180307NK01 | psB1C3\_BBa\_K20600 | 46.99 |
| 180307NK02 | psB1C3\_BBa\_B0015 | 21.51 |

## Restriction

Restriction of pSB1C3\_BBa\_K20600, pSB1C3\_BBa\_E0840 and pSB1C3.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | psB1C3\_BBa\_K206  00 | pSB1C3\_BBa\_E08  40 | pSB1C3 | pSB1C3 |
| DNA concentration | 46.99 ng/µL | 57 ng/µL | 141 ng/µL | 149 ng/µL |
| DNA | 2.13 µL | 17.54 µL | 21.28 µL | 20.13 µL |
| NE Buffer 2.1 | 2 µL | 5 µL | 5 µL | 5 µL |
| Fast AP | - | 0.5 µL | 0.5 µL | 0.5 µL |
| EcoRI-HF | 0.5 µL | 0.5 µL | - | 1 µL |
| XbaI | - | 0.5 µL | 1 µL | - |
| SpeI | 0.5 µL | - | - | - |
| PstI | - | - | 1 µL | 1 µL |
| H 2 O | 14.87 µL | 25.96 µL | 21.22 µL | 22.37 µL |

The restriction incubated for 80 min at 38°C. After that the restricted plasmids, PCR products and annealing product were stored at -20°C.

## Agarose gel electrophoresis

Agarose gel electrophoresis of PCRs of parts from E.coli and restricted pSB1C3 and pSB1C3\_BBa\_E0840. We made a 1.5% agarose gel was loaded with PCR products of 180307 and annealed BB\_1718018 in respective order. MlcRE, Pre-MlcRE-Suf and oHypB, Pre-oHybB-Suf produced expected bands. All other bands were negative, bands of products were excised.

![](https://static.igem.org/mediawiki/2018/1/1f/T--Hamburg--180308_PCR.JPG)

Then we made another 1% agarose gel. It was loaded restricted pSB1C3, pSB1C3\_BBa\_E0840 and pSB1C3\_BBa\_K206000, bands of the first two were excised.

![](https://static.igem.org/mediawiki/2018/d/d9/T--Hamburg--180308_Restriction.JPG)

The excised bands were used for gel extraction with Invisorb Fragment CleanUp kit. The DNA was lost in the process. Use of said kit was discontinued.

## PCR for amplification of parts by E.coli

Master mix for 30 preparations:

* 300 µL 5 x Phusion HF Buffer
* 30 µL dNTPs
* 30 µL Template DNA (E. coli cells)
* 15 µL Phusion DNA polymerase
* 1065 µL H2Odest

Added 1 µL primer to each 48 µL mastermix.

|  |  |  |  |
| --- | --- | --- | --- |
| No. | Amplified Part | Fw Primer | Rv Primer |
| 1 | MlcRE | Pre- MlcRE-Fw | MlcRE-Suf-Rv |
| 1 | MlcRE | MlcRE-Fw | MlcRE-Rv |
| 3 | IdhA | Pre-IdhA-Fw | IdhA-Suf |
| 4 | IdhA | IdhA-Fw | IdhA-Rv |
| 5 | sulA | Pre-sulA-Fw | sulA-Suf-Rv |
| 6 | sulA | sulA-Fw | sulA-Rv |
| 7 | BBa\_B0031\_IdhA | 31-IdhA-Fw | IdhA-Suf-Rv |
| 8 | BBa\_B0032\_IdhA | 32-IdhA-Fw | IdhA-Suf-Rv |
| 9 | BBa\_B0034\_IdhA | 34-IdhA-Fw | IdhA-Suf-Rv |
| 10 | BBa\_B0031\_IdhA | 31-sulA-Fw | sulA-Suf-Rv |
| 11 | BBa\_B0032\_IdhA | 32-sulA-Fw | sulA-Suf-Rv |
| 12 | BBa\_B0034\_IdhA | 31-sulA-Fw | sulA-Suf-Rv |
| 13 | oHypB Promotor | Pre-HypB-Fw | HypB-Suf-Rv |
| 14 | oHypB Promotor | HypB-Fw | HypB-Rv |

Annealing:

|  |  |  |  |
| --- | --- | --- | --- |
| No. | Amplified Part | Fw Primer | Rv Primer |
| 15 | BBa\_1718018 | dapA-Fw | dapA-Rv |

PCR program:

|  |  |
| --- | --- |
| 98°C | 3 min |
| 98°C | 10 s |
| 60°C | 30 s |
| 72°C | 40 s |
| Go to step 2 30x | |
| 72°C | 5 min |

Annealing program, afterwards chilled on ice:

|  |  |
| --- | --- |
| 98°C | 30 s |
| 50°C | 30 s |
| 72°C | 5 min |

## Restriction

Restriction of pSB1C3\_BBa\_K20600, pSB1C3\_BBa\_E0840 and pSB1C3.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | psB1C3\_BBa\_K20600 | pSB1C3\_BBa\_E0840 | pSB1C3 | pSB1C3 |
| DNA concentration | 46.99 ng/µL | 57 ng/µL | 141 ng/µL | 149 ng/µL |
| DNA | 2.13 µL | 17.54 µL | 21.28 µL | 20.13 µL |
| NE Buffer 2.1 | 2 µL | 5 µL | 5 µL | 5 µL |
| Fast AP | - | 0.5 µL | 0.5 µL | 0.5 µL |
| EcoRI-HF | 0.5 µL | 0.5 µL | - | 1 µL |
| XbaI | - | 0.5 µL | 1 µL | - |
| SpeI | 0.5 µL | - | - | - |
| PstI | - | - | 1 µL | 1 µL |
| H2Odest | 14.87 µL | 25.96 µL | 21.22 µL | 22.37 µL |

After the restriction the products incubated for 60 min (37°C) and afterwards stored on ice by 4°C.

## Agarose gel electrophoresis of PCR products

Agarose gel electrophoresis of PCRs of parts from E. coli from 180312. A 1.5% agarose gel was used and the products did produced neither expected nor defined bands and were discarded.

![](https://static.igem.org/mediawiki/2018/6/6c/T--Hamburg--180312_PCR1.JPG)
![](https://static.igem.org/mediawiki/2018/8/8b/T--Hamburg--180312_PCR2.JPG)

## Minipreparation

The miniprep was made by GeneJET Plasmid Miniprep Kit by Thermo Scientific with following differences: cells were pelleted by centrifugation for 3 min and 12.000 rpm instead of 4 min and 5.000 rpm, for elution 50 µL H2O instead of 50 µL elution buffer were used.

Miniprep of these parts:

|  |  |
| --- | --- |
| Part | Lot number |
| BBa\_E0840 | 180202NK08 |
| BBa\_I13504 | 180202NK09 |
| BBa\_I20270 | 180202NK10 |

## Nanodrop measurement

Measurement for λ = 260 nm

|  |  |  |
| --- | --- | --- |
| Lot number | Name | DNA-concentration [ng/µL] |
| 180315NB01 | BBa\_E0840 | 128.24 |
| 180315NB02 | BBA\_I13504 | 175.48 |
| 180315NB02 | BBa\_I20270 | 155.29 |

## Retaking the PCR from 180312

Retook the PCR from 180312 as described before.

## Restriction

Restriction of pSB1C3 (2x) and pSB1C3\_BBa\_E0840.

|  |  |  |  |
| --- | --- | --- | --- |
|  | pSB1C3 | pSB1C3 | pSB1C3\_BBa\_E0840 |
| DNA concentration | 176.00 ng/µL | 155.29 ng/µL | 128.00 ng/µL |
| DNA | 17.07 µL | 19.32 µL | 7.80 µL |
| NE Buffer 2.1 | 5.00 µL | 5.00 µL | 2.00 µL |
| EcoRI-HF | 1.00 µL | - | 0.50 µL |
| XbaI | - | 1.00 µL | 0.50 µL |
| SpeI | - | - | - |
| PstI | 1.00 µL | 1.00 µL | - |
| Fast AP | 0.50 µL | 0.50 µL | 0.50 µL |
| H2Odest | 25.42 µL | 23.18 µL | 8.70 µL |
| ad | 49.99 µL | 50.00 µL | 20.00 µL |

Incubated for 60 min by 37°C and afterwards stored on ice by 4°C.

## Gel extraction

The gel extraction was done using GeneJET Gel extraction Kit by Thermo Fisher.

## Restriction

The concentration of the parts from 180316 was measured by Nano Drop. The samples was pipetted together with a excel table. The table was not printed out. The samples incubated for 1 h by 37°C.

## Ligation

The concentration of the restricted parts was measured by Nano Drop. The concentrations were not noted. We did a mistake at the pipetting and cancelled the ligation.

## Retaking the PCR from 180312

Preparations of PCR tubes and PCR program were identical to the PCR from 180312.

![](https://static.igem.org/mediawiki/2018/a/aa/T--Hamburg--180320_PCR1.JPG)
![](https://static.igem.org/mediawiki/2018/a/a1/T--Hamburg--180320_PCR2.JPG)

The extracted DNA from successfully run PCRs via gel extraction with GeneJET Gel extraction Kit by Thermo Fisher.

 The purified DNAs got new lot numbers:

|  |  |
| --- | --- |
| Lot number | Part |
| 180320MW01 | Pre MlcRe suf |
| 180320MW02 | MlcRE without pre- and suffix |
| 180320MW03 | presulAsuf |
| 180320MW04 | sulA without pre- and suffix |
| 180320MW05 | BBa\_B0031sulA |
| 180320MW06 | BBa\_B0032sulA |
| 180320MW07 | BBa\_B0034sulA |
| 180320MW08 | preoHybBsuf |
| 180320MW09 | oHybB without pre- and suffix |

## Setting up of Minipreps of the ONC prepared on the 21st of March

The ONCs were purified with the “GeneJet Plasmid Miniprep Kit” of Thermo Fisher.

## Restriction and ligation

Restriction of vector-parts

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Part | pSB1C3 | pSB1C3 | pSB1C3-BBa\_E0840 | pSB1C3-K206000 |
| DNA | 17.1 µl | 17.1 µl | 7.8 µl | 18 µl |
| NEBBuffer 2.1 | 5 µl | 5 µl | 2 µl | 5 µl |
| EcoRI-HF | 1 µl | - | 0.5 µl | - |
| XbaI | - | 1 µl | 0.5 µl | - |
| SpeI | - | - | - | 0.5 µl |
| PstI | 1 µl | 1 µl | - | 0.5 µl |
| FastAP | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl |
| ddH2O | 25.4 µl | 25.4 µl | 8.7 µl | 25.5 µl |
| ad | 50 µl | 50 µl | 20 µl | 50 µl |

The samples were incubated at 37°C for 1 h and following 20 min at 80°C. Samples were applied on a 1.5% agarose gel and extracted through the “GeneJet Gel Extraction Kit” of Thermo Fisher.

![](https://static.igem.org/mediawiki/2018/5/5d/T--Hamburg--180321_Restriktion.JPG)

Restiction of the PCR products

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Part | MlcRE (1) | MlcRE (2) | sulA | BBa\_B0031-sulA | BBa\_B0032-sulA | BBa\_B0034-sulA | oHybB (1) | oHybB (2) |
| DNA | 10 µl | 10 µl | 20 µl | 20 µl | 20 µl | 20 µl | 20 µl | 20 µl |
| NEBBuffer 2.1 | 2 µl | 2 µl | 5 µl | 5 µl | 5 µl | 5 µl | 5 µl | 5 µl |
| EcoRI-HF | 0.5 µl | 0.5 µl | 0.5 µl | - | - | - | 0.5 µl | 0.5 µl |
| XbaI | - | - | - | 0.5 µl | 0.5 µl | 0.5 µl | - | - |
| SpeI | - | 0.5 µl | - | - | - | - | - | 0.5 µl |
| PstI | 0.5 µl | - | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl | - |
| FastAP | - | - | - | - | - | - | - | - |
| ddH2O | 7 µl | 7 µl | 24 µl | 24 µl | 24 µl | 24 µl | 24 µl | 24 µl |
| ad | 20 µl | 20 µl | 50 µl | 50 µl | 50 µl | 50 µl | 50 µl | 50 µl |

The samples were incubated at 37°C for 1 h and following 20 min at 80°C.

Ligation

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Product | pSB1C3-MlcRE(1) | pSB1C3-MlcRE(2)-BBa\_E0840 | pSB1C3-sulA | pSB1C3-BBa\_B0031-sulA | pSB1C3-BBa\_B0032-sulA |
| Vector | 3.01 µl | 7.7 µl | 1.86 µl | 1.97 µl | 1.97 µl |
| Part | 8.25 µl | 7.09 µl | 15.18 µl | 9.48 µl | 13.07 µl |
| T4 Buffer | 2 µl | 2 µl | 2 µl | 2 µl | 2 µl |
| T4 Ligase | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl |
| ddH2O | 6.24 µl | 2.71 µl | 0.46 µl | 6.06 µl | 2.46 µl |
| ad | 20 µl | 20 µl | 20 µl | 20 µl | 20 µl |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Product | pSB1C3-BBa\_B0034-sulA | pSB1C3-oHybB(1) | pSB1C3-oHybB(2)-E0840 | pSB1C3-oHybB(2) |
| Vector | 1.97 µl | 3.29 µl | 8.52 µl | 3.29 µl |
| Part | 22.56 µl | 9.21 µl | 8.26 µl | 9.94 µl |
| T4 Buffer | 5 µl | 2 µl | 2 µl | 2 µl |
| T4 Ligase | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl |
| ddH2O | 19.97 µl | 4.99 µl | 0.72 µl | 4.27 µl |
| ad | 50 µl | 20 µl | 20 µl | 20 µl |

The samples were incubated at 16°C overnight.

## Transformation of the ligations perfomed on the 21st of March

1. 150 µl Competent cell aliquots were thawed on ice for 15-30 min.
2. 10 µl DNA were added and mixed
3. Cells were incubated for 35 min on ice
4. Heat-shock: 42°C for 45 sec, then put back on ice for 2-5 min
5. Pre-warm 850 µl LB-Medium
6. Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
7. Cetrifuge 4500 rpm 2 min to collect cells.
8. Put cells on appropriate plates or medium
9. Incubation at 37°C overnight.

## Colony PCR of the transformation performed on the 22nd of March

Mastermix

|  |  |  |
| --- | --- | --- |
| Substance | 1x (volume in µl) | 60x (volume in µl) |
| 10x DreamTaq Buffer | 2 | 120 |
| dNTPs 10mM | 2 | 120 |
| MgCl2 50mM | 1.2 | 72 |
| Fw-Primer VF2 | 0.6 | 36 |
| Rv-Primer VR | 0.6 | 36 |
| Taq-Polymerase | 0.1 | 6 |
| ddH2O ad 20µl | 13.5 | 810 |

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Programm (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 95 | 180 |
| 94 | 30 |
| 55 | 30 |
| 72 | 90 |
| 72 | 600 |

PCR products were stored at 4°C over the weekend, no agarose gel was performed.

## Repetition of the colony PCR performed on the 29th of March

The colony PCR perfomed on the 29th of March had been applied on an 1% agarose gel but since the DNA-ladder had been forgotten the colony PCR was repeated.

Mastermix

|  |  |  |
| --- | --- | --- |
| Substance | 1x (volume in µl) | 60x (volume in µl) |
| 10x DreamTaq Buffer | 2 | 120 |
| dNTPs 10mM | 2 | 120 |
| MgCl2 50mM | 1.2 | 72 |
| Fw-Primer VF2 | 0.6 | 36 |
| Rv-Primer VR | 0.6 | 36 |
| Taq-Polymerase | 0.1 | 6 |
| ddH2O ad 20µl | 13.5 | 810 |

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Programm (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 95 | 180 |
| 94 | 30 |
| 55 | 30 |
| 72 | 90 |
| 72 | 600 |

![](https://static.igem.org/mediawiki/2018/c/c2/T--Hamburg--180403_cPCR.JPG)

The bands were accidentally cut out and purified by using the “GeneJet Gel Extraction Kit” from Thermo Fisher.

## PCR of multiple parts.

PCR were prepapared as followed:

|  |  |  |  |
| --- | --- | --- | --- |
| Tube | Part | Primer-fw | Primer-rv |
| 1 | IdhA | Pre\_IdhA-Fw | IdhA-Suf-Rev |
| 2 | IdhA | IdhA-Fw | IdhA-Rev |
| 3 | BBa\_B0031\_IdhA | 31-Idha-Fw | IdhA-Suf-Rev |
| 4 | BBa\_B0032\_IdhA | 32-Idha-Fw | IdhA-Suf-Rev |
| 5 | BBa\_B0034\_IdhA | 34-Idha-Fw | IdhA-Suf-Rev |
| 6 | BBa\_B0032\_sulA | 32-sulA-Fw | SulA-Suf-Rev |
| 7 | oHybB | Pre-HybB-Fw | HybB-Suf-Rev |
| 8 | oHybB | HybB-Fw | HybB-Rev |
| 9 (Annealing) | BBa\_I718018 | dapAp-Fw | dapAp-Rev |

Mastermix for 15 approaches were prepared as shown:

|  |  |
| --- | --- |
| 150 µL | 5x Phusion HF Buffer |
| 15 µL | dNTPs |
| 15 µL | Template DNA (DH5α) |
| 7.5 µL | Phusion DNA Polymerase |
| 532.5 µL | H2O |

This was aliquoted into 0.5 mL reaction tube to total volume of 48 µL. To each aliquot were added the specific primer pair, 1 µL forward and 1 µL reverse primer.

The PCR program was done as shown in the next table.

|  |  |  |
| --- | --- | --- |
| 1 | 98 °C | 3 min |
| 2 | 98 °C | 10 s |
| 3 | 60 °C | 30 s |
| 4 | 72 °C | 60 s |
| 5 | go to step 2 30 times | |
| 6 | 72 °C | 5 min |

For the Annealing we used the following program.

|  |  |  |
| --- | --- | --- |
| 1 | 98 °C | 30 s |
| 2 | 50 °C | 30 s |
| 3 | 60 °C | 5 min |

## Restriction of pSB1C3 and BBa\_K206000

For the digestion of pSB1C3 we used two approaches and for BBa\_K206000 one approach as shown in the following table.

|  |  |  |  |
| --- | --- | --- | --- |
|  | pSB1C3 (R1) | pSB1C3 (R2) | BBa\_K206000 (R3) |
| DNA content | 155.29 ng/µL | 155.29 ng/µL | 140.31 ng/µL |
| DNA used | 19.32 µL | 19.32 µL | 19.00 µL |
| NE-Buffer 2.1 | 5.00 µL | 5.00 µL | 5.00 µL |
| FAST-Ap | 0.50 µL | 0.50 µL | 0.50 µL |
| EcoRI-HF | 1.00 µL | 0.50 µL | - |
| XbaI | - | 1.00 µL | - |
| PstI | 1.00 µL | 1.00 µL | 0.50 µL |
| SpeI | - | - | 0.50 µL |
| H2O | 23.18 µL | 23.18 µL | 25.5 µL |

The restriction digests were incubated for 1 hour at 37 °C.

With the gel electroporation the correctness for the PCR and restriction can be shown. In this approach the PCR of Tube 6, 7 and 8 were correct and the restriction digest of all three constructs worked. For this 6 constructs a gel extraction with the Thermo Scientific Gel extraction Kit was done and the concentration was measured with a nanodrop.

|  |  |
| --- | --- |
| Probe | Concentration |
| Tube 6 | 17.863 ng/µL |
| Tube 7 | 29.722 ng/µL |
| Tube 8 | 30.555 ng/µL |
| R1 | 24.91 ng/µL |
| R2 | 6.813 ng/µL |
| R3 | 30.245 ng/µL |

## Miniprep of a colony PCR

For the miniprep of the colony PCR products these listed parts were used. The miniprep was done with the Kit from Thermo Scientific protocol A. The last centrifugation step was done by 8600 rpm for 2 min. The concentration was measured with a nanodrop.

|  |  |
| --- | --- |
| Lot number | Concentration |
| 180329DW02 | 72.183 ng/µL |
| 180329DW03 | 101.987 ng/µL |
| 180329DW05 | 9.721 ng/µL |
| 180329DW06 | 9.051 ng/µL |
| 180329DW09 | 131.545 ng/µL |
| 180329DW10 | 98.964 ng/µL |
| 180329DW14 | 25.731 ng/µL |
| 180329DW15 | 26.956 ng/µL |
| 180329DW21 | 75.207 ng/µL |
| 180329DW23 | 76.729 ng/µL |
| 180329DW26 | 18.672 ng/µL |
| 180329DW27 | 31.688 ng/µL |
| 180329DW28 | 139.269 ng/µL |
| 180329DW30 | 9.514 ng/µL |
| 180329DW39 | 93.788 ng/µL |
| 180329DW41 | 101.325 ng/µL |
| 180329DW44 | 101.745 ng/µL |
| 180329DW45 | 17.186 ng/µL |

The Parts were stored at -20 °C

## Repetition  PCR ldHA/Annealing

|  |  |  |
| --- | --- | --- |
| Tube | Part | Primer |
| 1 | ldhA | Pre-ldhA-Fw + ldhA-suf-Rv |
| 2 | ldhA | ldhA-Fw + ldhA-Rv |
| 3 | BBa\_B0031\_ldhA | 31-LdhA-Fw + ldhA-suf-Rv |
| 4 | BBa\_B0032\_ldhA | 32-ldhA-Fw + ldhA-suf-Rv |
| 5 | BBa\_B0034\_ldhA | 34-ldhA-Fw + ldhA-suf-Rv |
| 6 | BBa\_I718018 | dapAp-Fw + dapAp-Rv |

Mastermix for 15 uses:

* 150 µL 5x Phusion HF Buffer
* 15 µL dNTPs
* 15 µL Template DNA (E.coli cells DH5a)
* 5 µL Phusion DNA Polymerase
* 5 µL H2Odest

Mastermix 48 µL + each 1 µL Primer

PCR-Programm:

|  |  |
| --- | --- |
| 98°C | 3 min |
| 98°C | 10 s |
| 60°C | 30 s |
| 72°C | 90 s |
| GOTO Step 2 30x | |
| 72°C | 5 min |

Ligation-Programm

|  |  |
| --- | --- |
| 98°C | 3 min |
| 98°C | 30 s |
| 50°C | 30 s |
| 72°C | 5 min |
| GOTO Step 2 30x | |
| 72°C | 5 min |

## Gelelectrophoresis:

We made 1.5% Agarose-Gel. It ran for 40 min by 120 V.

![](https://static.igem.org/mediawiki/2018/5/51/T--Hamburg--180406_PCR.JPG)

After that we did a gelextraction with GeneJet Gel Extraction Kit by Thermofischer.

Extraction of: ldHA, Pre-ldhA, BBa\_B0031\_ldhA, BBa\_B0032\_ldhA, BBa\_B0034\_ldhA

Lotnumbers:

* 180406NK01 = ldhA with pre- and -suffix
* 180406NK02 = ldhA
* 180406NK03 = BBa\_B0031\_ldhA
* 180406NK04 = BBa\_B0032\_ldhA
* 180406NK05 = BBa\_B0034\_ldhA
* 180406NK06 = ldhA (reserve)
* Stored at -20°C

## Restriction of BBa\_B0032\_sulA and oHybB (Pre + Suffix)

|  |  |  |  |
| --- | --- | --- | --- |
|  | BBa\_B0032\_sulA | oHybB | oHybB |
| DNA | 5.6 µL | 3.36 µL | 3.33 µL |
| NE 2.1 | 2 µL | 2 µL | 2 µL |
| EcoR1 | - | 0.5 µL | 0.5 µL |
| Xba1 | 0.5 µL | - | - |
| Spe1 | - | - | 0.5 µL |
| Pst1 | 0.5 µL | 0.5 µL | - |
| FastAp | - | - | - |
| H2O | 11.4 µL | 13.64 µL | 13.67 µL |
| Total | 20 µL | 20 µL | 20 µL |

Incubated for 1 h by 37°C and stored at -20°C.

* 180406DK01 = BBa\_B0032\_sulA
* 180406DK02 = oHybB cut with EcoR1 and Pst1
* 180406DK03 = OHybB cut with EcoR1 and Spe1

## Ligation of psB1c3\_BBa-B0032\_sulA (L12) and psB1C3\_oHybB (L16)

|  |  |  |
| --- | --- | --- |
| Product | psB1C3\_BBa\_B0032\_sulA | psB1C3\_oHybB |
| Vector | psB1C3 (180406DK01) | oHybB (180406DK03) |
| Volume | 3.94 µL | 1.93 µL |
| Part | BBa\_B0032\_sulA (180406DK01) | oHybB (180406DK03) |
| Volume | 6 µL | 1.14 µL |
| T4 Buffer | 2 µL | 2 µL |
| T4 Ligase | 0.5 µL | 0.5 µL |
| H2O | 7.56 µL | 14.43 µL |
| Total | 20 µL | 20 µL |

Incubated for 1 h by 37°C and stored at -20°C.

New Lotnumbers:

* 180406BK01 = psB1C3\_BBa\_B0032\_sulA
* 180406BK02 = psB1C3\_oHybB

## PCR of BBa\_B0031SulA, BBa\_1718018 (Annealing)

After the scheme on page 18 a new Mastermix was pipetted together

|  |  |  |
| --- | --- | --- |
| Tube | Part | Primer |
| 1 | BBa\_B0031SulA | 31-SulA-FW + SulA-Suf-RV |
| 2 | BBa\_1718018 | DapAP-FW + DapAP-RV |

48 µL Mastermix per 1 µL Primer was used.

PCR-Program

|  |  |
| --- | --- |
| 98 °C | 3 min |
| 98 °C | 10 s |
| 60 °C | 30 s |
| 72 °C | 90 s |
| GOTO Step 2 30 x | |
| 72 °C | 5 min |

Annealing-Program

|  |  |
| --- | --- |
| 98 °C | 30 s |
| 50 °C | 40 s |
| 72°C | 5 min |

Restriction of LdhA

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Name | LdhA | BBa\_B0031\_LdhA | BBa\_0032\_LdhA | BBa\_0034\_LdhA |
| NE 2.1 buffer | 40 µL | 40 µL | 40 µL | 40 µL |
| EcoRI | 0.5 µL | - | - | - |
| XbaI | - | 0.5 µL | 0.5 µL | 0.5 µL |
| SpeI | - | - | - | - |
| PstI | 0.5 µL | 0.5 µL | 0.5 µL | 0.5 µL |
| Fast AP | - | - | - | - |
| H2O dest. | 4 µL | 4 µL | 4 µL | 4 µL |
| ges. | 50 µL | 50 µL | 50 µL | 50 µL |

Incubatated at 27 °C for 1h

|  |  |
| --- | --- |
| LdhA | 180409NK01 |
| 31LdhA | 180409NK02 |
| 32LdhA | 180409NK03 |
| 34LdhA | 180409NK04 |

## Gelelektrophorese

U  = 120 V

t = 30 min

![](https://static.igem.org/mediawiki/2018/8/8b/T--Hamburg--180409_PCR.JPG)

Gelextraction

|  |  |  |  |
| --- | --- | --- | --- |
|  | M(tube) | m(Agarose) | Added buffer |
| Annealing: | 0.9925 g | - | - |
| PCR: | 0.9906 g | 1.1417 g | 151.1 µL |

Gelextraction-Kit used: Thermo Scientific GeneJet Gel Extraction Kit #R0691,#R0692

Annealing did not work, expected band not found.

DNA-concentration BBa\_B0031\_SulA: 23.386 ng/µL.

## Repetition of the PCR of BBa\_I718018

Mastermix without template (for 3 PCRs):

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| 5x Phusion HF Buffer | 30 |
| dNTPs | 3 |
| Phusion DNA Polymerase | 1.5 |
| H2O | 109.5 |

Total: 48 µL Mastermix

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| dapAP-Fw | 1 |
| dapAP-Rev | 1 |

PCR-Programm (30 cycles)

|  |  |
| --- | --- |
| Temperature (C°) | Time (s) |
| 98 | 180 |
| 98 | 10 |
| 60 | 30 |
| 72 | 90 |
| 72 | 300 |

## Restriction of BBa\_B0031\_sulA mit XbaI and PstI

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| DNA | 4.28 |
| NF 2.1 | 2.00 |
| XbaI | 0.50 |
| PstI | 0.50 |
| H2O | 12.72 |

Total: 20.00 µL Mastermix

Restriction (180412BK01) was incubated at 37 °C for 1 h and stored at -20°C

## Ligation of pSB1C3-IdhA, pSB1C3-BBa\_0031-IdhA, pSB1C3-BBa\_B0032-IdhA and pSB1C3-BBa\_B0034-IdhA

pSB1C3(1) was cut with EcoR1-Hf and PstI; pSB1C3(2) was cut with XbaI and PstI

 Construct: pSB1C3-IdhA

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Vector pSB1C3 (1) | 0.67 |
| Part IdhA | 3.35 |
| T4 Buffer | 2.00 |
| T4 Ligase | 0.50 |
| H2O | 13.45 |

Total: 20 µL Ligated part name: 180412NK01

Construct: pSB1C3-BBa\_B0031-IdhA

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Vector pSB1C3 (2) | 2.49 |
| Part BBa\_B0031-IdhA | 5.72 |
| T4 Buffer | 2.00 |
| T4 Ligase | 0.50 |
| H2O | 9.35 |

Total: 20 µL Ligated part name: 180412NK02

Construct: pSB1C3-BBa\_B0032-IdhA

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Vector pSB1C3 (2) | 2.49 |
| Part BBa\_B0032-IdhA | 5.72 |
| T4 Buffer | 2.00 |
| T4 Ligase | 0.50 |
| H2O | 9.36 |

Total: 20 µL Ligated part name: 180412NK03

Construct: pSB1C3-BBa\_B0034-IdhA

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Vector pSB1C3 (2) | 2.43 |
| Part BBa\_B0034-IdhA | 4.77 |
| T4 Buffer | 2.00 |
| T4 Ligase | 0.50 |
| H2O | 10.30 |

Total: 20 µL Ligated part name: 180412NK04

Each ligated part was incubated at 37°C for 1h and was stored at -20°C.

## Repetition of the PCR of MlcRE with Pre and Suffix

Mastermix for (30 cycles) was prepared:

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| 5x Phusion HF Buffer | 300 |
| dNTPs | 30 |
| Template DNA (E. coli cells) | 30 |
| Phusion DNA Polymerase | 15 |
| H2O | 1065 |

Primers:

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| dapAP-Fw | 1 |
| dapAP-Rev | 1 |

For each PCR 48µL of the Mastermix and 1µL of the pair of primers was used.

PCR-Programm (30 cycles)

|  |  |
| --- | --- |
| Temperature (C°) | Time (s) |
| 98 | 180 |
| 98 | 10 |
| 60 | 30 |
| 72 | 90 |
| 72 | 300 |

## Ligation of pSB1C3-BBa\_K206000-BBa\_B0031-SulA

Construct: pSB1C3-BBa\_K206000-BBa\_B0031-sulA

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Vector pSB1C3-BBa\_K206000 | 1.00 |
| Part BBa\_B0031-IdhA | 4.77 |
| T4 Buffer | 2.00 |
| T4 Ligase | 0.50 |
| H2O | 12.60 |

Total: 20 µL Ligated part name: 180412NK05

Part was incubated at 37°C for 1h and was stored at -20°C.

## Preparation of media

LB-Agar: 40 g on 1 L

LB-Medium: 25 g on 1 L

## Gel electrophoresis of MlcRE with Pre and Suffix and BBa\_I718018

![](https://static.igem.org/mediawiki/2018/a/ab/T--Hamburg--1804012_PCR.JPG)

Gel electrophoresis with MlcRE with pre and suffix and BBa\_I718018  at 120 V, 30 min

## Gelextraction of MlcRE with pre-and suffix

Gel extraction of MlcRE with pre- and suffix was performed using GeneJet Gel Extraction kit (Thermo scientific). Concentration of DNA (180412BK02) was measured (8.079 ng/µL) and stored at .20 °C.

## Restriction of MlcRE with pre-and suffix

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| DNA | 12.38 |
| NF 2.1 | 2.00 |
| EcoR/-HF | 0.50 |
| SpeI | 0.50 |
| H2O | 4.62 |

Total: 20 µL

Restriction (180412BK09) was incubated at 37°C for 1 h.

## Ligation of pSB1C3-MIcRE-BBa\_B0031-IdhA, pSB1C3-MlcRE-BBa\_B0032-idhA, pSB1C3-MlcRE-BBa-B0034-IdhA

Each Ligation had a total Volume of 20 µL and was run at 16 °C overnight.

L3:

Construct: pSB1C3-MlcRE-BBa\_B0031-IdhA (180417AK01)

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Vector pSB1C3 (1) | 0.55 |
| Part 1: MlcRE | 2.07 |
| Part 2: 31 IdhA | 4.76 |
| T4 Buffer | 2.00 |
| T4 Ligase | 0.50 |
| H2Odest | 10.12 |

L4:

Construct: pSB1C3-MlcRE-BBa\_B0032-IdhA (180417AK02)

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Vector pSB1C3 (1) | 0.55 |
| Part 1: MlcRE | 2.07 |
| Part 2: 32 IdhA | 4.76 |
| T4 Buffer | 2.00 |
| T4 Ligase | 0.50 |
| H2Odest | 10.12 |

L5:

Construct: pSB1C3-MlcRE-BBa\_B0031-IdhA (180417AK03)

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Vector pSB1C3 (1) | 0.55 |
| Part 1: MlcRE | 2.07 |
| Part 2: 34 IdhA | 3.97 |
| T4 Buffer | 2.00 |
| T4 Ligase | 0.50 |
| H2Odest | 10.90 |

## Preparation of agar-Plates

1 L of LB-Agar was prepared and 1 mL Chloramphenicol (cAMP) was added.  44 agar-plates were prepared.

## Transformations of L3-L9, L24 preparation of agar-plates

Transformations were performed as follows:

DH5α were thawn on ice for 15 to 30 min. DNA was added and mixed. After 35 min a heat-shock at 42°C for 45 sek. was performed. While the cells were put back on ice for 5 min 850 µL of LB medium was pre-warmed. LB Medium was added to the cells and incubated at 37°C, 900 rpm for 1h followed by centrifugation at 4500 rpm for 2 min. Cells were put on agar-plates and grown over night at 37°C.

## Repetition of Transformations L3-L9, L24

Transformations were performed as follows:

DH5α were thawn on ice for 15 to 30 min. DNA was added and mixed. After 35 min a heat-shock at 42°C for 45 sek. was performed. While the cells were put back on ice for 5 min 850 µL of LB medium was pre-warmed. LB Medium was added to the cells and incubated at 37°C, 900 rpm for 1h followed by centrifugation at 4500 rpm for 2 min. Cells were put on agar-plates and grown over night at 37°C.

## Repetition of the PCR of MlcRE with Pre and Suffix

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| 5x Phusion HF Buffer | 10 |
| dNTPs | 1 |
| Phusion DNA Polymerase | 0.5 |
| H2O | 35.5 |
| Template DNA (MlcRE) | 1 |
| Pre-MlcRE-Fw | 1 |
| Suf-MlcRE-Rv | 1 |

## PCR purification using Gene Jet PCR Purification Kit (eluted with ddH2O)

Concentration of purified DNA (180419LP09) was measured (153.53 ng/µL) and the DNA stored at -20 °C.

## Gel electrophoresis of MlcRE (PCR Product)

![](https://static.igem.org/mediawiki/2018/1/1f/T--Hamburg--180419.JPG)

Gel electrophoresis with MlcRE at 120 V, 30 min (1.5 % Agarose-Gel). The band at 250 bp indicates a successful PCR.

## Repetition of Transformations L3-L9, L24, and Transformation of L12 and L16

Since the Transformations L3-L9 and L24 were overgrown, transformations were repeated as follows:

DH5α were thawn on ice for 15 to 30 min. DNA was added and mixed. After 35 min a heat-shock at 42°C for 45 sek. was performed. While the cells were put back on ice for 5 min 850 µL of LB medium was pre-warmed. LB Medium was added to the cells and incubated at 37°C, 900 rpm for 1h followed by centrifugation at 4500 rpm for 2 min. Cells were put on agar-plates and grown over night at 22°C.

For the transformations of L12 and L16 a different protocol was used:

DH5α were thawn on ice for 10 min. 10 µL DNA was added to 50 µL of the aliquot and mixed. After 30 min a heat-shock at 42°C for 45 sek. was performed. While the cells were put back on ice for 3 min 300 µL of LB medium was pre-warmed. LB Medium was added to the cells and incubated at 37°C, 900 rpm for 1h. Cells were put on agar-plates and grown over night at 37°C.

## Repetition of Ligations L12, L16 and L5

Each Ligation had a total Volume of 20 µL and was run for 1h, 400 rpm at 37 °C.

L5:

Construct: pSB1C3-MlcRE-BBa\_B0034-IdhA (180425LD01)

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Vector | 0.55 |
| Part 1 | 2.07 |
| Part 2 | 3.97 |
| T4 Buffer | 2.00 |
| T4 Ligase | 0.50 |
| H2Odest | 10.09 |

L12:

Construct: pSB1C3-BBa\_B0032-SulA (180425LD02)

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Vector | 3.94 |
| Part 1 | 6.00 |
| Part 2 | - |
| T4 Buffer | 2.00 |
| T4 Ligase | 0.50 |
| H2Odest | 7.50 |

L16:

Construct: pSB1C3-oHybB (180425LD03)

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Vector | 1.93 |
| Part 1 | 1.14 |
| Part 2 | - |
| T4 Buffer | 2.00 |
| T4 Ligase | 0.50 |
| H2Odest | 14.43 |

## Transformations of L3 (pSB1C3-MlcRE-B0031-IdhA) and L4 (pSB1c3-MlcRE-BBa-B0032-IdhA), L5, L12, and L16

DH5α were thawn on ice for 15 to 30 min. DNA was added and mixed. After 35 min a heat-shock at 42°C for 45 sek. was performed. While the cells were put back on ice for 5 min 850 µL of LB medium was pre-warmed. LB Medium was added to the cells and incubated at 37°C, 900 rpm for 1h followed by centrifugation at 4500 rpm for 2 min. Cells were put on agar-plates and grown over night at 37°C.

|  |  |
| --- | --- |
| LOT-Number | Ligation |
| 180425NK01 | L3 |
| 180425NK02 | L4 |
| 180425LD04 | L5 |
| 180425LD05 | L12 |
| 180425LD06 | L16 |

## Colony PCR of L 3, L4, L6-L9, L24

Mastermix:

|  |  |
| --- | --- |
| Substance | Volume (µL) |
| Taq-Buffer | 80 |
| dNTPs (10 mM) | 16 |
| MgCl2 (50 mM) | 24 |
| VF2 (10 µL) | 24 |
| VF (10 µL) | 24 |
| H2O | 632 |

Total: 800 µL Mastermix

Colonies were picked with pipette-tips and transferred into 1 mL LB-Medium tubes after they were put in 20 µL aliquots of the mastermix for the following PCR-reaction.

PCR-Programm (30 cycles)

|  |  |
| --- | --- |
| Temperature (C°) | Time (s) |
| 98 | 180 |
| 98 | 10 |
| 60 | 30 |
| 72 | 90 |
| 72 | 300 |

Gel-electrophoresis of the PCR-Products

![](https://static.igem.org/mediawiki/2018/1/1e/T--Hamburg--180426_cPCR1.JPG)
![](https://static.igem.org/mediawiki/2018/d/db/T--Hamburg--180426_cPCR2.JPG)

Following bands were positive: 180426NK01, 02, 03, 04, 20, 28, 30

## Culture pSB1c3 for Minipreps was prepared

Cryostock cells (psB1C3-BBa\_120270) were grown for 6 h at 37°C in 3 mL LB medium. 1 mL was then transferred twice to two 11 mL cultures of LB-Medium.

## Overnight cultures of PCR-Products were prepared

Overnight Cultures (3 mL of LB-Medium + 100 µL of colonies (in LB-medium)) of each of the following colonies:

180426NK01, 02, 03, 04, 20, 28, 30

were prepared. (37°C, 220 rpm)

## Minipreps of 180426NK01, 02, 03, 04, 20, 28, 30

Minipreps were performed using GeneJet Plasmid Miniprep Kit from Thermo Fischer

|  |  |
| --- | --- |
| LOT-Number | Concentration (ng/µL) |
| 180426NK01 | 44.431 |
| 180426NK02 | 124.428 |
| 180426NK03 | 38.912 |
| 180426NK04 | 69.166 |
| 180426NK20 | 63.040 |
| 180426NK28 | 85.514 |
| 180426NK30 | 80.416 |

## Set up of Midiprep from pSB1C3-BBa\_I20270

The culture set up on the the 2nd of March was used to set up another midiprep (11ml LB-Medium plus 1ml culture). The rest was purified with the “GeneJet Plasmid Midiprep Kit”of Thermo Fisher.

## Set up of Midiprep from pSB1C3-BBa\_I20270

The culture set up on the 4th of March was purified with the “GeneJet Plasmid Midiprep Kit” of Thermo Fisher.

Step 7: another 20 min were centrifuged

Step 15: step was repeated with the eluate

## Resuspension of kit-DNA

pSB1C3-BBa\_B0030: kit 2017 plate 4, well 4G

pSB1C3-BBa\_B0031: kit 2017 plate 2, well 2H

pSB1C3-BBa\_B0032: kit 2017 plate 2, well 2J

pSB1C3-BBa\_B0033: kit 2017 plate 2, well 2L

pSB1C3-BBa\_B0034: kit 2017 plate 4, well 1N

pSB1C3-BBa\_K554008: kit 2017 plate 6, well 11I

pSB1C3-BBa\_K554009: kit 2017 plate 1, well 21O

pSB1C3-BBa\_K554002: kit 2017 plate 1, well 21G

DNA was resuspended in 10 µl ddH2O and incubated for 15 min at room temperature.

## Transformation of the kit-DNA into DH5α

10. 50 µl Competent cell aliquots were thawed on ice for 40 min.
11. 3 µl DNA were added and mixed
12. Cells were incubated for 40 min on ice
13. Heat-shock: 42°C for 70 sec, then put back on ice for 7 min
14. Pre-warm 300 µl LB-Medium
15. Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
16. Cetrifuge 4500 rpm 2 min to collect cells.
17. Put cells on appropriate plates or medium
18. Incubation at 37°C overnight.

## Set up of ONCs

pSB1C3-BBa\_I20270 was set up as an ONC at 37°C

## Set up of chloramphenicol-stocks

340 mg chloramphenicol were added to 10 ml 96% EtOH. Aliquots of 1 ml were made.

## ONCs from the performed transformations of the 8th of March

Except the pSB1C3-BBa\_B0030 all plates showed colonies and ONCs got set up.

## Set up of Midiprep from pSB1C3-BBa\_I20270

The culture set up on the 8th of March was purified with the “GeneJet Plasmid Midiprep Kit” of Thermo Fisher.

## ONCs from the performed transformations of the 8th of March

Since the ONCs set up on the 9th of March weren’t purified, new ones were performed.

## Setting up Minipreps of the ONCs performed on the 13th of March

The ONCs were purified with the “GeneJet Plasmid Miniprep Kit” of Thermo Fisher.

## Transformation of the ready parts for the cryostocks (characterizations)

Protocol was by Zhang Ghong.

|  |  |  |
| --- | --- | --- |
| Lot number plasmid | Part | Lot number culture |
| 180329DW03 | pSB1C3-MlcRE | 180517LD01 |
| 180329DW05 | pSB1C3-MlcRE-BBa\_E0840 | 180517LD02 |
| 180329DW09 | pSB1C3-sulA | 180517LD03 |
| 180329DW14 | pSB1C3-BBa\_B0031\_sulA | 180517LD04 |
| 180329DW21 | pSB1C3-BBa\_B0034\_suA | 180517LD05 |
| 180329DW27 | pSB1C3-oHybB | 180517LD06 |
| 180329DW28 | pSB1C3-oHybB\_BBa\_E0840 | 180517LD07 |
| 180329DW41 | pSB1C3-BBa\_K20600\_BBa\_B0032\_sulA | 180517LD08 |
| 180329DW45 | pSB1C3- BBa\_K20600\_BBa\_B0034\_sulA | 180517LD09 |
| 180426NK20 | pSB1C3-BBa\_B0032-LdhA | 180517LD10 |
| 180426NK28 | pSB1C3- BBa\_B0034-LdhA | 180517LD11 |
| 180426NK30 | pSB1C3-BBa\_K20600\_BBa\_B0031\_sulA | 180517LD12 |
| 180509NF01 | pSB1C3-BBa\_I20270 | 180517LD13 |
| 180508OM01 | pSB1C3-BBa\_B0030 | 180517LD14 |

## Cryostocks and inoculate the precultures

We put 5 mL LB medium into culture tubes and added 5 µL Camp. Inoculated with 180517LD01 – LD14 and 180508OM05 – OM12. Stored over night by 37°C and 295 rpm.

## Setting up cryotocks

|  |  |  |
| --- | --- | --- |
| Lot number culture | Part | Lot number cryostock |
| 180517LD01 | pSB1C3-MlcRE | 180518BK01 |
| 180517LD02 | pSB1C3-MlcRE-BBa\_E840 | 180518BK02 |
| 180517LD03 | pSB1C3-sulA | 180518BK03 |
| 180517LD04 | pSB1C3-BBa\_B0031\_sulA | 180518BK04 |
| 180517LD05 | pSB1C3- BBa\_B0034\_sulA | 180518BK05 |
| 180517LD06 | pSB1C3-oHybB | 180518BK06 |
| 180517LD07 | pSB1C3-oHybB-BBa\_E0840 | 180518BK07 |
| 180517LD08 | pSB1C3-BBa\_K206000-BBa\_B0032-sulA | 180518BK08 |
| 180517LD09 | pSB1C3-BBa\_K206000-BBa\_B0034-sulA | 180518BK09 |
| 180517LD10 | pSB1C3-BBa\_B0032-sulA | 180518BK10 |
| 180517LD11 | pSB1C3-BBa\_B0034-sulA | 180518BK11 |
| 180517LD12 | pSB1C3-BBa\_K20600-BBa\_B0031-sulA | 180518BK12 |
| 180517LD13 | pSB1C3-BBa\_I20270 | 180518BK13 |
| 180508OM06 | pSB1C3-BBa\_B0031 | 180518BK14 |
| 180508OM09 | pSB1C3-BBa\_B0034 | 180518BK15 |
| 180508OM08 | pSB1C3-BBa\_B0033 | 180518BK16 |
| 180508OM10 | pSB1C3-BBa\_K554008 | 180518BK17 |
| 180508OM12 | pSB1C3-BBa\_K554002 | 180518BK18 |
| 180508OM11 | pSB1C3-BBa\_K554009 | 180518BK19 |
| 180508OM07 | pSB1C3-BBa\_B0032 | 180518BK20 |

600 µL of the culture medium were mixed with 200 µL  50% Glycerol and put them into tubes. Frozen the tubes in liquid nitrogen and stored at -80°C.

## Prepared new plates

Plated the plates with 500 µL cAMP and 500 mL LB medium

## Prepared primer

Made 100 µL Aliquots with 10 µM concentrated primer. Protocol was by Microsynth.

## PCR of the new Parts

Mastermix for 20 samples:

* 200 µL 5x HF Buffer
* 20 µL dNTPs
* 10 µL Phusion DNA-Polymerase
* 710 µL H2Odest
* 20 µL Template DNA (DH5α)

PCR program:

|  |  |
| --- | --- |
| 98°C | 3 min |
| 98°C | 10 s |
| 60°C | 30 s |
| 72°C | 90 s |
| Go to step 2 30x | |
| 72°C | 5 min |

PCR products:

|  |  |  |
| --- | --- | --- |
| Tube | Part | Length |
| 1 | ldhA | 1036 |
| 2 | BBa\_B0034-ldhA | 1054 |
| 3 | BBa\_B0031-ldhA | 1056 |
| 4 | BBa\_B0032-ldhA | 1055 |
| 5 | BBa\_B0032-sulA | 575 |
| 6 | BBa\_J45503 | 446 |
| 7 | cspD | 273 |
| 8 | accA | 1011 |
| 9 | accB | 522 |
| 10 | tesA | 678 |
| 11 | cbtA | 426 |
| 12 | mraz | 507 |
| 13 | leuABCD | 4748 |
| 14 | leuABCD6H | 4766 |
| 15 | OmpT | 1002 |
| 16 | MlcRE | 252 |

## 180522- 2

## Gelelectrophoresis

We made a 1% agarose gel.

![](https://static.igem.org/mediawiki/2018/6/60/T--Hamburg--180522_PCR.JPG)

## PCR Purification

PCR-purification of the PCR products with GeneJET PCR-purification Kit by Thermo Fischer.

## Determine the DNA concentration of the PCR products

|  |  |  |
| --- | --- | --- |
| Lot number | Part | DNA concentration [ng/µL] |
| 180522BK01 | BBa\_B0032-sulA | 31.01 |
| 180522BK02 | BBa\_J45503 | 23.895 |
| 180522BK03 | cspD | 25.537 |
| 180522BK04 | accB | 36.938 |
| 180522BK05 | cbtA | 37.221 |
| 180522BK06 | Mraz | 32.949 |
| 180522BK07 | MlcRE | 38.68 |

## Plated the cryostocks for the characterizations

|  |  |  |
| --- | --- | --- |
| Lot number cryostocks | Part | Lot number plate |
| 180518BK02 | pSB1C3-MlcRE-BBa\_E0840 | 180522NiF01 |
| 180518BK07 | pSB1C3-oHybB-BBa\_E0840 | 180522NiF02 |
| 180518BK08 | pSB1C3-BBa\_K20600-BBa\_B0032-sulA | 180522NiF03 |
| 180518BK09 | pSB1C3-BBa\_K20600-BBa\_B0034-sulA | 180522NiF04 |
| 180518BK12 | pSB1C3-BBa\_K20600-BBa\_B0031-sulA | 180522NiF05 |

## Plating Cryostocks for Characterization of MlcRE, oHybB and sulA

Cryostocks of multiple parts were brought onto LB-agar-Cmp plates:

|  |  |  |
| --- | --- | --- |
| Cryostock | Part | Plate |
| 180518BK02 | pSB1C3-MlcRE-BBa\_E0840 | 180522NF01 |
| 180518BK07 | pSB1C3-oHybB-BBa\_E0840 | 180522NF02 |
| 180518BK08 | pSB1C3-BBa\_K206000-BBa\_B0032-sulA | 180522NF03 |
| 180518BK09 | pSB1C3-BBa\_K206000-BBa\_B0034-sulA | 180522NF04 |
| 180518BK12 | pSB1C3-BBa\_K206000-BBa\_B0031-sulA | 180522NF05 |

## Liquid cultures for characterizations

10 mL Lb-Cmp liquid cultures were inoculated with colonies from 108522 plates:

|  |  |
| --- | --- |
| Plate | Part |
| 180522NF01 | pSB1C3-MlcRE-BBa\_E0840 |
| 180522NF02 | pSB1C3-oHybB-BBa\_E0840 |
| 180522NF03 | pSB1C3-BBa\_K206000-BBa\_B0032-sulA |
| 180522NF04 | pSB1C3-BBa\_K206000-BBa\_B0034-sulA |
| 180522NF05 | pSB1C3-BBa\_K206000-BBa\_B0031-sulA |

Additionally, cryostock of pSB1C3-BBa\_I20270 was used for an additional liquid culture.

## 180523 – 1

## Restriction and ligation of multiple parts

Restriction:

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | **Tube** | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** |
|  | **Part** | pSB1C3-BBa\_B0031 | pSB1C3-BBa\_B0032 | pSB1C3-BBa\_B0033 | pSB133-BBa\_E0840 | Bba\_B0032\_sulA | Bba\_J45503 | cspD | accB | cbtA | mraZ | pSB1C3-I20270 |
|  | **Lot#** | 180514NB01 | 180514NB07 | 180514NB03 | 180315NB01 | aus PCR | aus PCR | aus PCr | aus PCR | aus PCR | aus PCR | 180509NF01 |
|  | **ρ** | 153 ng/µL | 81 ng/µL | 81 ng/µL | 128 ng/µL | 31 ng/µL | 24 ng/µL | 26 ng/µL | 37 ng/µL | 37 ng/µL | 33 ng/µL | 166 ng/µL |
|  | Use |  |  |  |  |  |  |  |  |  |  |  |
|  | target m | 150 ng | 150 ng | 150 ng | 100 ng | 100 ng | 150 ng | 250 ng | 100 ng | 300 ng | 300 ng | 2000 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 0.98 µL | 1.86 µL | 1.85 µL | 0.78 µL | 3.22 µL | 6.28 µL | 9.79 µL | 2.71 µL | 8.06 µL | 9.10 µL | 12.05 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL |
| EcoRI-HF |  |  |  | 0.50 µL |  | 0.50 µL |  |  |  |  |  |
| XbaI |  |  |  | 0.50 µL | 0.50 µL |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| SpeI | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  |  |  |  |  |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  | 0.50 µL |
| H2O | 6.52 µL | 5.64 µL | 5.65 µL | 6.72 µL | 4.78 µL | 1.72 µL | 7.21 µL | 5.29 µL | 8.94 µL | 7.90 µL | 4.45 µL |
|  | ad | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 15 ng/µL | 15 ng/µL | 15 ng/µL | 10 ng/µL | 10 ng/µL | 15 ng/µL | 13 ng/µL | 10 ng/µL | 15 ng/µL | 15 ng/µL | 100 ng/µL |

Ligation:

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** |
| Product | | pSB1C3-Bba\_B0032-sulA | pSB1C3-Bba\_J45503-Bba\_E0840 | pSB1C3-BBa\_B0031-cspD | pSB1C3-BBa\_B0032-cspD | pSB1C3-Bba\_B0033-cspD | pSB1C3-cspD | pSB1C3-Bba\_B0032-cbtA | pSB1C3-Bba\_B0033-cbtA | pSB1C3-cbtA | pSB1C3-Bba\_B0033-mraZ | pSB1C3-mraZ |
|  | |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3-I20270 | pSB1C3-BBa\_E0840 | pSB1C3-BBa\_B0031 | pSB1C3-BBa\_B0032 | pSB1C3-BBa\_B0033 | pSB1C3-I20270 | pSB1C3-BBa\_B0032 | pSB1C3-BBa\_B0033 | pSB1C3-I20270 | pSB1C3-BBa\_B0033 | pSB1C3-I20270 |
| Tube | **11** | **4** | **1** | **2** | **3** | **11** | **2** | **3** | **11** | **3** | **11** |
| ρ | 24 ng/µL | 5 ng/µL | 4 ng/µL | 4 ng/µL | 36 ng/µL | 24 ng/µL | 4 ng/µL | 36 ng/µL | 24 ng/µL | 36 ng/µL | 24 ng/µL |
| Länge | 2989 bp | 2948 bp | 2084 bp | 2083 bp | 2081 bp | 2989 bp | 2038 bp | 2081 bp | 2989 bp | 2081 bp | 2989 bp |
| V | 1.43 µL | 8.65 µL | 10.69 µL | 9.84 µL | 1.21 µL | 2.19 µL | 7.46 µL | 0.92 µL | 1.73 µL | 0.81 µL | 1.55 µL |
| **Part 1** | Part 1 | Bba\_B0032\_sulA | Bba\_J45503 | cspD | cspD | cspD | cspD | cbtA | cbtA | cbtA | mraZ | mraZ |
| Tube | **5** | **6** | **7** | **7** | **7** | **7** | **9** | **9** | **9** | **10** | **10** |
| ρ | 10 ng/µL | 15 ng/µL | 13 ng/µL | 13 ng/µL | 13 ng/µL | 13 ng/µL | 15 ng/µL | 15 ng/µL | 15 ng/µL | 15 ng/µL | 15 ng/µL |
| Length | 575 bp | 446 bp | 273 bp | 273 bp | 273 bp | 273 bp | 426 bp | 426 bp | 426 bp | 507 bp | 507 bp |
| V | 6.59 µL | 4.01 µL | 4.36 µL | 4.36 µL | 4.36 µL | 3.67 µL | 4.48 µL | 4.48 µL | 3.92 µL | 4.73 µL | 4.19 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |  |  |  |
| Length |  |  |  |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |  |  |  |
| T4 Buffer | | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 0.48 µL | 4.84 µL | 2.45 µL | 3.30 µL | 2.93 µL | 2.64 µL | 5.56 µL | 3.11 µL | 2.85 µL | 2.96 µL | 2.76 µL |
| ad | | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |

## Transformation

Competent DH5a cells were thawed at 4°C on ice for 5 min. Cells were inoculated with Ligations from 180523 and incubated for 1 h at 4°C on ice. Heat shock was performed at 42°C for 60 s. Cells were chilled at 4°C on ice for 5 min. 850 µL LB medium was added, and cells were incubated at 37°C, 900 RPM on the thermomix for 1h. Cells were brought onto LB-agar-Cmp plates and incubated at 37°C overnight as:

|  |  |
| --- | --- |
| Part | Plate |
| pSB1C3-BBa\_B0032-sulA | 170524NB01 |
| pSB1C3-BBa\_J45503-BBa\_E0840 | 170524NB02 |
| pSB1C3-BBa\_B0031-cspD | 170524NB03 |
| pSB1C3-BBa\_B0032-cspD | 170524NB04 |
| pSB1C3-BBa\_B0033-cspD | 170524NB05 |
| pSB1C3-cspD | 170524NB06 |
| pSB1C3-BBa\_B0032-cbtA | 170524NB07 |
| pSB1C3-BBa\_B0033-cbtA | 170524NB08 |
| pSB1C3-cbtA | 170524NB09 |
| pSB1C3-BBa\_B0033-mraZ | 170524NB10 |
| pSB1C3-mraZ | 170524NB11 |

## Liquid cultures for characterization

150 µL of overnight cultures from 180523 were carried over in 5 mL fresh lb/Cmp medium and incubated overnight at 37°c, 270 RPM overnight.

## Cancellation of characterization

Characterization overnight cultures from 180524 were discarded.

## Characterization of ohybB and MlcRE

Set up precultures and incubated at 37°C and 210 rpm.

OD-measure at 11:30 am.

|  |  |
| --- | --- |
| Part | OD |
| ohybB (4°C) | 0.752 |
| MlcRe (0g/L) | 0.307 |
| DH5α (negative control) | 0.236 |
| GFP (positive control) | 0.385 |
| MlcRE (0.125 g/L) | 0.391 |
| MlcRE (0.25 g/L) | 0.358 |
| MlcRE (0.5 g/L) | 0.382 |
| MlcRE (1 g/L) | 0.427 |
| MlcRE (2 g/L) | 0.418 |

|  |  |  |
| --- | --- | --- |
| Part | Temperature (°C) | OD |
| ohybB | 10 | 0.727 |
| ohybB | 15 | 0.746 |
| ohybB | 20 | 0.709 |
| ohybB | 25 | 0.729 |
| ohybB | 30 | 0.782 |
| ohybB | 37 | 0.831 |

The ohybB series were diluted 1:1. All cultures were transferred at 4°C. Of the cultures MlcRE, DH5α and GFP were prepared new cultures. To all cultures were 6.5 mL LB-medium added. After another OD-measurement after 1 h the measure were cancelled. The culture tubes stored at 4°C over night.

## Characterization of MlcRE

6x 6 mL lb-Cmp medium was inoculated with DH5a/pSB1C3-MlcRE-BBa\_BE0840 and 2x 6 mL lb-Cmp was inoculated with DH5a or DH5a/pSB1C3-BBa\_I20270 and grown to OD600 = 0.3. Present GFP was inactivated under high light for 10 min. DH5a/pSB1C3-MlcRE-BBa\_E0840 samples were induced with 0, 0.125, 0.250, 0.500, 1.000 and 2.000 µg/mL glucose. At 0, 30, 60, and 90 min, 1ml was taken off the culture, and cells were fixed in 4% PFA by slight vortexing and incubation for 20 min at RT. Cells were spun down at 13000 g for 5 min, washed with PBS twice, and resuspended in 1.5 mL PBS. 1 mL was frozen away. Three wells of a 96-well plate reader plate were filled with 200 µL of one of the cultures each, and GFP fluorescence was measured on a plate reader:

MlcRE induces GFP transcription. Glucose inhibits MlcRE activity in a concentration-dependent manner.

## Colony PCR of 180524 transformation

Colonies of 180524 transformations were picked and, and colony PCR was set up employing a PCR master mix for 30 PCRs in 20 µL each:

|  |  |
| --- | --- |
| DreamTaq Buffer | 8 µL |
| dNTPs 25 mM each | 16 µL |
| 50 mM MgCl2 | 24 µL |
| 10 µM VF2 | 24 µL |
| 10 µM VR | 24 µL |
| DreamTaq | 1 µL |
| H2O | 632 µL |
| ad | 800 µL |

20 µL master mix was inoculated with a colony of 180524 transformation plates. PCR was carried out as follows:

|  |  |  |
| --- | --- | --- |
| 1 | 95°C | 3:00 |
| 2 | 94°C | 0:30 |
| 3 | 55°C | 0:30 |
| 4 | 72°C | 1:30 |
| 5 | GOTO 2, rep 30x |  |
| 6 | 72°C | 5:00 |
| 8 | 4°C | Hold |

## Agarose gel electrophoresis of cPCR from 180530

cPCRs were analysed on a 1% agarose gel:

![](https://static.igem.org/mediawiki/2018/0/0f/T--Hamburg--180531_cPCR1.JPG)
![](https://static.igem.org/mediawiki/2018/b/b7/T--Hamburg--180531_cPCR2.JPG)

Colonies with constructs displaying correct lengths were taken into 10 mL LB-Cmp overnight cultures at 37°C, 200 RPM.

## Transformation of DH5a with pSB1C3-BBa\_E0840

Competent DH5a cells were thawed at 4°C on ice for 5 min. Cells were inoculated with 200 pg pSB1C3-BBa\_E0240 from 2018 Distribution Kit Plate 2, well 24B and incubated for 1 h at 4°C on ice. Heat shock was performed at 42°C for 60 s. Cells were chilled at 4°C on ice for 5 min. 850 µL LB medium was added, and cells were incubated at 37°C, 900 RPM on the thermomix for 1h. Cells were brought onto LB-agar-Cmp plates and incubated at 37°C overnight.

## Miniprep of 180524 transformations

Plasmids from 180530 overnight cultures of positive colonies from 180524 transformations were purified using GeneJET PCR Purification Kit (ThermoFisher). Plasmids were stored at -20°C as:

|  |  |
| --- | --- |
| pSB1C3-BBa\_B0033-mraZ | 180531LP01 |
| pSB1C3-BBa\_B0033-cbtA | 180531LP02 |
| pSB1C3-BBa\_B0032-cbtA | 180531LP03 |
| pSB1C3-BBa\_B0033-cspD | 180531LP04 |
| pSB1C3-BBa\_B0032-cspD | 180531LP05 |
| pSB1C3-BBa\_B0032-sulA | 180531LP06 |

600 µL of each culture was used for glycerol stocks which were stored at -80°C as:

|  |  |
| --- | --- |
| DH5a/pSB1C3-BBa\_B0033-mraZ | 180531NF01 |
| DH5a/pSB1C3-BBa\_B0033-cbtA | 180531NF02 |
| DH5a/pSB1C3-BBa\_B0032-cbtA | 180531NF03 |
| DH5a/pSB1C3-BBa\_B0033-cspD | 180531NF04 |
| DH5a/pSB1C3-BBa\_B0032-cspD | 180531NF05 |
| DH5a/pSB1C3-BBa\_B0032-sulA | 180531NF06 |

## PCR of E. coli genomic basic parts

Basic parts were amplified from E. coli genomic DNA. A PCR master mix was set up as following:

|  |  |
| --- | --- |
| 5x Phusion HF Buffer | 200 µL |
| dNTPs | 20 µL |
| Phusion DNA Polymerase | 10 µL |
| H2O | 710 µL |
| DNA Template | 20 µL |

50 µL PCRs were set up using 48 µL master mix plus 1 µL forward and reverse primers each:

|  |  |  |
| --- | --- | --- |
| Product | Forward primer | Reverse primer |
| BBPre-ldhA-BBSuf | Pre\_ldhA\_f | ldhA\_Suf\_r |
| XbaI-BBa\_B0034-ldhA-BBSuf | 34\_ldhA\_f | ldhA\_Suf\_r |
| XbaI-BBa\_B0031-ldhA-BBSuf | 31\_ldhA\_f | ldhA\_Suf\_r |
| XbaI-BBa\_B0032-ldhA-BBSuf | 32\_ldhA\_f | ldhA\_Suf\_r |
| BBPre-BBa\_J45503-BBSuf | Pre\_hybB\_f | hybB\_Suf\_r |
| BBPre-cspD-BBSuf | Pre\_cspD\_f | cspD\_Suf\_r |
| BBPre-accA-BBSuf | Pre\_accA\_f | accA\_Suf\_r |
| BBPre-accB-BBSuf | Pre\_accB\_f | accB\_Suf\_r |
| BBPre-tesA-BBSuf | Pre\_tesA\_f | tesA\_Suf\_r |
| BBPre-cbtA-BBSuf | Pre\_cbtA\_f | cbtA\_Suf\_r |
| BBPre-mraZ-BBsuf | Pre\_mraZ\_f | mraZ\_Suf\_r |
| BBPre-LeuABCD-BBSuf | Pre\_LeuA\_f | LeuD\_Suf\_r |
| BBPre-LeuABCD-His-BBSuf | Pre\_LeuA\_f | LeuD\_His\_Suf\_r |
| BBPre-OmpT-BBSuf | Pre\_OmpT\_f | OmpT\_Suf\_r |
| BBPre-MlcRE-BBSuf | Pre\_MlcRE\_f | MlcRE\_Suf\_r |
| BBPre-BBa\_B0031-ldhA-BBSuf (amplified with Taq) | 31\_ldhA\_f | ldhA\_Suf\_r |

PCR was carried out using following protocol:

|  |  |  |
| --- | --- | --- |
| 1 | 98°C | 3:00 |
| 2 | 98°C | 0:10 |
| 3 | 60°C | 0:30 |
| 4 | 72°C | 2:00 |
| 5 | GOTO 2, rep 30x |  |
| 6 | 72°C | 5:00 |

PCR products were analysed by agarose gel electrophoresis:

![](https://static.igem.org/mediawiki/2018/d/d2/T--Hamburg--180604_PCR.JPG)

Correctly amplified products were purified using GeneJet PCR Purification Kit (ThermoFisher) and stored as:

|  |  |
| --- | --- |
| 180604NB01 | BBa\_J45503 |
| 180604NB02 | cspD |
| 180604NB03 | cbtA |
| 180604NB04 | mraZ |
| 180604NB05 | MlcRE |
| 180604NB06 | BBa\_B0031-ldhA |

## InterLab Day 1

Parts were resuspended in 10 µL H2O from 2018 Distribution Kit Plate 7:

|  |  |
| --- | --- |
| Part | Well |
| BBa\_R0040 | 2D |
| BBa\_I20270 | 2B |
| BBa\_J364000 | 2F |
| BBa\_J364001 | 2H |
| BBa\_J364002 | 2J |
| BBa\_J364007 | 2L |
| BBa\_J364008 | 2N |
| BBa\_J364009 | 2P |

Competent DH5a were thawed on ice for 10 min at 4°C on ice and split to 50 µL aliquots. Cells were inoculated with 3 µL DNA of each part and incubated for 30 min at 4°C on ice. Heat shock was performed at 42°C for 45 s and chilled at 4°C on ice for 5 minutes. 200 µL LB medium was added, and cells were incubated at 37°C, 500 RPM on the thermomix for 1 h. Cells were brought onto LB-agar-Cmp plates and incubated at 37°C overnight.

## InterLab Day 2

Plate reader was calibrated using Ludox, Silica Beads, and Fluorescin according to InterLab protocol.

2x5 mL lb-Cmp were inoculated with colonies from each 180605 InterLab transformation plate. Cultures were incubated at 37°C, 200 RPM overnight.

## PCR of the 2nd Generation from *e. coli*

All PCR Reactions were prepared as written below.

|  |  |
| --- | --- |
| 19 µL | H2O |
| 1 µL | DH5α |
| 2.5 µL | Fw-Primer |
| 2.5 µL | Rv-Primer |
| 25 µL | Q5 High-Fidelity 2X Master Mix |

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Tube | Part | Fw-Primer | Rv-Primer | Predicted length (bp) | Concentration (ng/µL) |
| 1 | ldhA | Pre-ldhA-fw | ldhA-Suf-rv | 1036 | 34.465 |
| 2 | BBa\_B0034-ldhA | 34-ldhA-fw | ldhA-Suf-rv | 1054 | 43.920 |
| 3 | BBa\_B0031-ldhA | 31-ldhA-fw | ldhA-Suf-rv | 1056 | 40.609 |
| 4 | BBa\_B0032-ldhA | 32-ldhA-fw | ldhA-Suf-rv | 1055 | 39.545 |
| 5 | BBa\_J45503 | Pre-45503-fw | 45503-Suf-rv | 446 | 41.660 |
| 6 | cspD | Pre-cspD-fw | cspD-Suf-rv | 273 | 46.327 |
| 7 | accA | Pre-accA-fw | accA-Suf-rv | 1011 | 35.789 |
| 8 | accB | Pre-accB-fw | accB-Suf-rv | 522 | X |
| 9 | tesA | Pre-tesA-fw | tesA-Suf-rv | 678 | 38.747 |
| 10 | cbtA | Pre-cbtA-fw | cbtA-Suf-rv | 426 | 50.447 |
| 11 | mraZ | Pre-mraZ-fw | mraZ-Suf-rv | 507 | X |
| 12 | LeuABCD | Pre-LeuA-fw | LeuD-Suf-rv | 4748 | X |
| 13 | LeuABCD6H | Pre-LeuA-fw | LeuD6H-Suf-rv | 4766 | X |
| 14 | OmpT | Pre-OmpT-fw | OmpT-Suf-rv | 1002 | X |
| 15 | MlcRE | Pre-MlcRE-fw | MlcRE-Suf-rv | 252 | 54.880 |

PCR was performed using following Cycler-Protocol

|  |  |  |  |
| --- | --- | --- | --- |
| Temperature (°C) | Time | Time (for LeuABCD) | Repeats |
| 98 | 2:00 | 2:00 | 1 |
| 98 | 0:10 | 0:10 | 30 |
| 60 | 0:30 | 0:30 | 30 |
| 72 | 1:00 | 3:00 | 30 |
| 72 | 2:00 | 2:00 | 1 |
| 4 | ∞ | ∞ | 1 |

A gel electrophoresis was performed using a 1%-Agarose-Gel, for LeuABCD a 0.7%-Agarose-Gel was prepared but got destroyed due to high voltage.

![](https://static.igem.org/mediawiki/2018/0/0b/T--Hamburg--180613_PCR.JPG)

PCR Purification was performed using the Thermo Scientific PCR Purification Kit. The PCR Products were stored at 4 °C.

## Restriction of PCR Products

The reaction was prepared as seen in the table below, incubated at 37 °C for 60 minutes and heat inactivated at 80 °C for 20 minutes.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** |  | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** | **15** | **16** | **17** | **18** | **19** |
|  | Part | pSB1C3-BBa\_B0031 | pSB1C3-BBa\_B0032 |  | pSB133-BBa\_K206000 | pSB1C3-I20270 | pSB1C3-I20270 | pSB1C3-BBa\_E0840 | pSB1C3-I20270 | BBa\_J45503 | cspD | accA | accB | cbtA | mraZ | ldhA | BBa\_B0031-ldhA | BBa\_B0032-ldhA | BBa\_B0034-ldhA | MlcRE |
|  | Lot# | 180514NB01 | 180514NB07 |  | 180321NB01 | 180509NF01 | 180509NF01 | 180315NB01 | 180509NF01 | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR |
|  | **ρ** | 153 ng/µL | 81 ng/µL |  | 140 ng/µL | 166 ng/µL | 166 ng/µL | 128 ng/µL | 166 ng/µL | 42 ng/µL | 46 ng/µL | 30 ng/µL | 36 ng/µL | 39 ng/µL | 50 ng/µL | 34 ng/µL | 41 ng/µL | 40 ng/µL | 44 ng/µL | 55 ng/µL |
|  | Use |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | target m | 200 ng | 200 ng | 200 ng | 200 ng | 500 ng |  |  |  | 200 ng | 200 ng | 100 ng | 200 ng | 150 ng | 300 ng | 250 ng | 200 ng | 200 ng | 200 ng | 100 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 1.31 µL | 2.48 µL |  | 1.43 µL | 6.02 µL | 0.60 µL | 0.78 µL | 0.60 µL | 4.80 µL | 4.32 µL | 3.39 µL | 5.59 µL | 3.87 µL | 5.95 µL | 7.30 µL | 10.00 µL | 10.00 µL | 10.00 µL | 2.00 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL |  | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL |
| EcoRI-HF |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  | 0.50 µL |
| XbaI |  |  |  |  | 0.50 µL |  | 0.50 µL |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |
| SpeI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  | 0.50 µL |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |  |  |
| H2O | 6.19 µL | 5.02 µL |  | 6.07 µL | 10.48 µL | 6.90 µL | 6.72 µL | 6.90 µL | 3.20 µL | 3.68 µL | 4.61 µL | 2.41 µL | 4.13 µL | 2.05 µL | 9.70 µL | 7.00 µL | 7.00 µL | 7.00 µL | 6.00 µL |
|  | ad | 10.00 µL | 10.00 µL |  | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 20 ng/µL | 20 ng/µL |  | 20 ng/µL | 50 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 15 ng/µL | 30 ng/µL | 13 ng/µL | 20 ng/µL | 20 ng/µL | 22 ng/µL | 11 ng/µL |

The gel for the vectors melted. Restrictions needed to be repeated.

## Repetition of the 180614-Restriction

The reaction was prepared as seen in the table below, incubated at 37 °C for 60 minutes and heat inactivated at 80 °C for 20 minutes.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** |  | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** | **15** | **16** | **17** | **18** | **19** |
|  | Part | pSB1C3-BBa\_B0031 | pSB1C3-BBa\_B0032 |  | pSB133-BBa\_K206000 | pSB1C3-I20270 | pSB1C3-I20270 | pSB1C3-BBa\_E0840 | pSB1C3-I20270 | BBa\_J45503 | cspD | accA | accB | cbtA | mraZ | ldhA | BBa\_B0031-ldhA | BBa\_B0032-ldhA | BBa\_B0034-ldhA | MlcRE |
|  | Lot# | 180514NB01 | 180514NB07 |  | 180321NB01 | 180509NF01 | 180509NF01 | 180315NB01 | 180509NF01 | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR |
|  | **ρ** | 153 ng/µL | 81 ng/µL |  | 140 ng/µL | 166 ng/µL | 166 ng/µL | 128 ng/µL | 166 ng/µL | 42 ng/µL | 46 ng/µL | 30 ng/µL | 36 ng/µL | 39 ng/µL | 50 ng/µL | 34 ng/µL | 41 ng/µL | 40 ng/µL | 44 ng/µL | 55 ng/µL |
|  | Use |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | target m | 200 ng | 200 ng | 200 ng | 200 ng | 500 ng |  |  |  | 200 ng | 200 ng | 100 ng | 200 ng | 150 ng | 300 ng | 250 ng | 200 ng | 200 ng | 200 ng | 100 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 1.31 µL | 2.48 µL |  | 1.43 µL | 6.02 µL | 0.60 µL | 0.78 µL | 0.60 µL | 4.80 µL | 4.32 µL | 3.39 µL | 5.59 µL | 3.87 µL | 5.95 µL | 7.30 µL | 10.00 µL | 10.00 µL | 10.00 µL | 2.00 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL |  | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL |
| EcoRI-HF |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  | 0.50 µL |
| XbaI |  |  |  |  | 0.50 µL |  | 0.50 µL |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |
| SpeI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  | 0.50 µL |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |  |  |
| H2O | 6.19 µL | 5.02 µL |  | 6.07 µL | 10.48 µL | 6.90 µL | 6.72 µL | 6.90 µL | 3.20 µL | 3.68 µL | 4.61 µL | 2.41 µL | 4.13 µL | 2.05 µL | 9.70 µL | 7.00 µL | 7.00 µL | 7.00 µL | 6.00 µL |
|  | ad | 10.00 µL | 10.00 µL |  | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 20 ng/µL | 20 ng/µL |  | 20 ng/µL | 50 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 15 ng/µL | 30 ng/µL | 13 ng/µL | 20 ng/µL | 20 ng/µL | 22 ng/µL | 11 ng/µL |
|  |  |  |  |  |  | 24 |  |  |  | 15 | 13 ng/µL |  | 10 | 15 | 15 |  |  |  |  |  |

For the Restrictions 1-7 a 1%-Agarose gel was prepared and 4, 5 and 7 were extracted using the Thermo Scientific Gel Extraction Kit.

![](https://static.igem.org/mediawiki/2018/8/87/T--Hamburg--180618_Restriktion_%28pSB1C3-I20270%29.JPG)

The concentration of tube 4 was 4.03 ng/µL, the concentration of tube 5 was 17.013 ng/µL and the concentration of tube 7 was 5.901 ng/µL.

## Ligation of the restricted DNA

Following Ligation were performed at 37 °C for 60 minutes.

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **1** | **4** | **6** | **L7** | **L12** | **L13** | **L15** | **L16** | **L19** |
| Product | | pSB1C3-ldhA | pSB1C3-BBa\_B0034-ldhA | pSB1C3-BBa\_K20600-BBa\_B0031-ldhA | pSB1C3-BBa\_K20600-BBa\_B0032-ldhA | pSB1C3-BBa\_E0840-BBa\_J45503 | pSB1C3-cspD | pSB1C3-accA | pSB1C3-accB | pSB1C3-cbtA |
|  | |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3-I20270 | pSB1C3-I20270 | pSB133-BBa\_K206000 | pSB133-BBa\_K206000 | pSB1C3-BBa\_E0840 | pSB1C3-I20270 | pSB1C3-I20270 | pSB1C3-I20270 | pSB1C3-I20270 |
| Tube | **5** | **5** | **4** | **4** | **7** | **5** | **5** | **5** | **5** |
| ρ | 50 ng/µL | 50 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL |
| Länge | 2989 bp | 2989 bp | 2150 bp | 2150 bp | 2948 bp | 2989 bp | 2989 bp | 2989 bp | 2989 bp |
| V | 0.44 µL | 1.05 µL | 2.20 µL | 1.46 µL | 3.68 µL | 0.74 µL | 0.74 µL | 0.73 µL | 0.83 µL |
| **Part 1** | Part 1 | ldhA | BBa\_B0034-ldhA | BBa\_B0031-ldhA | BBa\_B0032-ldhA | BBa\_J45503 | cspD | accA | accB | cbtA |
| Tube | **15** | **18** | **16** | **17** | **9** | **10** | **11** | **12** | **13** |
| ρ | 13 ng/µL | 22 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 15 ng/µL |
| Länge | 1050 bp | 273 bp | 273 bp | 522 bp | 507 bp | 507 bp | 507 bp | 522 bp | 426 bp |
| V | 6.19 µL | 2.17 µL | 2.76 µL | 3.58 µL | 3.16 µL | 3.15 µL | 6.29 µL | 3.18 µL | 3.92 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |  |
| T4 Buffer | | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 1.87 µL | 5.28 µL | 3.54 µL | 3.46 µL | 1.66 µL | 4.61 µL | 1.47 µL | 4.59 µL | 3.76 µL |
| ad | | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |

## Transformation of the 180618 Ligations

Transformation of the ligation reactions of the day before were carried out using the Zhang protocol.

## cPCR of the transformation of the 19th June

The Master Mix for 40 reactions was prepared according to following table

|  |  |
| --- | --- |
| chemical | Volume (µL) |
| 10X Taq buffer | 80 |
| dNTP 10 mM | 16 |
| 50 mM MgCl2 | 24 |
| Fw-Primer VF2 | 48 |
| Rv-Primer VR | 48 |
| Taq DNA Polymerase (5 u/µL) | 1 |
| H2O | 583 |

1% Agarose gel was prepared for gel electrophoresis.

![](https://static.igem.org/mediawiki/2018/f/fa/T--Hamburg--180620_cPCR1.JPG)
![](https://static.igem.org/mediawiki/2018/d/d6/T--Hamburg--180620_cPCR2.JPG)

## PCR of the 2nd Generation from *e. coli* and test restriction

All PCR Reactions were prepared as written below.

|  |  |
| --- | --- |
| 19 µL | H2O |
| 1 µL | DH5α |
| 2.5 µL | Fw-Primer |
| 2.5 µL | Rv-Primer |
| 25 µL | Q5 High-Fidelity 2X Master Mix |

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Tube | Part | Fw-Primer | Rv-Primer | Predicted length (bp) | Concentration (ng/µL) |
| 1 | ldhA | Pre-ldhA-fw | ldhA-Suf-rv | 1036 | 34.465 |
| 2 | BBa\_B0034-ldhA | 34-ldhA-fw | ldhA-Suf-rv | 1054 | 43.920 |
| 3 | BBa\_B0031-ldhA | 31-ldhA-fw | ldhA-Suf-rv | 1056 | 40.609 |
| 4 | BBa\_B0032-ldhA | 32-ldhA-fw | ldhA-Suf-rv | 1055 | 39.545 |
| 5 | BBa\_J45503 | Pre-45503-fw | 45503-Suf-rv | 446 | 41.660 |
| 6 | cspD | Pre-cspD-fw | cspD-Suf-rv | 273 | 46.327 |
| 7 | accA | Pre-accA-fw | accA-Suf-rv | 1011 | 35.789 |
| 8 | accB | Pre-accB-fw | accB-Suf-rv | 522 | X |
| 9 | tesA | Pre-tesA-fw | tesA-Suf-rv | 678 | 38.747 |
| 10 | cbtA | Pre-cbtA-fw | cbtA-Suf-rv | 426 | 50.447 |
| 11 | mraZ | Pre-mraZ-fw | mraZ-Suf-rv | 507 | X |
| 12 | LeuABCD | Pre-LeuA-fw | LeuD-Suf-rv | 4748 | X |
| 13 | LeuABCD6H | Pre-LeuA-fw | LeuD6H-Suf-rv | 4766 | X |
| 14 | OmpT | Pre-OmpT-fw | OmpT-Suf-rv | 1002 | X |
| 15 | MlcRE | Pre-MlcRE-fw | MlcRE-Suf-rv | 252 | 54.880 |

PCR was performed using following Cycler-Protocol

|  |  |  |  |
| --- | --- | --- | --- |
| Temperature (°C) | Time | Time (for LeuABCD) | Repeats |
| 98 | 2:00 | 2:00 | 1 |
| 98 | 0:10 | 0:10 | 30 |
| 60 | 0:30 | 0:30 | 30 |
| 72 | 1:00 | 4:00 | 30 |
| 72 | 2:00 | 5:00 | 1 |
| 4 | ∞ | ∞ | 1 |

A 1% Agarose gel was prepared for gel electrophoresis.

![](https://static.igem.org/mediawiki/2018/f/fa/T--Hamburg--180621_PCR_und_Restriktion.JPG)

The test restrction in the picture above was set up to check the activity of our restriction enzymes. The reactions were each set up with 11.5 µL H2O, 6 µL of pSB1C3-I20270, 2 µL of NEB 2.1 buffer and 0.5 µL of restriction enzyme. Each tube was incubated at 37 °C for an hour and heat inacticated at 80 °C for 20 minutes.

## Repetition of the restriction and the ligation of the 14th June

Restriction and ligation were performed by pipetting following tables. Restriction was performed at 37 °C for 1 hour and heat inactivated for 20 minutes at 80 °C.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **2** | **4** | **5** | **7** | **9** | **10** | **11** | **12** | **13** | **14** | **16** | **17** | **18** |
|  | Part | pSB1C3-BBa\_B0032 | pSB133-BBa\_K206000 | pSB1C3-I20270 | pSB1C3-BBa\_E0840 | BBa\_J45503 | cspD | accA | accB | cbtA | mraZ | BBa\_B0031-ldhA | BBa\_B0032-ldhA | BBa\_B0034-ldhA |
|  | Lot# | 180514NB07 | 180321NB01 | 180509NF01 | 180315NB01 | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR |
|  | **ρ** | 81 ng/µL | 140 ng/µL | 166 ng/µL | 128 ng/µL | 42 ng/µL | 46 ng/µL | 30 ng/µL | 36 ng/µL | 39 ng/µL | 50 ng/µL | 41 ng/µL | 40 ng/µL | 44 ng/µL |
|  | Use |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | target m | 200 ng | 200 ng | 500 ng |  | 200 ng | 200 ng | 100 ng | 200 ng | 150 ng | 300 ng | 200 ng | 200 ng | 200 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 2.48 µL | 1.43 µL | 6.02 µL | 0.78 µL | 4.80 µL | 4.32 µL | 3.39 µL | 5.59 µL | 3.87 µL | 5.95 µL | 10.00 µL | 10.00 µL | 10.00 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL |
| EcoRI-HF |  |  |  | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |
| XbaI |  |  | 0.50 µL | 0.50 µL |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| SpeI | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  |  |  |  |  |  |  |  |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |
| H2O | 5.02 µL | 6.07 µL | 10.48 µL | 6.72 µL | 3.20 µL | 3.68 µL | 4.61 µL | 2.41 µL | 4.13 µL | 2.05 µL | 7.00 µL | 7.00 µL | 7.00 µL |
|  | ad | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL |
|  | Länge Produkt |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 5.205 ng/µL | 22.086 ng/µL | 39.7 ng/µL | 3.07 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 15 ng/µL | 30 ng/µL | 20 ng/µL | 20 ng/µL | 22 ng/µL |

Gel extraction with the Thermo Scientific gel extraction kit was performed for tube 2, 4, 5 and 7

Ligation was performed over the weekend at 4 °C.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **4** | **5** | **6** | **L7** | **L8** | **L12** | **L13** | **L15** | **L16** | **L17** | **L19** | **L21** | **L22** |
| Product | | pSB1C3-BBa\_B0034-ldhA |  | pSB1C3-BBa\_K20600-BBa\_B0031-ldhA | pSB1C3-BBa\_K20600-BBa\_B0032-ldhA | pSB133-BBa\_K206000-BBa\_B0034-ldhA | pSB1C3-BBa\_E0840-BBa\_J45503 | pSB1C3-cspD | pSB1C3-accA | pSB1C3-accB | pSB1C3-BBa\_B0032-accB | pSB1C3-cbtA | pSB1C3-BBa\_B0032-mraZ | pSB1C3-mraZ |
|  | |  |  |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3-I20270 |  | pSB133-BBa\_K206000 | pSB133-BBa\_K206000 | pSB133-BBa\_K206000 | pSB1C3-BBa\_E0840 | pSB1C3-I20270 | pSB1C3-I20270 | pSB1C3-I20270 | pSB1C3-BBa\_B0032 | pSB1C3-I20270 | pSB1C3-BBa\_B0032 | pSB1C3-I20270 |
| Tube | **5** |  | **4** | **4** | **4** | **7** | **5** | **5** | **5** | **2** | **5** | **2** | **5** |
| ρ | 50 ng/µL |  | 20 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 20 ng/µL | 50 ng/µL | 20 ng/µL | 50 ng/µL |
| Länge | 2989 bp |  | 2150 bp | 2150 bp | 2150 bp | 2948 bp | 2989 bp | 2989 bp | 2989 bp | 2082 bp | 2989 bp | 2082 bp | 2989 bp |
| V | 1.05 µL |  | 2.20 µL | 1.46 µL | 1.68 µL | 3.68 µL | 0.74 µL | 0.74 µL | 0.73 µL | 1.43 µL | 0.83 µL | 1.46 µL | 0.74 µL |
| **Part 1** | Part 1 | BBa\_B0034-ldhA |  | BBa\_B0031-ldhA | BBa\_B0032-ldhA | BBa\_B0034-ldhA | BBa\_J45503 | cspD | accA | accB | accB | cbtA | mraZ | mraZ |
| Tube | **18** |  | **16** | **17** | **18** | **9** | **10** | **11** | **12** | **12** | **13** | **14** | **14** |
| ρ | 22 ng/µL |  | 20 ng/µL | 20 ng/µL | 22 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 20 ng/µL | 15 ng/µL | 30 ng/µL | 30 ng/µL |
| Länge | 273 bp |  | 273 bp | 522 bp | 426 bp | 507 bp | 507 bp | 507 bp | 522 bp | 522 bp | 426 bp | 507 bp | 507 bp |
| V | 2.17 µL |  | 2.76 µL | 3.58 µL | 3.03 µL | 3.16 µL | 3.15 µL | 6.29 µL | 3.18 µL | 3.57 µL | 3.92 µL | 2.36 µL | 2.10 µL |
| T4 Buffer | | 1.00 µL |  | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 5.28 µL |  | 3.54 µL | 3.46 µL | 3.80 µL | 1.66 µL | 4.61 µL | 1.47 µL | 4.59 µL | 3.50 µL | 3.76 µL | 4.68 µL | 5.66 µL |
| ad | | 10.00 µL |  | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |

## PCR parts Generation 2 (missing parts)

Mastermix:

* 10 µL HF buffer
* 1 µL dNTPs
* 0.5 µL Phusion Polymerase
* 1 µL DNA
* 1.5 µL DMSO
* 1 µL fw primer
* 1 µL rv Primer
* 34 µL H2Odest

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Tube | Part | Fw primer | Rv primer | Length | DNA concentration [ng/µL] |
| 1 | BBa\_B0031-ldhA | 31-ldhA-fw | ldhA-suf-rv | 1056 | 44.465 |
| 2 | BBa\_345503 | Pre-45503-fw | 45503-suf-rv | 446 | 44.646 |
| 3 | tesA | Pre-tesA-fw | tesA-suf-rv | 678 | - |
| 4 | leuABCD | Pre-leuA-fw | leuD-suf-rv | 4748 | - |
| 5 | leuABCD6H | Pre-leuA-fw | leuD6H-suf-rv | 4766 | - |
| 6 | ompT | Pre-ompT-fw | ompT-suf-rv | 1002 | - |

PCR program:

|  |  |
| --- | --- |
| BBa\_B0031\_ldhA 1.1 | |
| BBa\_345503 1.2 | |
| 98°C | 2 min |
| 98°C | 10 s |
| 60°C | 30 s |
| 72°C | 1 min |
| Go to step 2 30x | |
| 72°C | 2 min |

|  |  |
| --- | --- |
| tesA 2.1 | |
| ompT 2.2 | |
| 98°C | 2 min |
| 98°C | 10 s |
| 55°C | 30 s |
| 72°C | 1 min |
| Go to step 2 25x | |
| 72°C | 2 min |

|  |  |
| --- | --- |
| leuABCD 3.1 | |
| leuABCD6H 3.2 | |
| 98°C | 2 min |
| 98°C | 10 s |
| 55°C | 30 s |
| 72°C | 4 min |
| Go to step 2 30x | |
| 72°C | 5 min |

|  |  |
| --- | --- |
| leuABCD with Phusion 4.1 and DMSO | |
| leuABCD6H 4.2 and DMSO | |
| 98°C | 3 min |
| 98°C | 10 s |
| 60°C | 30 s |
| 72°C | 4 min |
| Go to step 2 30x | |
| 72°C | 5 min |

## Gelelectrophoresis

We made a 1% agarose gel.

![](https://static.igem.org/mediawiki/2018/3/30/T--Hamburg--180625_PCR.JPG)

## PCR-Purification

PCR-purification of the PCR products with GeneJET PCR-purification Kit by Thermo Fischer. Only BBa\_B0031\_ldhA and BBa\_345503 had worked.

## Transformation of the ligations from 180622

The transformation was made by iGEM Transformation Protocol. Tube 6 and 8 were not transformed, because they were identically with tube 4 and 21.

## cPCR of the transformation from 180625

Mastermix for 50 samples:

* 100 µL Taq buffer
* 20 µL dNTPs
* 30 µL MgCl2
* 30 µL VF2
* 30 µL VR
* 1 µL Taq polymerase
* 789 µL H2Odest

|  |  |
| --- | --- |
| Tube | Part |
| 1-3 | pSB1C3-MlcRE |
| 4-7 | pSB1C3-BBa\_B0032-ldhA |
| 8-11 | pSB1C3-BBa\_B0034-ldhA |
| 12-15 | pSB1C3-cspD |
| 16-19 | pSB1C3-accA |
| 20-23 | pSB1C3-accB |
| 24-27 | pSB1C3-cbtA |
| 28-31 | pSB1C3-BBa\_B00-cbtA |
| 32-35 | pSB1C3-mraz |
| 36-38 | pSB1C3-ldhA |

## Gelelectrophoresis

We made a 1% agarose gel.

![](https://static.igem.org/mediawiki/2018/3/3c/T--Hamburg--180626_cPCR1.JPG)
![](https://static.igem.org/mediawiki/2018/9/94/T--Hamburg--180626cPCR2.JPG)

## 180627 – 1

## Miniprep and cryostocks of multiple parts

Cryostocks:

Add 600 µL of cell culture and 200 µL of 50 % Glycerol in a 1.5 mL tube.

Stocks are at -80 °C stalled as:

|  |  |
| --- | --- |
| Part | Lot-No. |
| pSB1C3-BBa\_B0032-ldhA | 180627LP01 |
| pSB1C3-BBa\_E0840 | 180627LP02 |
| pSB1C3-cbtA | 180627LP03 |

Miniprep:

DNA was prepared using the GeneJet Kit No. K0503 from ThermoScientific, with two elution steps with water.

DNA is stored at -20 °C and stalled as:

|  |  |  |
| --- | --- | --- |
| Part | Lot-No. | Concentration [ng/µL] |
| pSB1C3-BBa\_B0032-ldhA | 180627LP04 | 75.97 |
| pSB1C3-BBa\_E0840 | 180627LP05 | 91.69 |
| pSB1C3-cbtA | 180627LP06 | 65.50 |

## PCR: leuABCD, ompT, tesA, psB1C3-Backbone and PCR Purification

PCR-Mastermix for leuABCD, ompT, tesA

|  |  |
| --- | --- |
| Q5-Mastermix | 10 µL |
| Primer fw | 0.5 µL |
| Primer rev | 0.5 µL |
| Template | 0.5 µL |
| H20 ad 20 µL | 8.5 µL |

PCR-Mastermix for pSB1C3-Backbone

|  |  |
| --- | --- |
| Q5-Mastermix | 25 µL |
| Primer fw SB-prep-3p | 1 µL |
| Primer rev SB-prep 2Ea | 1 µL |
| Template | 0.3 µL |
| H20 ad 50 µL | 22.7 µL |

|  |  |
| --- | --- |
| Tube No. | Part |
| 1 | leuABCD |
| 2 | leuABCD-H |
| 3 | ompT |
| 4 | tesA |
| 5 | pSB1C3 |

PCR-program Tube 1.2

|  |  |  |
| --- | --- | --- |
| Step No. | Temperature | Time |
| 1 | 98 ° C | 2:00 |
| 2 | 98 ° C | 0:10 |
| 3 | 50 ° C | 0:30 |
| 4 | 72 ° C | 3:00 |
| 5 | Go to step 2 (30X) |  |
| 6 | 72 ° C | 2:00 |
| 7 | 4 ° C | ∞ |

PCR-program Tube 3.4,5

|  |  |  |
| --- | --- | --- |
| Step No. | Temperature | Time |
| 1 | 98 ° C | 2:00 |
| 2 | 98 ° C | 0:10 |
| 3 | 50 ° C | 0:30 |
| 4 | 72 ° C | 3:00 |
| 5 | Go to step 2 (30X) |  |
| 6 | 72 ° C | 2:00 |
| 7 | 4 ° C | ∞ |

Gel electrophoresis was performed using a 1% agarose gel

![](https://static.igem.org/mediawiki/2018/7/76/T--Hamburg--180628_PCR.JPG)

After gel electrophoresis only lane 5 (psB1C3) showed a band.

psB1C3 PCR product was purified with the Gene-Jet PCR-Purification kit.

|  |  |  |
| --- | --- | --- |
| Part | Lot No. | DNA conc |
| psB1C3-Backbone | 180628NB01 | 96.6 ng/µL |

## Restriction and ligation of multiple Parts

Restriction Table

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Tube** | **1** | **2** | **4** | **7** | **9** | **10** | **11** | **12** | **13** | **14** | **15** | **16** | **17** | **18** | **1** | **25** |
| **Part** | pSB1C3-Bba\_B0031 | pSB1C3-Bba\_B0032 | pSB1C3-Bba\_K206000 | pSB1C3-Bba\_E0840 | Bba\_J45503 | cspD | accA | accB | cbtA | mreZ | ldhA | Bba\_B0031-ldhA | Bba\_B0032\_ldhA | Bba\_B0034\_ldhA | pSB1C3 | pSB1C3 |
| **Lot#** | 180514NB01 | 180514NB07 | 180321NB01 | 180315NB01 | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | 180628NB01 | 180628NB01 |
| **ρ** | 153 ng/µL | 81 ng/µL | 47 ng/µL | 128 ng/µL | 42 ng/µL | 44 ng/µL | 30 ng/µL | 36 ng/µL | 39 ng/µL | 50 ng/µL | 34 ng/µL | 41 ng/µL | 40 ng/µL | 40 ng/µL | 97 ng/µL | 97 ng/µL |
| Use |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| target m | 200 ng | 100 ng | 200 ng |  | 200 ng | 200 ng | 100 ng | 200 ng | 150 ng | 300 ng | 250 ng | 100 ng | 100 ng | 100 ng |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| DNA | 1.31 µL | 1.24 µL | 4.26 µL | 0.78 µL | 4.80 µL | 4.32 µL | 3.39 µL | 5.59 µL | 3.87 µL | 5.95 µL | 6.00 µL | 4.00 µL | 4.00 µL | 4.00 µL | 1.04 µL | 1.04 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF |  |  |  | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |  | 0.50 µL |
| XbaI |  |  |  | 0.50 µL |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |
| SpeI | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL |  |  |  |  |  |  |  |  |  |  | 0.50 µL |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |  |  |  |
| H2O | 6.19 µL | 6.26 µL | 3.24 µL | 6.72 µL | 3.20 µL | 3.68 µL | 4.61 µL | 2.41 µL | 4.13 µL | 2.05 µL | 2.00 µL | 4.00 µL | 4.00 µL | 4.00 µL | 6.96 µL | 6.96 µL |
| ad | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Länge Produkt |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| ρ final | 20 ng/µL | 10 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 15 ng/µL | 30 ng/µL | 21 ng/µL | 16 ng/µL | 16 ng/µL | 18 ng/µL | 10 ng/µL | 10 ng/µL |

Sequencing results from the 26th of august 2018 showed that EcoR1 was falsely added to restriction No. 10.

Ligation Table

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** | **L12** | **L13** | **L14** |
| Product | | pSB1C3-ldhA | pSB1C3-Bba\_B0031-ldhA | pSB1C3-Bba\_K206000-Bba\_B0031-ldhA | pSB1C3-Bba\_K206000-Bba\_B0032\_ldhA | pSB1C3-Bba\_K206000-Bba\_B0034\_ldhA | pSB1C3-Bba\_J45503 | pSB1C3-Bba\_E0840-Bba\_J45503 | pSB1C3-cspD | pSB1C3-Bba\_B0031-cspD | pSB1C3-accA | pSB1C3-Bba\_B0032-accB | pSB1C3-Bba\_B0031-cbtA | pSB1C3-Bba\_B0031-mraZ | pSB1C3-Bba\_B0032-mraZ |
|  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3 | pSB1C3-Bba\_B0031 | pSB1C3-Bba\_K206000 | pSB1C3-Bba\_K206000 | pSB1C3-Bba\_K206000 | pSB1C3 | pSB1C3-Bba\_E0840 | pSB1C3 | pSB1C3-Bba\_B0031 | pSB1C3 | pSB1C3-Bba\_B0032 | pSB1C3-Bba\_B0031 | pSB1C3-Bba\_B0031 | pSB1C3-Bba\_B0032 |
| Tube | **24** | **1** | **4** | **4** | **4** | **25** | **7** | **24** | **1** | **24** | **2** | **1** | **1** | **2** |
| ρ | 10 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 20 ng/µL | 10 ng/µL | 10 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL |
| Länge | 2989 bp | 2081 bp | 2150 bp | 2150 bp | 2150 bp | 2989 bp | 2948 bp | 2070 bp | 2081 bp | 2989 bp | 2082 bp | 2081 bp | 2081 bp | 2082 bp |
| V | 2.22 µL | 1.59 µL | 2.20 µL | 1.46 µL | 1.68 µL | 4.12 µL | 3.68 µL | 4.27 µL | 1.46 µL | 3.71 µL | 2.85 µL | 1.64 µL | 1.46 µL | 2.91 µL |
| **Part 1** | Part 1 | ldhA | ldhA | Bba\_B0031-ldhA | Bba\_B0032\_ldhA | Bba\_B0034\_ldhA | Bba\_J45503 | Bba\_J45503 | cspD | cspD | accA | accB | cbtA | mraZ | mraZ |
| Tube | **15** | **15** | **16** | **17** | **18** | **9** | **9** | **10** | **10** | **11** | **12** | **13** | **14** | **14** |
| ρ | 21 ng/µL | 21 ng/µL | 16 ng/µL | 16 ng/µL | 18 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 15 ng/µL | 30 ng/µL | 30 ng/µL |
| Länge | 1050 bp | 446 bp | 273 bp | 273 bp | 426 bp | 426 bp | 507 bp | 273 bp | 507 bp | 507 bp | 522 bp | 426 bp | 507 bp | 507 bp |
| V | 3.76 µL | 3.30 µL | 3.44 µL | 4.48 µL | 3.78 µL | 2.94 µL | 3.16 µL | 2.84 µL | 3.54 µL | 6.29 µL | 3.57 µL | 4.48 µL | 2.36 µL | 2.36 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| T4 Buffer | | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 2.52 µL | 3.61 µL | 2.85 µL | 2.56 µL | 1.44 µL | 1.44 µL | 1.44 µL | 1.38 µL | 3.50 µL | 7.50 µL | 2.08 µL | 2.38 µL | 4.68 µL | 3.23 µL |
| ad | | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |

## Restriction and ligation of multiple Parts

Restriction Table

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Tube** | **1** | **2** | **4** | **7** | **9** | **10** | **11** | **12** | **13** | **14** | **15** | **16** | **17** | **18** | **1** | **25** |
| **Part** | pSB1C3-Bba\_B0031 | pSB1C3-Bba\_B0032 | pSB1C3-Bba\_K206000 | pSB1C3-Bba\_E0840 | Bba\_J45503 | cspD | accA | accB | cbtA | mreZ | ldhA | Bba\_B0031-ldhA | Bba\_B0032\_ldhA | Bba\_B0034\_ldhA | pSB1C3 | pSB1C3 |
| **Lot#** | 180514NB01 | 180514NB07 | 180321NB01 | 180315NB01 | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | 180628NB01 | 180628NB01 |
| **ρ** | 153 ng/µL | 81 ng/µL | 47 ng/µL | 128 ng/µL | 42 ng/µL | 44 ng/µL | 30 ng/µL | 36 ng/µL | 39 ng/µL | 50 ng/µL | 34 ng/µL | 41 ng/µL | 40 ng/µL | 40 ng/µL | 97 ng/µL | 97 ng/µL |
| Use |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| target m | 200 ng | 100 ng | 200 ng |  | 200 ng | 200 ng | 100 ng | 200 ng | 150 ng | 300 ng | 250 ng | 100 ng | 100 ng | 100 ng |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| DNA | 1.31 µL | 1.24 µL | 4.26 µL | 0.78 µL | 4.80 µL | 4.32 µL | 3.39 µL | 5.59 µL | 3.87 µL | 5.95 µL | 6.00 µL | 4.00 µL | 4.00 µL | 4.00 µL | 1.04 µL | 1.04 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF |  |  |  | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |  | 0.50 µL |
| XbaI |  |  |  | 0.50 µL |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |
| SpeI | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL |  |  |  |  |  |  |  |  |  |  | 0.50 µL |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |  |  |  |
| H2O | 6.19 µL | 6.26 µL | 3.24 µL | 6.72 µL | 3.20 µL | 3.68 µL | 4.61 µL | 2.41 µL | 4.13 µL | 2.05 µL | 2.00 µL | 4.00 µL | 4.00 µL | 4.00 µL | 6.96 µL | 6.96 µL |
| ad | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Länge Produkt |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| ρ final | 20 ng/µL | 10 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 15 ng/µL | 30 ng/µL | 21 ng/µL | 16 ng/µL | 16 ng/µL | 18 ng/µL | 10 ng/µL | 10 ng/µL |

Sequencing results from the 26th of august 2018 showed that EcoR1 was falsely added to restriction No. 10.

Ligation Table

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** | **L12** | **L13** | **L14** |
| Product | | pSB1C3-ldhA | pSB1C3-Bba\_B0031-ldhA | pSB1C3-Bba\_K206000-Bba\_B0031-ldhA | pSB1C3-Bba\_K206000-Bba\_B0032\_ldhA | pSB1C3-Bba\_K206000-Bba\_B0034\_ldhA | pSB1C3-Bba\_J45503 | pSB1C3-Bba\_E0840-Bba\_J45503 | pSB1C3-cspD | pSB1C3-Bba\_B0031-cspD | pSB1C3-accA | pSB1C3-Bba\_B0032-accB | pSB1C3-Bba\_B0031-cbtA | pSB1C3-Bba\_B0031-mraZ | pSB1C3-Bba\_B0032-mraZ |
|  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3 | pSB1C3-Bba\_B0031 | pSB1C3-Bba\_K206000 | pSB1C3-Bba\_K206000 | pSB1C3-Bba\_K206000 | pSB1C3 | pSB1C3-Bba\_E0840 | pSB1C3 | pSB1C3-Bba\_B0031 | pSB1C3 | pSB1C3-Bba\_B0032 | pSB1C3-Bba\_B0031 | pSB1C3-Bba\_B0031 | pSB1C3-Bba\_B0032 |
| Tube | **24** | **1** | **4** | **4** | **4** | **25** | **7** | **24** | **1** | **24** | **2** | **1** | **1** | **2** |
| ρ | 10 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 20 ng/µL | 10 ng/µL | 10 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL |
| Länge | 2989 bp | 2081 bp | 2150 bp | 2150 bp | 2150 bp | 2989 bp | 2948 bp | 2070 bp | 2081 bp | 2989 bp | 2082 bp | 2081 bp | 2081 bp | 2082 bp |
| V | 2.22 µL | 1.59 µL | 2.20 µL | 1.46 µL | 1.68 µL | 4.12 µL | 3.68 µL | 4.27 µL | 1.46 µL | 3.71 µL | 2.85 µL | 1.64 µL | 1.46 µL | 2.91 µL |
| **Part 1** | Part 1 | ldhA | ldhA | Bba\_B0031-ldhA | Bba\_B0032\_ldhA | Bba\_B0034\_ldhA | Bba\_J45503 | Bba\_J45503 | cspD | cspD | accA | accB | cbtA | mraZ | mraZ |
| Tube | **15** | **15** | **16** | **17** | **18** | **9** | **9** | **10** | **10** | **11** | **12** | **13** | **14** | **14** |
| ρ | 21 ng/µL | 21 ng/µL | 16 ng/µL | 16 ng/µL | 18 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 15 ng/µL | 30 ng/µL | 30 ng/µL |
| Länge | 1050 bp | 446 bp | 273 bp | 273 bp | 426 bp | 426 bp | 507 bp | 273 bp | 507 bp | 507 bp | 522 bp | 426 bp | 507 bp | 507 bp |
| V | 3.76 µL | 3.30 µL | 3.44 µL | 4.48 µL | 3.78 µL | 2.94 µL | 3.16 µL | 2.84 µL | 3.54 µL | 6.29 µL | 3.57 µL | 4.48 µL | 2.36 µL | 2.36 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| T4 Buffer | | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 2.52 µL | 3.61 µL | 2.85 µL | 2.56 µL | 1.44 µL | 1.44 µL | 1.44 µL | 1.38 µL | 3.50 µL | 7.50 µL | 2.08 µL | 2.38 µL | 4.68 µL | 3.23 µL |
| ad | | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |

## Restriction and Ligation of PCR Gen II and Transformation

Restriction:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Tube** |  | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** | **15** | **16** | **17** | **18** | **19** | **20** | **21** | **22** | **23** | **24** | **25** |
| **Part** | pSB1C3-BBa\_B0030 | pSB1C3-BBa\_B0031 | pSB1C3-BBa\_B0032 | pSB1C3-BBa\_B0031 | pSB133-BBa\_K206000 | pSB1C3-I20270 | pSB1C3-I20270 | pSB1C3-BBa\_E0840 | pSB1C3-I20270 | BBa\_J45503 | cspD | accA | accB | cbtA | mraZ | ldhA | BBa\_B0031-ldhA | BBa\_B0032-ldhA | BBa\_B0034-ldhA | MlcRE | ldhA | cspD | accB | mraZ | pSB1C3 | pSB1C3 |
| **Lot#** |  | 180514NB01 | 180514NB07 | 180514NB01 | 180321NB01 | 180509NF01 | 180509NF01 | 180315NB01 | 180509NF01 | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | aus PCR | 180628NB01 | 180628NB01 |
| **ρ** |  | 153 ng/µL | 81 ng/µL | 153 ng/µL | 47 ng/µL | 166 ng/µL | 166 ng/µL | 128 ng/µL | 166 ng/µL | 42 ng/µL | 46 ng/µL | 30 ng/µL | 36 ng/µL | 39 ng/µL | 50 ng/µL | 34 ng/µL | 41 ng/µL | 40 ng/µL | 44 ng/µL | 55 ng/µL | 34 ng/µL | 46 ng/µL | 36 ng/µL | 50 ng/µL | 97 ng/µL | 97 ng/µL |
| Use |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| target m | 200 ng | 200 ng | 100 ng | 200 ng | 200 ng | 500 ng |  |  |  | 200 ng | 200 ng | 100 ng | 200 ng | 150 ng | 300 ng | 250 ng | 100 ng | 100 ng | 100 ng | 100 ng | 250 ng | 200 ng | 200 ng | 300 ng |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| DNA |  | 1.31 µL | 1.24 µL | 1.31 µL | 4.26 µL | 6.02 µL | 0.60 µL | 0.78 µL | 0.60 µL | 4.80 µL | 4.32 µL | 3.39 µL | 5.59 µL | 3.87 µL | 5.95 µL | 6.00 µL | 4.00 µL | 4.00 µL | 4.00 µL | 2.00 µL | 7.30 µL | 4.32 µL | 5.59 µL | 5.95 µL | 1.04 µL | 1.04 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  | 0.50 µL |
| XbaI | 0.50 µL |  |  | 0.50 µL |  |  |  | 0.50 µL |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL |  | 0.50 µL |  |
| SpeI |  | 0.50 µL | 0.50 µL |  | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  | 0.50 µL |  |  |  |  |  | 0.50 µL |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| H2O | 7.50 µL | 6.19 µL | 6.26 µL | 6.19 µL | 3.24 µL | 10.48 µL | 6.90 µL | 6.72 µL | 6.90 µL | 3.20 µL | 3.68 µL | 4.61 µL | 2.41 µL | 4.13 µL | 2.05 µL | 2.00 µL | 4.00 µL | 4.00 µL | 4.00 µL | 6.00 µL | 9.70 µL | 3.68 µL | 2.41 µL | 2.05 µL | 6.96 µL | 6.96 µL |
| ad | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Länge Produkt |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| ρ final | 0 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 20 ng/µL | 50 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 15 ng/µL | 30 ng/µL | 21 ng/µL | 16 ng/µL | 16 ng/µL | 18 ng/µL | 11 ng/µL | 13 ng/µL | 20 ng/µL | 20 ng/µL | 30 ng/µL | 10 ng/µL | 10 ng/µL |
|  |  |  |  |  |  | 24 |  |  |  | 15 | 13 ng/µL |  | 10 | 15 | 15 |  |  |  |  |  |  | 13 ng/µL | 10 | 15 |  |  |

The parts were restricted at 37°C for 1 h and after that stored at 80°C and 20 min.

Ligation:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| pSB1C3-ldhA | pSB1C3-BBa\_B0031-ldhA | pSB1C3-BBa\_B0032-ldhA | pSB1C3-BBa\_B0034-ldhA |  | pSB1C3-BBa\_K20600-BBa\_B0031-ldhA | pSB1C3-BBa\_K20600-BBa\_B0032-ldhA | pSB133-BBa\_K206000-BBa\_B0034-ldhA |  |  | pSB1C3-BBa\_J45503 | pSB1C3-BBa\_E0840-BBa\_J45503 | pSB1C3-cspD | pSB1C3-BBa\_B0031-cspD | pSB1C3-accA | pSB1C3-accB | pSB1C3-BBa\_B0032-accB | pSB1C3-BBa\_B0031-cbtA | pSB1C3-cbtA | pSB1C3-BBa\_B0031-mraZ | pSB1C3-BBa\_B0032-mraZ | pSB1C3-mraZ | pSB1C3-MlcRE | pSB1C3-MlcRE-BBa\_B0032-ldhA | pSB1C3-MlcRE-BBa\_B0034-ldhA | pSB1C3-BBa\_B0030-accA |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| pSB1C3 | pSB1C3-BBa\_B0031 | pSB1C3-BBa\_B0032 | pSB1C3-I20270 |  | pSB133-BBa\_K206000 | pSB133-BBa\_K206000 | pSB133-BBa\_K206000 |  |  | pSB1C3 | pSB1C3-BBa\_E0840 | pSB1C3 | pSB1C3-BBa\_B0031 | pSB1C3 | pSB1C3 | pSB1C3-BBa\_B0032 | pSB1C3-BBa\_B0031 | pSB1C3 | pSB1C3-BBa\_B0031 | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I20270 | pSB1C3-I20270 | pSB1C3-I20270 | pSB1C3-BBa\_B0030 |
| **24** | **1** | **2** | **5** |  | **4** | **4** | **4** |  |  | **25** | **7** | **24** | **1** | **24** | **24** | **2** | **1** | **24** | **1** | **2** | **24** | **6** | **6** | **6** |  |
| 10 ng/µL | 20 ng/µL | 10 ng/µL | 50 ng/µL |  | 20 ng/µL | 20 ng/µL | 20 ng/µL |  |  | 10 ng/µL | 10 ng/µL | 10 ng/µL | 20 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |  |
| 2989 bp | 2081 bp | 2084 bp | 2989 bp |  | 2150 bp | 2150 bp | 2150 bp |  |  | 2989 bp | 2948 bp | 2989 bp | 2081 bp | 2989 bp | 2989 bp | 2082 bp | 2081 bp | 2989 bp | 2081 bp | 2082 bp | 2989 bp | 2989 bp | 2989 bp | 2989 bp | 2085 bp |
| 2.22 µL | 1.59 µL | 4.33 µL | 1.05 µL |  | 2.20 µL | 1.46 µL | 1.68 µL |  |  | 4.12 µL | 3.68 µL | 3.71 µL | 1.46 µL | 3.71 µL | 3.64 µL | 2.85 µL | 1.64 µL | 4.12 µL | 1.46 µL | 2.91 µL | 3.71 µL | 1.86 µL | 1.86 µL | 1.86 µL |  |
| ldhA | ldhA | ldhA | BBa\_B0034-ldhA |  | BBa\_B0031-ldhA | BBa\_B0032-ldhA | BBa\_B0034-ldhA |  |  | BBa\_J45503 | BBa\_J45503 | cspD | cspD | accA | accB | accB | cbtA | cbtA | mraZ | mraZ | mraZ | MlcRE | MlcRE | MlcRE |  |
| **15** | **15** | **15** | **18** |  | **16** | **17** | **18** |  |  | **9** | **9** | **10** | **10** | **11** | **12** | **12** | **13** | **13** | **14** | **14** | **14** | **19** | **19** | **19** |  |
| 21 ng/µL | 21 ng/µL | 21 ng/µL | 18 ng/µL |  | 16 ng/µL | 16 ng/µL | 18 ng/µL |  |  | 20 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 10 ng/µL | 20 ng/µL | 20 ng/µL | 15 ng/µL | 15 ng/µL | 30 ng/µL | 30 ng/µL | 30 ng/µL | 11 ng/µL | 11 ng/µL | 11 ng/µL |  |
| 1050 bp | 446 bp | 273 bp | 273 bp |  | 273 bp | 522 bp | 426 bp |  |  | 426 bp | 507 bp | 507 bp | 507 bp | 507 bp | 522 bp | 522 bp | 426 bp | 426 bp | 507 bp | 507 bp | 507 bp | 252 bp | 252 bp | 252 bp |  |
| 3.76 µL | 3.30 µL | 2.74 µL | 2.72 µL |  | 3.44 µL | 4.48 µL | 3.78 µL |  |  | 2.94 µL | 3.16 µL | 3.15 µL | 3.54 µL | 6.29 µL | 3.18 µL | 3.57 µL | 4.48 µL | 3.92 µL | 2.36 µL | 2.36 µL | 2.10 µL | 1.43 µL | 1.43 µL | 1.43 µL |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | BBa\_B0032-ldhA | BBa\_B0034-ldhA | accA |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | **16** | **17** | **18** |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 16 ng/µL | 16 ng/µL | 18 ng/µL |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 1054 bp | 1055 bp | 1056 bp | 507 bp |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 4.48 µL | 4.60 µL | 4.14 µL |  |
| 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |  | 1.00 µL | 1.00 µL | 1.00 µL |  |  | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |  |
| 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |
| 2.52 µL | 3.61 µL | 1.43 µL | 4.74 µL |  | 2.85 µL | 2.56 µL | 3.04 µL |  |  | 1.44 µL | 1.66 µL | 1.65 µL | 3.50 µL | 7.50 µL | 1.68 µL | 2.07 µL | 2.38 µL | 0.46 µL | 4.68 µL | 3.23 µL | 2.69 µL | 0.73 µL | 0.61 µL | 1.07 µL |  |
| 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |  | 10.00 µL | 10.00 µL | 10.00 µL |  |  | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |  |

The parts were ligated at 37°C for 1 h.

## Transformation

Transformation protocol by Zheng.

Lotbumbers of the plates: 180629NF01-14

Stored at room temperature over the weekend.

## Colony-PCR (Gen.II Parts)

|  |  |  |  |
| --- | --- | --- | --- |
| 1-3 | 180629NiF01 | pSB1C3-ldhA | X |
| 4-6 | 180629NiF02 | pSB1C3-BBa\_B0031-ldhA | ✓6 |
| 7-9 | 180629NiF03 | pSB1C3-BBa\_K206000-BBa\_B0031-ldhA | X |
| 10-12 | 180629NiF04 | pSB1C3-BBa\_K206000-BBa\_B0032-ldhA | ✓11-13 |
| 13-15 | 180629NiF05 | pSB1C3-BBa\_K206000-BBa\_B0034-ldhA | ✓15 |
| 16-18 | 180629NiF06 | pSB1C3-BBa\_J45503 | X |
| 19-21 | 180629NiF07 | pSB1C3-BBa\_E0840-BBa\_J45503 | ✓20 |
| 22-24 | 180629NiF08 | pSB1C3-cspD | ✓22 |
| 25-27 | 180629NiF09 | pSB1C3-BBa\_B0031-cspD | X |
| 28-30 | 180629NiF10 | pSB1C3-accA | X |
| 31-33 | 180629NiF11 | pSB1C3-BBa\_B0032-accB | ✓32 |
| 34-36 | 180629NiF12 | pSB1C3- BBa\_B0031-cbtA | X |
| 37-39 | 180629NiF13 | pSB1C3- BBa\_B0031-mraz | X |
| 40-42 | 180629NiF14 | pSB1C3- BBa\_B0032-mraz | ✓40-42 |

 For 45 Uses

PCR-Program

|  |  |
| --- | --- |
| 95 °C | 3 min |
| 95 °C | 30 s |
| 55 °C | 30 s |
| 72 °C | 1:30 min |
| 72 °C | 5 min |

Lot-Numbers

|  |
| --- |
| 6 à 180702NB01: psB1C3-BBa\_B0031-ldhA |
| 11 à 180702NB02: psB1C3-BBa\_K206000-BBa-B0032-ldhA |
| 12 à 180702NB03: psB1C3-BBa\_K206000-BBa-B0032-ldhA |
| 13 à 180702NB04: psB1C3-BBa\_K206000-BBa-B0032-ldhA |
| 15 à 180702NB05: psB1C3-BBa\_ K206000-BBa-B0034-ldhA |
| 20 à 180702NB06: psB1C3-BBa\_E0840-JU5503 |
| 22 à 180702NB07 psB1C3-cspD |
| 32 à 180702NB08: psB1C3-BBa\_B0032-accB |
| 40 à 180702NB09: psB1C3-BBa\_B0032-mraz |
| 41 à 180702NB10: psB1C3-BBa\_B0032-mraz |
| 42 à 180702NB11: psB1C3-BBa\_B0032-mraz |

## <https://static.igem.org/mediawiki/2018/7/7a/T--Hamburg--180702_cPCR1.JPG>

## Colony-PCR (Gen.II Parts)

|  |  |  |  |
| --- | --- | --- | --- |
| 1-3 | 180629NiF01 | pSB1C3-ldhA | X |
| 4-6 | 180629NiF02 | pSB1C3-BBa\_B0031-ldhA | ✓6 |
| 7-9 | 180629NiF03 | pSB1C3-BBa\_K206000-BBa\_B0031-ldhA | X |
| 10-12 | 180629NiF04 | pSB1C3-BBa\_K206000-BBa\_B0032-ldhA | ✓11-13 |
| 13-15 | 180629NiF05 | pSB1C3-BBa\_K206000-BBa\_B0034-ldhA | ✓15 |
| 16-18 | 180629NiF06 | pSB1C3-BBa\_J45503 | X |
| 19-21 | 180629NiF07 | pSB1C3-BBa\_E0840-BBa\_J45503 | ✓20 |
| 22-24 | 180629NiF08 | pSB1C3-cspD | ✓22 |
| 25-27 | 180629NiF09 | pSB1C3-BBa\_B0031-cspD | X |
| 28-30 | 180629NiF10 | pSB1C3-accA | X |
| 31-33 | 180629NiF11 | pSB1C3-BBa\_B0032-accB | ✓32 |
| 34-36 | 180629NiF12 | pSB1C3- BBa\_B0031-cbtA | X |
| 37-39 | 180629NiF13 | pSB1C3- BBa\_B0031-mraz | X |
| 40-42 | 180629NiF14 | pSB1C3- BBa\_B0032-mraz | ✓40-42 |

For 45 Uses

PCR-Program

|  |  |
| --- | --- |
| 95 °C | 3 min |
| 95 °C | 30 s |
| 55 °C | 30 s |
| 72 °C | 1:30 min |
| 72 °C | 5 min |

## <https://static.igem.org/mediawiki/2018/5/59/T--Hamburg--180702_cPCR2.JPG>

## 180704 – 1

## oHybB characterisation

Cultures for characterisation were set up at an OD of 0.1 employing over night cultures from the day before.

Cultures were incubated for 6 hours at different temperatures. The optical density was measured each hour. At each time point 1 mL of culture was taken and added to 600 µL of PFA and stored in the fridge until measurement.

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | 18 °C | | | | 27 °C | | | | 37 °C | | | |
| t [h] | DH5α (-) | I20270 (+) | oHybB | J45503 | DH5α (-) | I20270 (+) | oHybB | J45503 | DH5α (-) | I20270 (+) | oHybB | J45503 |
| 0 | 0.134 | 0.074 | 0.100 | 0.102 | 0.140 | 0.068 | 0.102 | 0.108 | 0.098 | 0.082 | 0.194 | 0.110 |
| 1 | 0.252 | 0.060 | 0.098 | 0.098 | 0.196 | 0.084 | 0.170 | 0.170 | 0.430 | 0.260 | 0.490 | 0.340 |
| 2 | 0.210 | 0.088 | 0.144 | 0.160 | 0.836 | 0.400 | 0.924 | 0.788 | 1.376 | 0.756 | 1.448 | 0.980 |
| 3 | 0.304 | 0.112 | 0.208 | 0.210 | 0.952 | 0.372 | 0.920 | 0.784 | 3.120 | 2.008 | 3.544 | 2.352 |
| 4 | 0.440 | 0.164 | 0.312 | 0.282 | 1.792 | 0.832 | 1.624 | 1.416 | 3.920 | - | 3.890 | 4.410 |
| 5 | 0.658 | 0.202 | 0.430 | 0.404 | 2.920 | 2.010 | 2.030 | 2.440 | 5.540 | - | 5.360 | 6.300 |
| 6 | 1.240 | 0.352 | 0.736 | 0.680 | 4.700 | 3.240 | 4.640 | 4.920 | 7.200 | - | 6.960 | 8.500 |

GFP measurement was carried out the next day.

## Miniprep of pSB1C3\_BBa\_I20270

Miniprep was performed using GeneJet Plasmid Miniprep Kit from Thermo Fischer

|  |  |
| --- | --- |
| LOT-Number | Concentration (ng/µL) |
| 180507LP01 | 6.136 |

## Evaluation of oHybB-characterization

Plate-reader measured with following load-schematic:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | 18°C | | | | | 27°C | | | | 37°C | | | |
|  |  | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| t1 | A | - | + | oHybB | J45503 | - | + | oHybB | J45503 | - | + | oHybB | J45503 |
| t2 | B | - | + | oHybB | J45503 | - | + | oHybB | J45503 | - | + | oHybB | J45503 |
| t3 | C | - | + | oHybB | J45503 | - | + | oHybB | J45503 | - | + | oHybB | J45503 |
| t4 | D | - | + | oHybB | J45503 | - | + | oHybB | J45503 | - | + | oHybB | J45503 |
| t5 | E | - | + | oHybB | J45503 | - | + | oHybB | J45503 | - | + | oHybB | J45503 |
| t6 | F | - | + | oHybB | J45503 | - | + | oHybB | J45503 | - | + | oHybB | J45503 |

-  = DH5a

+ = pSB1C3-I20270

Load-schematic for all temperatures:

The rows G and H were added to the plates 27°C and 37°C as a reference. There was no positive control from 4 at 37°C. There was no positive and negative control with 37°C at t6.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| t1 | A | - | - | - | + | + | + | oHybB | oHybB | oHybB | J45503 | J45503 | J45503 |
| t2 | B | - | - | - | + | + | + | oHybB | oHybB | oHybB | J45503 | J45503 | J45503 |
| t3 | C | - | - | - | + | + | + | oHybB | oHybB | oHybB | J45503 | J45503 | J45503 |
| t4 | D | - | - | - | + | + | + | oHybB | oHybB | oHybB | J45503 | J45503 | J45503 |
| t5 | E | - | - | - | + | + | + | oHybB | oHybB | oHybB | J45503 | J45503 | J45503 |
| t6 | F | - | - | - | + | + | + | oHybB | oHybB | oHybB | J45503 | J45503 | J45503 |
| 18°C | G | + tx | + tx | + tx |  |  |  |  |  |  |  |  |  |
| H |  |  |  |  |  |  |  |  |  |  |  |  |

## TD-PCR (PCR-GGA)

PCR of LeuABCD, LeuABCD6H and tesA was performed.

Two different master mixes  were set up.

Mastermix1

|  |  |
| --- | --- |
| Substances | 1x volume (in µl) |
| Q5-master mix | 2.5 |
| F-Primer | 2.5 |
| R-Primer | 2.5 |
| DNA (DH5α) | 1 |
| DMSO | 1.5 |
| ddH2O | 7.5 |

Mastermix 2

|  |  |
| --- | --- |
| Substances | 1x volume (in µl) |
| 10x Taq buffer | 2 |
| Taq Polymerase | 0.5 |
| F-Primer | 1.2 |
| R-Primer | 1.2 |
| DNA (DH5α) | 1 |
| dNTPs | 0.4 |
| 50 mM MgCl2 | 0.6 |
| DMSO | 1.5 |
| ddH2O | 13 |

PCR-Program

|  |  |
| --- | --- |
| 95 °C | 3 min |
| 95 °C | 30 s |
| 60 °C | 30 s |
| 72 °C | 1:30 min |
| 72 °C | 5 min |

Start-Annealing was set to 60 °C. Every cycle the temperature was set down 2 °C until 40 °C was reached following 20 cycles at 40°C.

## PCR Golden Gate Assembly (GGA)

14x PCR-set ups with 50 μl with Q5-Polymerase (NEB)

10 μM 2.5 μL fw-Primer, 10 μM 2.5 μL rv-Primer, 25 μL Q5 2x Master Mix

Template DNA: 0.1 μL Plasmid or 0.5 μL DH5α, 20 μL H2O

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Product | Fw-Primer | Rv-Primer | Pred. length | Template | End length |
| BsaI30cbtABsaI | BsaI30cbtA | cbtA\_BsaI\_r | 394 | cbtA |  |
| BsaI30sspDBsaI | BsaI30sspD | sspD\_BsaI\_r | 244 | sspD |  |
| BsaI30mrazBsaI | BsaI30mraz | Mraz\_BsaI\_r | 408 | mraZ |  |
| BsaI30SulABsaI | BsaISulA | SulA\_Suf\_Bsa | 550 | SulA |  |
| BsaI32cbtABsaI | BsaI32cbtA | cbtA\_BsaI\_r | 394 | cbtA |  |
| BsaI32sspDBsaI | BsaI32sspD | sspD\_BsaI\_r | 244 | sspD |  |
| BsaI32mrazBsaI | BsaI32mraz | Mraz\_BsaI\_r | 408 | mraZ |  |
| BsaI32SulABsaI | BsaISulA | SulA\_Suf\_Bsa | 550 | pSB1C3SulA |  |
| BsaI34cbtABsaI | BsaI34cbtA | cbtA\_BsaI\_r | 394 | cbtA |  |
| BsaI34sspDBsaI | BsaI34sspD | sspD\_BsaI\_r | 244 | sspD |  |
| BsaI34mrazBsaI | BsaI34mraz | Mraz\_BsaI\_r | 408 | mraZ |  |
| BsaI34SulABsaI | BsaISulA | SulA\_Suf\_Bsa | 550 | pSB1C3SulA |  |
| dapAP | dapAP\_s\_f | dapAP\_s\_r | 120 | synthetic |  |
| dapAP\_BsaI | BsaI\_dapAP\_f | dapAP\_s\_r | 120 | DH5α |  |

PCR-Progamm:

|  |  |
| --- | --- |
| Temperature | Time |
| 98 °C | 0:30 (2:00 für DH5α) |
| 98 °C | 0:10 |
| 55 °C | 0:20 |
| 72 °C | 1:00 (Go to step 2, repeat 30 times) |
| 72 °C | 2:00 |

Tubes 1-14 à Lotnumber 180706BK01-14

![](https://static.igem.org/mediawiki/2018/b/bf/T--Hamburg--180706_PCR_GGA.JPG)

## PCR for amplification of RNA-Operator G120

Mastermix:

* 25 µL Q5 2xMastermix
* 5 µL Primer Syn fw
* 5 µL Primer Syn rv
* 5 µL Template DNA of IDT
* 50 µL H2Odest

PCR-Programm:

|  |  |
| --- | --- |
| 98°C | 30 s |
| 98°C | 10 s |
| 63°C | 30 s |
| 72°C | 30 s |
| Go to step 2 x30 | |
| 72°C | 2 min |
| 4°C |  |

## Agarose-Gel:

We made 1% Agarose-gel. It ran for 45 min by 80 V. Following that the sample was extracted with GeneJET Gelextraction Kit.

![](https://static.igem.org/mediawiki/2018/0/03/T--Hamburg--180709_PCR.JPG)

## Restriction

|  |  |  |
| --- | --- | --- |
| Vektor | psB1C3 (180628NB01) | RNA G120 Operator (180709NB01) |
| ρ | 96.6 ng/µL | 144.037 ng/µL |
| VDNA | 1.04 µL | 0.69 µL |
| NEB Buffer | 1 µL | 1 µL |
| EcoR1 | 0.5 µL | 0.5 µL |
| Pst1 | 0.5 µL | 0.5 µL |
| Fast AP | 0.5 µL | - |
| H2Odest | 6.46 µL | 7.31 µL |

We incubated for 1 h by 37°C and inactivated for 10 min by 80°C.

## Ligation

|  |  |
| --- | --- |
| psB1C3 | 3.05 µL |
| RNA G120 Operator | 6.95 µL |
| T4-Buffer | 1 µL |
| T4-Ligase | 0.5 µL |
| H2Odest | - |
| Total | 11.5 µL |

The Parts were incubated over night by 37°C.

## Heat shock Transformation of RNA G120

Protocol by Zhang

180710NB01 = negativ control

180710NB02 = RNA G120 (Ligation)

180710NB03 = Test-Trafo (pSB1C3-BBa\_B0032)

## Preparation of Agar-plates and Medium

Agar-plates:

* 16 g Agar
* 400 mL H2Odest

Medium:

* 25 g Agar
* 1 L H2Odest
* All with cAMP

Additive 400 µM DAP :

* 0767 g on 1 L medium
* 0309 g on 0.4 L Agar

## PCR-dapAP

Mastermix:

* 25 µL Q5 2xMix
* 20 µL H2Odest
* 5 µL Bsa1-dapAP-fw Primer
* 5 µL dapAP-Bsa1-rv Primer
* 1 µL dapAP\_s\_f
* 1 µL dap\_s\_r

PCR Program:

|  |  |
| --- | --- |
| 98°C | 2 min |
| 98°C | 10 s |
| 55°C | 20 s |
| 72°C | 30 s |
| Go to step 2 30x | |
| 72°C | 2 min |

## Agarose-Gel

We made a 1% Agarose-gel.

![](https://static.igem.org/mediawiki/2018/4/45/T--Hamburg--180710_PCR.JPG)

## Golden Gate Assembly (Restriction and Ligation)

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Tube | H2O | GGABulter | GGAM | pGGA | 180706BK… |
| 1 | 14 µL | 2 µL | 1 µL | 1 µL | 0.15 µL (01-12) |
| 2 | 14 µL | 2 µL | 1 µL | 1 µL | 0.15 µL (01-12) |
| 3 | 14 µL | 2 µL | 1 µL | 1 µL | 0.8 µL (12), 0.55 µL (05), 0.4 µL (06), 0.43 µL (07) |
| 4 | 14 µL | 2 µL | 1 µL | 1 µL | 0.55 µL (04), 0.55 µL (05), 0.4 µL (06), 0.43 µL (07) |
| 5 | 14 µL | 2 µL | 1 µL | 1 µL | 0.8 µL (12), 0.4 µL (01), 0.3 µL (02), 0.3 µL (03) |

GGA Program:

|  |  |
| --- | --- |
| 37°C | 1 min |
| 16°C | 1 min |
| Go to step 1 30x | |
| 55°C | 5 min |

## Transformation

Protocol by Zhang:

* 180710BK02 = Tube 1
* 180710BK03 = Tube 2
* 180710BK04 = Tube 3
* 180710BK05 = Tube 4
* 180710BK06 = Tube 5

## 180711 – 1

## cPCR – of trafos from GGA of the growth inhibition with different ribosome binding sites and RNA G120

cPCR was performed as the next step of the transformations of the previous day. Colonies were picked and used for the cPCR as follows:

               Plate                     No. of Colonies Tube No.

               180710NB02       4                            01 – 04
                180710BK02       9                            05 – 13
                180710BK03       14                          14 – 27
                180710BK04       4                            28 – 31
                180710BK05       4                            32 – 35
                180710BK06       4                            36 – 39

Master Mix was set up for 40 reactions with:

 80 µL of 10x Taq Buffer
 16 µL of 10 nM DNTPs
 14 µL of 50 nM MgCl2
 2 µL of Taq DNA Polymerase
 631 µL of H20

Forward and reversed primers were added with 0.6 µL each. For the GGA cPCR the primers used were GGA\_cPCR\_f (forward) and GGA\_cPCR\_r (reversed). For the cPCR for RNA G120 the primers used were Vf2 (forward) and Vr (reversed).

PCR was carried out according to the following protocol:

step       T (°C)     t (min)

1             95           3:00
 2             95           0:20
 3             56           0:30
 4             72           2:30
 5             72           5:00
 6             6             hold

               Cycle from step 2 to 4 was repeated 30 times.

Results:

![](https://static.igem.org/mediawiki/2018/b/bf/T--Hamburg--180711-1.JPG)

cPCR of RNA-G120 operator worked and we got a part with the desired length.

cPCR of the colonies from the GGA colonies didn’t work well. Just one colonie (lane 32) had a part with the desired length (~ 2000 bp).

## Restriktion, Ligation, Transformation, RNAG120-Operator

Restriktion:

|  |  |  |
| --- | --- | --- |
|  | RNA-Operator 180709NB01 | pSB1C3-E084018020BN03 |
| cDNA | 144.037 ng/μL | 57 ng/μL |
| VDNA | 0.69 μL | 1.75 μL |
| NEB-Buffer | 1 μL | 1 μL |
| EcoRI | 0.5 μL | 0.5 μL |
| SpeI | 0.5 μL |  |
| XbaI |  | 0.5 μL |
| FastAP |  | 0.5 μL |
| H2O | 7.31 μL | 5.75 μL |
| Total | 10 μL | 10 μL |

* 1 h, 37 °C + 20 min 80 °C heat shock deactivation

GGA Miniprep-Cryostock (rRNA G120) (check cPCR from 180711)

Culture 32: 180712BK01 Plasmid (c = 169.243 ng/μL)

                   180712BK02 Cryostock

Culture 2:  180712BK03 Plasmid (c = 93.328 ng/μL)

                  180712BK04 Cryostock

## PCR

1 – kivD

2 – accC

3 – accD

4 – BjaII-OmpT-SiteF-A6

Approach:

|  |
| --- |
| 20 μL ddH2O |
| 25 μL Q5-Mix |
| 2.5 μL Fw-Primer |
| 2.5 μL Rev-Primer |
| 0.2 μL Template |
| Total of: 50.2 μL |

Vectorrestriction:

|  |  |  |  |
| --- | --- | --- | --- |
|  | pSB1C3\_BBa\_320270 | BBa\_E0840 | pSB1C3\_RNA\_G120 |
| DNA | 12.05 μL | 6 μL | 6 μL |
| NEB 2.1 | 2 μL | 1 μL | 1 μL |
| EcoRI | 0.4 μL | 0.2 μL | 0.2 μL |
| PstI | 0.4 μL | 0.2 μL | 0.2 μL (SpeI) |
| FastAP | 0.5 μL | 0.5 μL | 0.5 μL |
| H2O | 4.65 μL | 2.1 μL | 2.1 μL |

Run Samples from PCR and restriction on 1%-agarosegel:

## Gelextraction

Actions performed according to ThermoFischers GeneJet GelEx-Kit.

|  |  |  |
| --- | --- | --- |
| DNA | concentration | Lot-Nr. |
| pSB1C3 | 30 ng/μL | 180716LP01 |
| pSB1C3\_BBa\_E0840 | 3.9 ng/μL | 180716LP02 |
| RNAG120-Operator-MIcRE | 5.9 ng/μL | 180716LP03 |
| kivD | 15.2 ng/μL | 180716LP04 |
| accC | 10 ng/μL | 180716LP05 |
| accD | 7.5 ng/μL | 180716LP06 |
| BjaII-OmpT-SiteF-A6 | 110 ng/μL | 180716LP07 |

Ligation:

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | L2 | L4 | L5 | L6 | L7 |
| DNA | pSB1C3\_E0840  (9.29 μL) | pSB1C3   (0.4 μL) | pSB1C3   (1.47 μL) | pSB1C3 (0.04 μL) | pSB1C3 (0.62 μL) |
| DNA | RNAG120 (10.81 μL) | kivD   (6.7 μL) | BjaII-OmpT-SiteF-A6 (0.5 μL) | accC   (8.67 μL) | accD   (10.82 μL) |
| T4-Buffer | 3 μL | 1 μL | 1 μL | 2 μL | 2 μL |
| T4-Ligase | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL |
| H2O | 6.4 μL | 1.4 μL | 6.54 μL | 8.39 μL | 6.02 μL |

Incubate over-night at 16 °C

## Tranformation of multiple parts

ransformation the ligations of the previous day (1) and parts of the distribution kit (2).

* pSB1C3-E804-RNAG120
   pSB1C3-hivD
   pSB1C3-BjalT-OmpT Flag
   pSB1C3-accC
   pSB1C3-accD
* BBa\_K577893
   BBa\_R0010

The transformation was carried out according to the protocol of Zhang (see 180418)

## Characterisation of the growth inhibition module

Growth inhibition module: pGGA-BBa\_B0032-cbtA-BBa\_B0032-cspD-BBa\_B0032-mraZ-BBa\_B0030-sulA

Characterisation of the growth inhibition module with DH5α without the part as a negative control (DH5α (-)). Diaminopimelic acid (DAP) used for repression of the growth inhibition at a concentration of 40 µM and at high concentration of 8.3 mM (DAP\*).

Initial OD measurements

               O/n culture         OD         µL taken to get an OD of 0.01 in 20 µL media

               DH5α (-)               1.78       112.35
                DH5α                    1.23       162.60
                DH5α + DAP        1.18       169.49
                DH5α + DAP\*      1.33       150.38

For the characterisation all cultures started with an OD of 0.01 in 20 mL.

For the characterisation the cultures were incubated at 37 °C for 6 hours, with a measurement of the OD at each full hour. The results were as follows:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| t [h] | DH5α (-) | DH5α | DH5α + DAP | DH5α + DAP\* |
| 0 | 0.01 | 0.01 | 0.01 | 0.01 |
| 1 | 0.133 | 0.009 | 0.011 | 0.025 |
| 2 | 0.034 | 0.022 | 0.021 | 0.058 |
| 3 | 0.172 | 0.027 | 0.030 | 0.064 |
| 4 | 0.711 | 0.156 | 0.148 | 0.303 |
| 5 | 1.162 | 0.28 | 0.205 | 0.678 |
| 6 | 1.422 | 0.593 | 0.459 | 1.145 |

Results:

Dap-repressed and unrepressed samples showed similar growth behaviour. However, at high dap concentrations the cultures grew faster. Cells without growth-inhibiting plasmid grew best.

## Colony PCR of the transformation from 17.07.18

A cPCR was performed of the transformation from the previous day. A PCR Mastermix was created:

|  |  |
| --- | --- |
| Taq 10x Buffer | 50 µL |
| dNTPs mM | 10 µL |
| MgCl2 50mM | 15 µL |
| Taq DNA Polymerase | 1 µL |
| VF2-Primer | 15 µL |
| VR-Primer | 15 µL |
| H2O | 394 µL |

Colonies were picked as follows:

|  |  |
| --- | --- |
| Part | Tube No. |
| pSB1C3-BBa\_R0010 | 1-4 |
| pSB1C3-BBa\_K577881 | 5-8 |
| pSB1C3-BBa\_E0240 | 9-12 |
| pSB1C3-BBa\_kivD | 13-16 |
| pSB1C3-BBa\_BjaIT-OmpTFlag | 17-20 |
| pSB1C3-BBa\_accd | 21-24 |

![](https://static.igem.org/mediawiki/2018/c/c5/T--Hamburg--180718_cPCR.JPG)

## 180720

## Miniprep and Cryostocks

Cryostocks were created by adding 200 µL 50% glycose to 600µL LB-Medium

|  |  |
| --- | --- |
| Part | Lot No. |
| pSB1C3-BBa\_R0010 | 180720BK01 |
| pSB1C3-BBa\_1577881 | 180720BK02 |

Minipreps were performed using the Thermo Fischer Kit

|  |  |  |
| --- | --- | --- |
| Part | Lot No. | Concentration [ng/µL] |
| pSB1C3-BBa\_R00010 | 180720BK03 | 80.454 |
| pSB1C3-BBa\_1572881 | 180720BK04 | 188.945 |

## PCR of synthetic Part

Approach: 20 μL H2O, 25 μL Q5-Mastermix, 2.5 μL Syn\_f-Primer, 2.5 μL Syn\_r-Primer,

DNA-Template from Table:

|  |  |  |
| --- | --- | --- |
| Lot-Nr. | concentration | Part |
| 180723BK01 | 171.027 ng/ μL | ADH2 |
| 180723BK02 | 117.758 ng/ μL | accC |
| 180723BK03 | 115.077 ng/ μL | kivD |
| 180723BK04 | 105.878 ng/ μL | OmpA-NnAOX1a |

Purifictation with ThermoFischer-Kit

PCR-Programm:

|  |  |
| --- | --- |
| Temperature | Time |
| 98 °C | 20 s |
| 98 °C | 10 s |
| 55 °C | 20 s (Go to Step 1, repeat 30 times) |
| 72 °C | 1:20 min |
| 72 °C | 2:00 min |

Restriction of pSB1C3 (linear, PCR-product) + 1 % Agarosegel

|  |  |
| --- | --- |
| 180628NB01 à pSB1C3 DNA | 15 μL |
| NEB 2.1 | 2 μL |
| EcoRI-HF | 0.5 μL |
| PstI | 0.5 μL |
| FastAP | 0.5 μL |
| H2O | 1.5 μL |

Incubate at 37 °C for 60 minutes, then at 80 °C for heat shock deactivation.

## Transformation of

Transformation was performed according to the protocol of Zhang (see 180418).

               Part                                      Lot-No.

               pSB1C3-ADH2                                   180724BK01
                pSB1C3-accC                                    180724BK02
                pSB1C3-kivD                                     180724BK03
                pSB1C3-OmpA-AOX1a                    180724BK04
                pSB1C3-BBA\_K410000-CmR         180724LP02

## cPCR of the Transformation  from 180724

Mastermix:

* 25 µL Taq 10x Buffer
* 5 µL DNTPs 10 mM
* 5 µL MgCl2 50 µM
* 5 µL Taq-Polymerase
* 5 µL Fw-Primer
* 5 µL Rv-Primer
* 196 µL H2Odest

PCR-Programm

|  |  |
| --- | --- |
| 98°C | 2 min |
| 98°C | 10 s |
| 65°C | 20 s |
| 72°C | 2 min |
| Go to step 2 30x | |
| 72°C | 5 min |

## Interlab- Protocol Colony farming units

We used I20270 2018 Plate 7 Well 4B, R0040 2018 Plate 7 Well 4D. First we resuspended the DNA and transformated each with 1 µL in DH5a.

## Repetition of the cPCR

Mastermix: see above

|  |  |  |
| --- | --- | --- |
| Tube | Part | Size [bp] |
| 1-5 | ADH2 | ~1000 |
| 6-10 | Accc | ~1000 |
| 11-14 | KivD | ~1600 |
| 15-19 | OmpA-Aox1a | ~1200 |

cPCR-program:

|  |  |
| --- | --- |
| 95°C | 3 min |
| 95°C | 30 s |
| 55°C | 30 s |
| 72°C | 2 min |
| Go to step 2 30x | |
| 72°C | 5 min |

## Interlab

Pecked each of 2 colonies and put in a 7 mL LB-cAMP and stored 1 h at 37°C and 200 rpm.

OD-Measure by 600 nm:

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | A1 | 1.1 | 1.2 | 1.3 | A2 | 2.1 | 2.2 | 2.3 |
| OD | 0.2892 | 0.152 | 0.158 | 0.156 | 0.2216 | 0.153 | 0.153 | 0.152 |

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | B1 | 1.1 | 1.2 | 1.3 | B2 | 2.1 | 2.2 | 2.3 |
| OD | 0.262 | 0.143 | 0.145 | 0.156 | 0.2936 | 0.174 | 0.183 | 0.173 |

|  |  |  |
| --- | --- | --- |
| Tube | C1 | C2 |
| OD | 0.048 | 0.048 |

## Minipreps for sequencing

|  |  |
| --- | --- |
| Lotnumber | Part |
| 180518BK02 | psB1C3-MIcRE-BBa-E0840 |
| 180531NF01 | psB1C3-BBa\_B0033-mraz |
| 180531NF02 | psB1C3-BBa\_B0033-cbtA |
| 180531NF03 | psB1C3-BBa\_B0032-cbtA |
| 180531NF04 | psB1C3-BBa\_B0033-cspD |
| 180531NF06 | psB1C3-BBa\_B0032-sulA |
| 180518BK05 | psB1C3-BBa\_B0031-sulA |
| 180518NK06 | psB1C3-oHybB |
| 180518BK09 | psB1C3-BBa\_K206000-BBa\_B0034-sulA |

Set the over-night-cultures of 180724LP02 (psB1C3-BBa\_K410000-CmR) for miniprep and cryostocks.

We did the over-night-cultures of the cPCR.

* 180726LP01 = kivD (Colony 12)
* 180726LP02 = OmpA-Aox (Colony 17)
* 180726LP03 = ADH2 (Colony 1)
* 180726LP04 = accC (Colony 10)

Stored at 37°C and 200 rpm in 7 mL LB+cAMP.

## Miniprep

We did the Miniprep with the GeneJet Kit. Then we measuered the DNA-Concentration with the Nanodrop.

|  |  |  |
| --- | --- | --- |
| Lotnumber | Part | DNA-concentration [ng/µL] |
| 180726LP01 | kivD (Colony 12) | 107.29 |
| 180726LP02 | OmpA (Colony 17) | 146.835 |
| 180726LP03 | ADH2 (Colony 1) | 114.015 |
| 180726LP04 | accC (Colony 10) | 143.41 |
| 180724LP02 | psB1C3-BBa\_K410000\_CmR | 147.586 |

We made cryostocks of all cultures. Then mixed 650 µL of the cell culture with 216.666 µL Glycerol and stored that at -80°C.

## PCR for GGA and Over-night-cultures (OVC) for sequencing

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Part | Fw-Primer | Rv-Primer | Template | Lotnumber | DNA-concentration [ng/µL] | Tube |
| accA | Bsa1\_accA\_f | Bsa1\_accA\_r | 180622NB05 | 180730BK01 | 174.035 | 1 |
| accB | Bsa1\_accB\_f | Bsa1\_accB\_r | 180703BK05 | 180730BK02 | 92.21 | 2 |
| accC | Bsa1\_accC\_f | Bsa1\_accC\_r | 180726LP04 | 180730BK03 | 17.536 | 3 |
| accD | Bsa1\_accD\_f | Bsa1\_accD\_r | IDT | 180730BK04 | 189.443 | 4 |
| tesA | Bsa1\_tesA\_f | Bsa1\_tesA\_r | e.coli | 180730BK05 | 111.71 | 5 |
| ADH2 | Bsa1\_ADH2\_f | Bsa1\_ADH2\_r | 180726LP02 | 180730BK06 | 62.293 | 6 |
| hivD | Bsa1\_hivD\_f | Bsa1\_hivD\_r | 180726LP01 | 180730BK07 | 91.148 | 7 |
| LeuA | Bsa1\_LeuA\_f | Bsa1\_LeuA\_r | e.coli | 180730BK08 | 88.001 | 8 |
| LeuB | Bsa1\_LeuB\_f | Bsa1\_LeuB\_r | e.coli | 180730BK09 | 61.115 | 9 |
| LeuC | Bsa1\_LeuC\_f | Bsa1\_LeuC\_r | e.coli | 180730BK10 | 93.537 | 10 |
| LeuD | Bsa1\_LeuD\_f | Bsa1\_LeuD\_r | e.coli | 180730BK11 | 109.97 | 11 |
| BjaIT | Bsa1\_BjaIT\_f | Bsa1\_BjaIT\_r | IDT | 180730BK12 | 127.138 | 12 |
| HlyA | Bsa1\_HlyA\_f | Bsa1\_HlyA\_r | pSB1C3-K554002 | 180730BK13 | 39.76 | 13 |
| pBAD | Bsa1\_pBAD\_f | Bsa1\_pBAD\_r | pSB1C3-K206000 | 180730BK14 | 99.724 | 14 |
| HlyB | Bsa1\_HlyB\_f | Bsa1\_HlyB\_r | pSB1C3-K554013 | 180730BK15 | 205.409 | 15 |
| OmpT | Bsa1\_OmpT\_f | Bsa1\_OmpT\_r | e.coli | 180730BK16 | 48.8 | 16 |
| TolC | Bsa1\_TolC\_f | Bsa1\_TolC\_r | pSB1C3-K554013 | 180730BK17 | 207.256 | 17 |
| Exer.s | Bsa1\_HlyB\_f | Bsa1\_TolC\_r | pSB1C3-K554013 | 180730BK18 | 207.123 | 18 |

Mastermix:

* 20 µL H2Odest
* 5 µL each Primer (fw and rv)
* 3 µL template
* 25 µL Q5 Mastermix

PCR-Programm:

|  |  |
| --- | --- |
| 98°C | 20 s |
| 98°C | 10 s |
| 55°C | 20 s |
| 72°C | 2 min |
| Go to step 2 30x | |
| 72°C | 2 min |

## Gelelectrophoresis:

We made a 1% agarose-gel. It ran over 45 min by 100 V.

## <https://static.igem.org/mediawiki/2018/7/73/T--Hamburg--180730_PCR_GGA.JPG>

## Over-night-cultures (OVC) for sequencing:

The Cryostocks were of the OVC from 180726. We made a new LB-Medium with cAMP and water down to 1:1000. 50 ml LB-Medium and 50 µL cAMP were used. We put 5 mL of the medium into culture tubes and added 50 µL of the cryostocks.

## Mini-prep of the over-night-cultures (OVC) for the sequencing:

The mini-prep was implemented with GeneJet Plasmid Mini-prep kit. The DNA we eluted with H2Odest.

|  |  |  |
| --- | --- | --- |
| Lot-number | Part | DNA-concentration [ng/µL] |
| 180731NF01 | pSB1C3\_MlcRE\_BBa\_E0840 | 195.617 |
| 180731NF02 | pSB1C3\_BBa\_B0033\_mraz | 143.368 |
| 180731NF03 | pSB1C3\_BBa\_B0033\_cbtA | 174.778 |
| 180731NF04 | pSB1C3\_BBa\_B0032\_cbtA | 173.534 |
| 180731NF05 | pSB1C3\_BBa\_B0033\_cspD | 110.056 |
| 180731NF06 | pSB1C3\_BBa\_B0032\_sulA | 152.544 |
| 180731NF07 | pSB1C3\_BBa\_B0031\_sulA | 167.888 |
| 180731NF08 | pSB1C3\_oHybB | 134.928 |
| 180731NF09 | pSB1C3\_BBa\_K206000\_BBa\_B0034\_sulA | 139.008 |

## Golden Gate Assembly

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| A | Calculation | Length [bp] | Density [ng/µL] | Volume [µL] | 3MB |
|  | Promotor | 161 | 99.724 | 0.1113930013 | pBAD |
| Part 1 | 1050 | 62.293 | 1.163005508 | ADH2 |
| Part 2 | 1650 | 91.148 | 1.24901749 | kivD |
| Part 3 | 1590 | 88.001 | 1.246640513 | LeuA |
| Part 4 | 1100 | 61.115 | 1.241871294 | LeuB |
| Part 5 | 1410 | 93.537 | 1.040081557 | LeuC |
| Part 6 | 630 | 109.97 | 0.3952738135 | LeuD |
| Buffer |  |  | 2 |  |
| Mix |  |  | 1 |  |
| pGGA |  |  | 1 |  |
| H2Odest |  |  | 9.552716824 |  |

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| B | Calculation | Length [bp] | Density [ng/µL] | Volume [µL] | Toxin |
|  | Promotor | 161 | 99.724 | 0.1113930013 | pBAD |
| Part 1 | 290 | 127.138 | 0.1573817398 | BjaIT |
| Part 2 | 210 | 39.76 | 0.3644220428 | HlyA |
| Buffer |  |  | 2 |  |
| Mix |  |  | 1 |  |
| pGGA |  |  | 1 |  |
| H2Odest |  |  | 15.36680322 |  |

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| C | Calculation | | Length [bp] | | Density [ng/µL] | | Volume [µL] | | Excretion 1 | |
|  | Promotor | | 161 | | 99.724 | | 0.1113930013 | | pBAD | |
| Part 1 | | 5164 | | 207.123 | | 1.72024231 | | HlyB+HlyD+TolC | |
| Part 2 | | 975 | | 48.8 | | 1.378530924 | | ompT | |
| Buffer | |  | |  | | 2 | |  | |
| Mix | |  | |  | | 1 | |  | |
| pGGA | |  | |  | | 1 | |  | |
| H2Odest | |  | |  | | 12.78983376 | |  | |
| D | | Calculation | | Length [bp] | | Density [ng/µL] | | Volume [µL] | | Excretion 2 |
|  | | Promotor | | 161 | | 99.724 | | 0.1113930013 | | pBAD |
| Part 1 | | 3872 | | 205.409 | | 1.30061153 | | HlyB+HlyD |
| Part 2 | | 1296 | | 207.256 | | 0.4314491411 | | TolC |
| Part 3 | | 975 | | 48.8 | | 1.378530924 | | ompT |
| Buffer | |  | |  | | 2 | |  |
| Mix | |  | |  | | 1 | |  |
| pGGA | |  | |  | | 1 | |  |
| H2Odest | |  | |  | | 12.7780154 | |  |
|  |  |  |  |  |  |  |  |  |  |  |

|  |  |
| --- | --- |
| 3MB, Toxin, Excretion | |
| 37°C | 1 min |
| 16°C | 1 min |
| Go to step 1 30x | |
| 55°C | 5 min |

|  |  |
| --- | --- |
| Excretion 1 | |
| 37°C | 1 min |
| Go to step 1 30x | |
| 16°C | 1 min |

## Sequencing

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Seq.ID | Lotnumber | Part | Conc. [ng/µL] | Length total [bp] | Part [bp] | Primer | V Part [µL] | V Wasser [µL] |
| 98EC87 | 180329DW03 | pSB1C3-MlcRE | 102 | 341 | 201 | vf2 | 0.98 | 4.02 |
| 98EC86 | 180731NF01 | pSB1C3-MlcRE-BBa\_E0840 | 195.6 | 1.225 | 1.085 | vf2 | 0.51 | 4.49 |
| 98EC90 | 180731NF01 | pSB1C3-MlcRE-BBa\_E0840 | 195.6 | 1.259 | 1.085 | vr | 0.51 | 4.49 |
| 98EC89 | 180329DW09 | pSB1C3-sulA | 131.5 | 650 | 510 | vf2 | 0.76 | 4.24 |
| 98EC88 | 180731NF07 | pSB1C3-BBa\_B0031-sulA | 167.9 | 670 | 530 | vf2 | 0.6 | 4.4 |
| 98EC91 | 180329DW21 | pSB1C3-BBa\_B0034-sulA | 75.2 | 668 | 528 | vf2 | 1.33 | 3.67 |
| 98EC92 | 180731NF008 | pSB1C3-oHybB | 135 | 313 | 173 | vf2 | 0.74 | 4.26 |
| 98EC93 | 180329DW28 | pSB1C3-BBa\_E0840-oHybB | 139.3 | 1.197 | 1.057 | vf2 | 0.72 | 4.28 |
| 98EC94 | 180329DW28 | pSB1C3-BBa\_E0840-oHybB | 139.3 | 1.231 | 1.057 | vr | 0.72 | 4.28 |
| 98EC95 | 180329DW41 | pSB1C3-BBa\_K206000-BBa\_B0032-sulA | 101.3 | 805 | 665 | vf2 | 0.99 | 4.01 |
| 98EC96 | 180731NF09 | pSB1C3-BBa\_K206000-BBa\_B0034-sulA | 139 | 804 | 664 | vf2 | 0.72 | 4.28 |
| 98EC97 | 180426NK28 | pSB1C3-BBa\_B0034-ldhA | 85.5 | 1.194 | 1054 | vf2 | 1.17 | 3.83 |
| 98EC98 | 180426NK28 | pSB1C3-BBa\_B0034-ldhA | 85.5 | 1.228 | 1054 | vr | 1.17 | 3.83 |
| 98EC99 | 180426NK30 | pSB1C3-BBa\_K206000-BBa\_B0031-sulA | 80.5 | 806 | 666 | vf2 | 1.24 | 3.76 |
| 98ED00 | 180731NF02 | pSB1C3-BBa\_B0033-mraz | 143.4 | 615 | 475 | vf2 | 0.7 | 4.3 |
| 98ED01 | 180731NF03 | pSB1C3-BBa\_B0033-cbtA | 174.8 | 532 | 392 | vf2 | 0.57 | 4.43 |
| 98ED02 | 180731NF04 | pSB1C3-BBa\_B0032-cbtA | 173.5 | 534 | 394 | vf2 | 0.58 | 4.42 |
| 98ED03 | 180731NF05 | pSB1C3-BBa\_B0033-cspD | 110.1 | 379 | 239 | vf2 | 0.91 | 4.09 |
| 98ED04 | 180531LP05 | pSB1C3-BBa\_B0032-cspD | 53.5 | 381 | 241 | vf2 | 1.87 | 3.13 |
| 98ED05 | 180731NF06 | pSB1C3-BBa\_B0032-sulA | 152.5 | 669 | 529 | vf2 | 0.66 | 4.34 |
| 98ED06 | 180627LP01 | pSB1C3-BBa\_B0032-ldhA | 76 | 1.195 | 1055 | vf2 | 1.32 | 3.68 |
| 98ED07 | 180627LP01 | pSB1C3-BBa\_B0032-ldhA | 76 | 1.229 | 1055 | vr | 1.32 | 3.68 |
| 98ED08 | 180627LP03 | pSB1C3-cbtA | 65.5 | 515 | 375 | vf2 | 1.53 | 3.47 |
| 98ED09 | 180703BK01 | pSB1C3-BBa\_K206000-BBa\_B0032-ldhA | 153 | 1.331 | 1191 | vf2 | 0.65 | 4.35 |
| 98ED10 | 180703BK01 | pSB1C3-BBa\_K206000-BBa\_B0032-ldhA | 153 | 1.365 | 1191 | vr | 0.65 | 4.35 |
| 98ED11 | 180703BK02 | pSB1C3-BBa\_K206000-BBa\_B0034-ldhA | 167 | 1.330 | 1190 | vf2 | 0.6 | 4.4 |
| 98ED12 | 180703BK02 | pSB1C3-BBa\_K206000-BBa\_B0034-ldhA | 167 | 1.364 | 1190 | vr | 0.6 | 4.4 |
| 98ED13 | 180703BK04 | pSB1C3-cspD | 129 | 362 | 222 | vf2 | 0.78 | 4.22 |
| 98ED14 | 180703BK05 | pSB1C3-BBa\_B0032-accB | 212 | 630 | 490 | vf2 | 0.47 | 4.53 |
| 98ED15 | 180703BK06 | pSB1C3-BBa\_B0032-mraz | 250 | 617 | 477 | vf2 | 0.4 | 4.6 |
| 98ED16 | 180712BK01 | pGGA-dapAP-30cbtA-30cspD-30mraz-34sulA | 169 | 1.940 | 1800 | Goldengate\_cPCR\_r | 0.59 | 4.41 |
| 98ED17 | 180712BK01 | pGGA-dapAP-30cbtA-30cspD-30mraz-34sulA | 169 | - | 1800 | Goldengate\_cPCR\_r | 0.59 | 4.41 |
| 98ED18 | 180712BK03 | pSB1C3-RnaG120-Operator + MlcRE | 93 | 473 | 333 | vf2 | 1.08 | 3.92 |
| 98ED19 | 180724LP01 | pSB1C3-BBa\_K410000 | 100 | 1.657 | 1517 | vf2 | 1 | 4 |
| 98ED20 | 180724LP01 | pSB1C3-BBa\_K410000 | 100 | 1.691 | 1517 | vr | 1 | 4 |

As seen on the table we diluted the template DNA concentration. We added 5 µL Primer (3µM). The total volume were 10 µL.

## Transformation GGA and BBa\_I13453

The DNA was resuspended with the Distribution Kit. Of the NEB 10-beta cells we made 50 µL aliquots. There was 2 µL NE assembly reaction added and 30 min on ice incubated. After that we made a heatshock for 30 s by 42°C. Then we put the samples 5 min on ice. We added 950 µL LB-medium and incubate for 1 h by 37°C and 500 rpm. Later we centrifugated for 2 min by 10000 rpm. We discarded 850 µL of the supernatant, resuspended and plated the cells.

For BBa\_I13453 we did the same steps except we used 75 µL DH5a cells, 1 µL assembly reaction and the heatshock was for 45 s.

## cPCR of the transformation from 180731

Mastermix for 20 samples:

* 40 µL 10x Taq buffer
* 8 µL dNTPs (10 mM)
* 12 µL MgCl2
* 1 µL Taq polymerase
* 5 µL H2Odest
* 12 µL GGA\_cPCR\_f
* 12 µL GGA-cPCR\_r

|  |  |
| --- | --- |
| Tube | Part |
| 1-5 | 3MB |
| 6-10 | Toxin |
| 11-15 | Excretion 1 |
| 16-19 | Excretion 2 |

PCR program:

|  |  |
| --- | --- |
| 95°C | 3 min |
| 95°C | 30 s |
| 55°C | 30 s |
| 72°C | 8 min |
| Go to step 2 30x | |
| 72°C | 5 min |

## Setting up overnight cultures

Setting up overnight cultures of tube 6-8 (Toxin).

|  |  |  |
| --- | --- | --- |
| Tube | Lot number | Part |
| 6 | 180801LP01 | pSB1C3-pBAD-BjaIT-HlyA |
| 7 | 180801LP02 | pSB1C3-pBAD-BjaIT-HlyA |
| 8 | 180801LP03 | pSB1C3-pBAD-BjaIT-HlyA |
| - | 180801LP04 | pSB1C3-BBa\_I13454 |

## Gelelectrophoresis

We made a 1% agarose gel.

![](https://static.igem.org/mediawiki/2018/c/c9/T--Hamburg--180801_cPCR_GGA.JPG)

## cPCR of the transformation from 180731

Mastermix for 20 samples:

* 40 µL 10x Taq buffer
* 8 µL dNTPs (10 mM)
* 12 µL MgCl2
* 1 µL Taq polymerase
* 5 µL H2Odest
* 12 µL GGA\_cPCR\_f
* 12 µL GGA-cPCR\_r

|  |  |  |
| --- | --- | --- |
| Part | Part | PCR tube |
| 3MB | pBAD |  |
| 3MB | ADH2 | 1-4 |
| 3MB | kivD | 5-8 |
| 3MB | leuA | 9-12 |
| 3MB | leuB | 13-16 |
| 3MB | leuC | 17-20 |
| 3MB | leuD | 21-24 |
| Toxin | pBAD |  |
| Toxin | BjaIT | 25-28 |
| Toxin | HlyA |  |
| Ex 1 | pBAD |  |
| Ex 1 | TolC | 29-32 |
| Ex 1 | ompT | 33-36 |
| Ex 2 | pBAD |  |
| Ex 2 | HlyB | 37-40 |
| Ex 2 | TolC | 41-44 |
| Ex 2 | ompT | 44-48 |

PCR program:

|  |  |
| --- | --- |
| 95°C | 2 min |
| 95°C | 30 s |
| 55°C | 30 s |
| 72°C | 3 min |
| Go to step 2 30x | |
| 72°C | 5 min |

## Gelelectrophoresis

We made a 1% agarose gel.

![](https://static.igem.org/mediawiki/2018/5/5b/T--Hamburg--180802_cPCR_GGA.JPG)
![](https://static.igem.org/mediawiki/2018/9/95/T--Hamburg--180802_cPCR_GGA2.JPG)

## Purification of I13457

Mini prep was made with GeneJET Miniprep Kit by Thermo Fischer.

## Setting up cryostocks

We mixed 600 µL of I13457 with 200 µL 50% glycerol and stored at -80°C.

## Inoculate overnight cultures

Inoculate tube 25 and 28 and stored overnight at 37°C.

## Miniprep BjalT and HylA

The miniprep was made with GeneJET Miniprep Purification Kit by Thermo Fischer.

180803NB01 = Cryostock

180803NB02 = Plasmid = pSB1C3-pBAD-BjalT-HylA; c = 257.7 ng/µL

## cPCR of the transformation from 180731

Mastermix:

* 80 µL Taq buffer
* 16 µL dNTPs
* 24 µL MgCl2
* 2 µL Taq Polymerase
* 630 µL H2Odest
* 6 µL of each primer

|  |  |  |  |
| --- | --- | --- | --- |
| Tube | Part | Fw primer | Rv primer |
| 01-20 | 3MB | LeuD\_f | cPCR\_GGA\_r |
| 21-30 | Excretion 2 | ompT\_f | cPCR\_GGA\_r |
| 31-38 | Excretion 1 | ompT\_f | cPCR\_GGA\_r |

 PCR program was deleted.

## Gelelectrophoresis

We made a 1% agarose gel.

## <https://static.igem.org/mediawiki/2018/4/4d/T--Hamburg--180803_cPCR_GGA.JPG>

## PCR for GGAs and Basic Parts

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Part | tube | fw\_Primer | rv\_Primer | template | pred. Length | concentration [ng/µL] |
| Bsa\_accD | 1 | Bsa\_accD\_f | Bsa\_accD\_rv | 180730BK04 | 950 | 11.558 |
| Bsa\_kivD | 2 | Bsa\_kivD\_f | Bsa\_kivD\_r | 180730BK07 | 1700 | 11.619 |
| Bsa\_pBAD | 3 | Bsa\_dapAP\_f | Bsa\_pBAD\_r | 180730BK14 | 200 | 16.782 |
| Bsa\_HlyB | 4 | Bsa\_HlyB\_f | Bsa\_HlyB\_r | 180730BK15 | 3850 | 7.68 |
| Bsa\_OmpT | 5 | Bsa\_OmpT\_f | Bsa\_OmpT\_r | 180730BK16 | 1000 | 9.895 |
| Bsa\_34-cbtA | 6 | Bsa\_34-cbtA\_f | cbtA\_Bsa\_r | 180622NB07 | 400 | 58.154 |
| Bsa\_34-cspD | 7 | Bsa\_34-cspD\_f | cspD\_Bsa\_r | 180622NB04 | 250 | 65.224 |
| Bsa\_34-mraz | 8 | Bsa\_34-mraz\_f | mraz\_Bsa\_-r | 180622NB08 | 500 | 66.294 |
| Bsa\_34-sulA | 9 | Bsa\_34-sulA\_f | sulA\_Bsa\_r | 180329DW09 | 580 | 87.334 |
| OmpT | 10 | OmpT\_fz | OmpT\_r2 | e.coli | 1000 | 60.291 |
| 33\_killR | 11 | 33\_killR\_f | killR\_r | e.coli | 280 | 58.983 |

Mastermix:

* 20 µL H2Odest
* 25 µL Q5 Master Mix
* 5 µL of each Primer

PCR Program:

|  |  |  |
| --- | --- | --- |
|  | | Tube 4 |
| 98°C | 2 min | 20 s |
| 98°C | 10 s |  |
| 55°C | 20s |  |
| 72°C | 1:30 min | 2:30 min |
| Go to step 2 30x | |  |
| 72°C | 2 min |  |

## Gelelectrophoresis

We made a 1% Agarose-gel. It ran for 45 min by 100 V.

![](https://static.igem.org/mediawiki/2018/b/be/T--Hamburg--180806_cPCR_GGA%2BOmpT%2CkilR.JPG)

Tube 1-5 were extracted by GeneJet Gelextraction Kit. Tube 6-11 were purificated with GeneJet PCR Purification Kit.

## Goldengate Assembly

PCR-Tube-marking:

* 3MB = 1
* Excretion = 2
* Miristic Acid = 3
* Groth inhibition = 4

GGA-Program:

|  |  |
| --- | --- |
| 37°C | 5 min |
| 16°C | 10 min |
| Go to step 1 30x | |
| 55°C | 5 min |

## 180807

## Transformation of GGAs from 180806

The transformation was carried out by NEB-GGA-Protocol with NEB 10-Beta-cells. We put each 80 µL per plate.

|  |  |
| --- | --- |
| Lotnumber | Part |
| 180807BK01 | 3MB |
| 180807BK02 | Excretion |
| 180807BK03 | Myristic Acid |
| 180807BK04 | Growth Inhibition |

## cPCR of trafo on 180807

We made two Master mixes.

Master Mix 1 for 25 samples:

* 50 µL Dream Taq Buffer
* 10 µL dNTPs
* 15 µL MgCl2
* 25 µL Dream Tag Polymerase
* 15 µL GGA\_cPCR\_fw
* 15 µL GGA\_cPCR\_rv
* 75 µL H2Odest

The plates was sealed with Parafilm and stored in the fridge.

Master Mix 2:

* 40 µL Dream Taq Buffer
* 8 µL dNTPs
* 12 µL MgCl2
* 1 µL Dream Taq Polymerase
* 12 µL GGA\_cPCR\_fw
* 12 µL psal\_pBAD\_rv
* 315 µL H2Odest

PCR-Program:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| PCR 1 | |  | PCR 2 | |
| 95°C | 3 min |  | 95°C | 3 min |
| 95°C | 30 s |  | 95°C | 30 s |
| 55°C | 30 s |  | 55°C | 30 s |
| 72°C | 8 min |  | 72°C | 1 min |
| Go to step 2 30x | |  | Go to step 2 30x | |
| 72°C | 10 min |  | 72°C | 2 min |

New Labels

PCR 1:

* BK01 = 3MB 1-5
* BK02 = EX 1-5
* BK03 = MA 1-5
* BK04 = GI 1-5 à No PCR 2-sample, because the plasmid is too small.

PCR 2:

* BK01 = 3MB 1-5
* BK02 = EX 1-5
* BK03 = MA 1-5

![](https://static.igem.org/mediawiki/2018/7/75/T--Hamburg--180808_cPCR_GGA.JPG)

## cPCR of 3MB#1, Myristic Acid#1 (MA#1), and Myristic Acid#4 (MA#4)

Master Mix for 20 samples:

* 40 µL Dream Taq Buffer
* 8 µL dNTPs
* 12 µL MgCl2
* 1 µL Dream Taq
* 2 µL of each primer
* 315 µL H2Odest

|  |  |  |  |
| --- | --- | --- | --- |
| Tube | Part | Part-components | Primer |
| 1 | 3MB | ADH2 | fw-GGa-cPCR |
| 2 | 3MB | kivD, AD2 | fw-GGa-cPCR |
| 3 | 3MB | LeuA, kivD, ADH2 | fw-GGa-cPCR |
| 4 | 3MB | LeuB, LeuC, LeuD | GGA-cPCR-rv |
| 5 | 3MB | LeuC, LeuD | GGA-cPCR-rv |
| 6 | 3MB | LeuD | GGA-cPCR-rv |
| 7 | MA#1 | accA | GGA-cPCR-fw |
| 8 | MA#1 | accB, accA | GGA-cPCR-fw |
| 9 | MA#1 | accC, accD, tesA | GGA-cPCR-rv |
| 10 | MA#1 | accD, tesA | GGA-cPCR-rv |
| 11 | MA#1 | tesA | GGA-cPCR-rv |
| 12 | MA#4 | accA | GGA-cPCR-fw |
| 13 | MA#4 | accB, accA | GGA-cPCR-fw |
| 14 | MA#4 | accC, accD, tesA | GGA-cPCR-rv |
| 15 | MA#4 | accD, tesA | GGA-cPCR-rv |
| 16 | MA#4 | tesA | GGA-cPCR-rv |

cPCR Program:

|  |  |
| --- | --- |
| 95°C | 3 min |
| 95°C | 30 s |
| 55°C | 30 s |
| 72°C | 5 min |
| Go to step 2 30x | |
| 72°C | 10 min |

![](https://static.igem.org/mediawiki/2018/0/01/T--Hamburg--180809_cPCR_GGA.JPG)

## PCR for 3A-Assembly + GGA

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Part | Tube | fw-Primer | rv-Primer | Template | Length [bp] |
| pSB1C3 | 1 | SB-prep-2Ea | SB-prep-3P1 | pSB1C3-I20270 | 2000 |
| accD | 2 | Pre\_Syn\_f | Syn\_Suf\_r | IDT | 950 |
| tesA | 3 | Pre\_tesA\_f | tesA\_Suf\_r | e.coli | 650 |
| Bsa\_accD | 4 | - | - | 180806DK01 | 950 |
| Bsa\_kivD | 5 | - | - | 180806DK02 | 1650 |
| Bsa\_HlyB | 6 | - | - | 180806DK04 | 3800 |
| Bsa\_OmpT | 7 | - | - | 180806DK05 | 1000 |

Mastermix:

* 25 µL Q5 Mastermix
* 20 µL H2Odest
* 5 µL of each Primer
* 25 µL template

Note: We made a new Mastermix, because it contained white flake. Mastermix was used by sample 4-7.

PCR-Program:

|  |  |  |  |
| --- | --- | --- | --- |
| 98°C | 2 min |  | 20 s (HyB + tesA) |
| 98°C | 10 s |  |  |
| 55°C | 20 s |  |  |
| 72°C | 1:20 min |  | 2 min (Hyb + tesA) |
| Go to step 2 30x | | | |
| 72°C | 2 min |  |  |

## Gelelectrophoresis

We made a 1%-Agarose gel. It ran for 40 min by 80 V. We put 10 µL of the PCR-product with 2 µL loading dye into the gel.

## Restriction of remaining 3A-Assembly + GGA

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Calculation** | Länge (bp) | Dichte (ng/µL) | Volumen (µL) | 3MB |
| Promotor | 161 | 99 | 0.5 | pBAD |
| Part 1 | 1050 | 62.293 | 2.90751377 | ADH2 |
| Part 2 | 1650 | 91 | 3.127622148 | kivD |
| Part 3 | 1590 | 88.001 | 3.116601282 | LeuA |
| Part 4 | 1100 | 61.115 | 3.104678235 | LeuB |
| Part 5 | 1410 | 93.537 | 2.600203891 | LeuC |
| Part 6 | 630 | 109.97 | 0.9881845337 | LeuD |
| Buffer |  |  | 2 |  |
| Mix |  |  | 1 |  |
| pGGA |  |  | 1 |  |
| H2O |  |  | 0 |  |
|  |  |  |  |  |
|  |  |  |  |  |
|  |  |  |  |  |
| **Calculation** | Länge (bp) | Dichte (ng/µL) | Volumen (µL) | Excretion |
| Promotor | 161 | 99 | 0.5610381645 | pBAD |
| Part 1 | 3872 | 205 | 6.516032042 | HlyB+HlyD |
| Part 2 | 1296 | 207.256 | 2.157245705 | TolC |
| Part 3 | 975 | 49 | 6.864521338 | ompT |
| Buffer |  |  | 2 |  |
| Mix |  |  | 1 |  |
| pGGA |  |  | 1 |  |
| H2O |  |  | 0 |  |
|  |  |  |  |  |
| **Calculation** | Länge (bp) | Dichte (ng/µL) | Volumen (µL) | Myristic Acid |
| Promotor | 161 | 99.724 | 0.569650063 | pBAD |
| Part 1 | 993 | 174.035 | 1.968404615 | accA |
| Part 2 | 489 | 92.21 | 1.829500619 | accB |
| Part 3 | 1358 | 108.9 | 4.302031775 | accC |
| Part 4 | 925 | 189 | 1.688424527 | accD |
| Part 5 | 679 | 111.71 | 2.096908336 | tesA |
| Buffer |  |  | 2 |  |
| Mix |  |  | 1 |  |
| pGGA |  |  | 1 |  |
| H2O |  |  | 3.557765122 |  |
|  |  |  |  |  |
| **Calculation** | Länge (bp) | Dichte (ng/µL) | Volumen (µL) | Growth Inhibition |
| Promotor | 161 | 99 | 0.5610381645 | pBAD |
| Part 1 | 410 | 58.154 | 2.432237546 | 34cbtA |
| Part 2 | 260 | 65.224 | 1.375205632 | 34cspD |
| Part 3 | 500 | 66.294 | 2.601941356 | 34mraZ |
| Part 4 | 565 | 87.334 | 2.231859337 | 34sulA |
| Buffer |  |  | 2 |  |
| Mix |  |  | 1 |  |
| pGGA |  |  | 1 |  |
| H2O |  |  | 6.797717965 |  |

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **3** | **5** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** |  | Tube | **6** | **8** | **10** |
|  | Part | pSB1C3-BBa\_K206000 | pSB1C3-I13453 | pSB1C3-NOTMlcRE | pSB1C3-E0840 | pSB1C3-E0840 | pSB1C3-BBa\_B0032-cbtA | pSB1C3-BBa\_B0033-cbtA | pSB1C3-BBa\_B0032-cspD | pSB1C3-BBa\_B0033-cspD | pSB1C3-BBa\_B0032-mraZ | pSB1C3-BBa\_B0033-mraZ |  | Part | pSB1C3-BBa\_K206001 | pSB1C3-I13454 | pSB1C3-NOTMlcRE |
|  | Lot# | 180307NK01 | 180802BK02 | 180712BK03 | 180627LP02 | 180627LP02 | 180731NF04 | 180731NF03 | 180531LP05 | 180731NF05 | 180703BK06 | 180731NF02 |  | Lot# | 180307NK02 | 180802BK03 | 180712BK04 |
|  | **ρ** | 47 ng/µL | 90 ng/µL | 93 ng/µL | 92 ng/µL | 92 ng/µL | 174 ng/µL | 175 ng/µL | 54 ng/µL | 110 ng/µL | 250 ng/µL | 143 ng/µL |  | **ρ** | 48 ng/µL | 91 ng/µL | 94 ng/µL |
|  | Use | vector | vector | vector | vector |  |  |  |  |  |  |  |  | Use | vector | vector | vector |
|  | target m | 2000 ng | 2000 ng |  | 2000 ng |  |  |  |  |  |  |  |  | target m |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 25.00 µL | 8.00 µL | 12.50 µL | 12.81 µL | 11.00 µL | 10.00 µL | 10.00 µL | 11.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | Volumes | DNA | 26.00 µL | 9.00 µL | 13.50 µL |
| NEBuffer 2.1 | 3.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | NEBuffer 2.2 |  |  |  |
| EcoRI-HF |  |  |  | 0.50 µL |  |  |  |  |  |  |  | EcoRI-HF |  |  |  |
| XbaI |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | XbaI |  |  |  |
| SpeI | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  | SpeI | 0.50 µL | 0.50 µL | 0.50 µL |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | PstI | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  | FastAP |  |  |  |
| H2O | 0.50 µL | 8.50 µL | 4.00 µL | 3.69 µL | 6.00 µL | 7.00 µL | 7.00 µL | 6.00 µL | 7.00 µL | 7.00 µL | 7.00 µL | H2O |  |  |  |
|  | ad | 30.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL |  | ad |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt |  |  |  |  |  |  |  |  |  |  |  |  | Länge Produkt |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 39 ng/µL | 36 ng/µL | 58 ng/µL | 59 ng/µL | 51 ng/µL | 87 ng/µL | 87 ng/µL | 29 ng/µL | 55 ng/µL | 125 ng/µL | 72 ng/µL |  | ρ final |  |  |  |

## Gelelectrophoresis

We made a 1% Agarose-Gel. It ran for 1 h by 100 V.

![](https://static.igem.org/mediawiki/2018/4/47/T--Hamburg--180814_Restriktion_PCR.JPG)

## Gelextraction of the restrictions and PCR-Product accD

The Gelextraction was made by GeneJET Gelextraction Kit. After that we measuered the DNA-concentration via Nano Drop.

|  |  |  |
| --- | --- | --- |
| Tube | Part | DNA-concentration [ng/µL] |
| 1 | pSB1C3-K206000 | 5.3 |
| 3 | pSB1C3-I13453 | 10.6 |
| 5 | pSB1C3-NOTMlcRE | 8.3 |
| 7 | pSB1C3-E0840 | 11.3 |
| 8 | E0840 | 2 |
| 9 | 32-cbtA | 1.8 |
| 10 | 33-cbtA | 4.1 |
| 11 | 32-cspD | 10 |
| 12 | 33-cspD | 3.3 |
| 13 | 32-mraZ | 9.6 |
| 14 | 33-mraZ | 32.5 |
| 23 | AOX1 | 20.7 |
|  | accD (180814OM01) | 7.6 |

## GGA Assembly + Ligation

GGA Assembly program:

|  |  |
| --- | --- |
| 37°C | 5 min |
| 16°C | 10 min |
| Go to step 1 40x | |
| 55°C | 5 min |

|  |  |
| --- | --- |
| Tube | Part |
| 1 | 3MB |
| 2 | Excretion |
| 3 | Myristic Acid |
| 4 | Growth Inhibition |

The ligation ran for 16 h by 16°C.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | **1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** | **L12** | **L13** | **L14** | **L15** | **L16** | **L17** | **L18** | **L19** | **L20** | **L21** |
| Tube | pSB1C3-BBa\_K206000-pSB1C3-BBa\_B0032-cbtA | pSB1C3-BBa\_K206000-pSB1C3-BBa\_B0033-cbtA | pSB1C3-BBa\_K206000-pSB1C3-BBa\_B0032-cspD | pSB1C3-BBa\_K206000-pSB1C3-BBa\_B0033-cspD | pSB1C3-BBa\_K206000-pSB1C3-BBa\_B0032-mraZ | pSB1C3-BBa\_K206000-pSB1C3-BBa\_B0033-mraZ | pSB1C3-I13453-pSB1C3-BBa\_B0032-cbtA | pSB1C3-I13453-pSB1C3-BBa\_B0033-cbtA | pSB1C3-I13453-pSB1C3-BBa\_B0032-cspD | pSB1C3-I13453-pSB1C3-BBa\_B0033-cspD | pSB1C3-I13453-pSB1C3-BBa\_B0032-mraZ | pSB1C3-I13453-pSB1C3-BBa\_B0033-mraZ | pSB1C3-NOTMlcRE-pSB1C3-E0840 | pSB1C3-E0840- | pSB1C3- | pSB1C3- | pSB1C3- | pSB1C3-BBa\_K206000- | pSB1C3-I13453- | pSB1C3-E0840- | pSB1C3- |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-NOTMlcRE | pSB1C3-E0840 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3-I13453 | pSB1C3-E0840 | pSB1C3 |
| Tube | **1** | **1** | **1** | **1** | **1** | **1** | **3** | **3** | **3** | **3** | **3** | **3** | **5** | **7** | **17** | **17** | **17** | **1** | **3** | **7** | **17** |
| Tube | 5 ng/µL | 5 ng/µL | 5 ng/µL | 5 ng/µL | 5 ng/µL | 5 ng/µL | 11 ng/µL | 11 ng/µL | 11 ng/µL | 11 ng/µL | 11 ng/µL | 11 ng/µL | 8 ng/µL | 59 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 5 ng/µL | 11 ng/µL | 11 ng/µL | 10 ng/µL |
| Tube | 2200 bp | 2200 bp | 2200 bp | 2200 bp | 2200 bp | 2200 bp | 2200 bp | 2200 bp | 2200 bp | 2200 bp | 2200 bp | 2200 bp | 2500 bp |  |  |  | 2000 bp | 2200 bp | 2200 bp | 2800 bp | 2000 bp |
| Tube | 2.50 µL | 2.50 µL | 3.00 µL | 3.00 µL | 2.00 µL | 2.00 µL | 2.50 µL | 2.50 µL | 3.00 µL | 3.00 µL | 2.00 µL | 2.00 µL | 2.87 µL |  |  |  | 1.67 µL | 1.50 µL | 1.57 µL | 3.64 µL | 4.44 µL |
| Tube | pSB1C3-BBa\_B0032-cbtA | pSB1C3-BBa\_B0033-cbtA | pSB1C3-BBa\_B0032-cspD | pSB1C3-BBa\_B0033-cspD | pSB1C3-BBa\_B0032-mraZ | pSB1C3-BBa\_B0033-mraZ | pSB1C3-BBa\_B0032-cbtA | pSB1C3-BBa\_B0033-cbtA | pSB1C3-BBa\_B0032-cspD | pSB1C3-BBa\_B0033-cspD | pSB1C3-BBa\_B0032-mraZ | pSB1C3-BBa\_B0033-mraZ | pSB1C3-E0840 |  |  |  |  |  |  |  |  |
| Tube | **9** | **10** | **11** | **12** | **13** | **14** | **9** | **10** | **11** | **12** | **13** | **14** | **8** |  | **21** | **22** | **24** | **23** | **23** | **25** | **26** |
| Tube | 2 ng/µL | 4 ng/µL | 10 ng/µL | 3 ng/µL | 10 ng/µL | 33 ng/µL | 2 ng/µL | 4 ng/µL | 10 ng/µL | 3 ng/µL | 10 ng/µL | 33 ng/µL | 2 ng/µL |  |  |  |  | 21 ng/µL |  |  |  |
| Tube | 400 bp | 400 bp | 250 bp | 250 bp | 500 bp | 500 bp | 400 bp | 400 bp | 250 bp | 250 bp | 500 bp | 500 bp | 800 bp |  |  |  | 1000 bp | 1100 bp | 1100 bp | 400 bp | 250 bp |
| Tube | 9.00 µL | 9.00 µL | 6.00 µL | 10.00 µL | 7.23 µL | 6.00 µL | 9.00 µL | 9.00 µL | 5.32 µL | 9.00 µL | 7.23 µL | 9.00 µL | 9.00 µL |  |  |  |  | 9.00 µL | 9.00 µL |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL |
| Tube | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| Tube | 6.00 µL | 6.00 µL | 8.50 µL | 4.50 µL | 8.27 µL | 0.50 µL | 6.00 µL | 6.00 µL | 0.18 µL | 5.50 µL | 8.27 µL | 6.50 µL | 5.63 µL | 8.50 µL | 8.50 µL | 8.50 µL | 6.83 µL | 7.00 µL | 6.93 µL | 4.86 µL | 4.06 µL |
| Tube | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL |

## Solving the Nootkatone DNA of Düsseldorf

We cut circles out of paper with the seperated plasmids. Then we put them in 1.5 µL tubes and overlayed with 100 µL Elution Buffer. After that we vortexed the tubes, incubated for 5 min and vortexed again. Afterwards we did a transformation and stored the residue of the mix (Plasmid + Buffer) at -20°C (180814BK01-04).

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | # | Sample ID | User name | Date and time | DNA-concentration [ng/µL] | A260 (Abs) | A280 (Abs) | 260/280 | 260/230 | Sample Type | Factor | Abx |
|  | 208 | h2o | Nanodrop | 02.08.2018 11:57:03 | 0.7 | 0.015 | 0.02 | 0.74 | 0.16 | DNA | 50 |  |
| 1 | 209 | ADH | Nanodrop | 02.08.2018 11:57:41 | 392.3 | 7.846 | 4.178 | 1.88 | 1.84 | DNA | 50 | Cam |
| 2 | 210 | ValS | Nanodrop | 02.08.2018 11:58:22 | 268.6 | 5.373 | 2.935 | 1.83 | 1.37 | DNA | 50 | Cam |
| 3 | 211 | AIPLF | Nanodrop | 02.08.2018 11:58:52 | 678 | 13.559 | 7.325 | 1.85 | 2.12 | DNA | 50 | Amp |
| 4 | 212 | AIPLF-pts1 | Nanodrop | 02.08.2018 11:59:21 | 486.1 | 9.721 | 5.25 | 1.85 | 1.89 | DNA | 50 | Amp |

## Transformation of the ligations, GGAs and Nootkatone DNA (Düsseldorf 2017)

The transformation, ligation and Nootkatone was made by protocol by Zhang and the GGAs by protocol Golden Gate Assembly.

## Colony-PCR of the ligations and GGAs of 180814

Mastermix (GGAs) 40x :

* 80 µL Dream Taq buffer
* 16 µL dNTPs
* 24 µL MgCl2
* 4 µL Dream Taq-Polymerase
* 628 µL H2Odest

Primer in the GGAs:

* 6 µL GGA\_cPCR\_f
* 6 µL Bsa1\_pBAD\_r

Primer in the ligation:

* 6 µL VF2
* 6 µL VR

![](https://static.igem.org/mediawiki/2018/a/aa/T--Hamburg--180816_cPCR_GGA%2B3A.JPG)

## Ligation

|  |  |
| --- | --- |
| Tube | Part |
| 1 | L3 |
| 2-6 | L4 |
| 7-9 | L17 |
| 10-18 | L21 |

##

## GGAs

|  |  |
| --- | --- |
| Tube | Part |
| 15-20 | 3MB |
| 21-26 | Excretion |
| 27-32 | Myristic Acid |
| 33-38 | Growth Inhibition |

We made over night cultures of 1, 2, 10, 16, 18, 24, 27, 35, 38. We put 5 mL of each culture in LB-Medium and added 5 µL Camp.

## Miniprep of the overnight cultures from 180816

|  |  |  |
| --- | --- | --- |
| Lotnumber | Part | DNA-concentration [ng/µL] |
| 180817LD01 | cPCR 1 | 117 |
| 180817LD02 | cPCR 2 | 132 |
| 180817LD03 | cPCR 10 | 66 |
| 180817LD04 | cPCR 16 | 110 |
| 180817LD05 | cPCR 18 | 181 |
| 180817LD06 | cPCR 24 | 113 |
| 180817LD07 | cPCR 27 | 266 |
| 180817LD08 | cPCR 35 | 10 |
| 180817LD09 | cPCR 38 | 191 |
| 180817LD10 | pSB1C3-E0240 | 163 |
| 180817LD11 | pSB1C3-ADH #1 | 185 |
| 180817LD12 | pSB1C3-ValS #2 | 79 |
| 180817LD13 | pSB1C3-AlPLF #3 | 453 |
| 180817LD14 | pSB1C3-AlPLF-pts1 #4 | 326 |

## Control-digestion (01-09)

* 5 µL DNA
* 2 µL 2.1 Buffer
* 5 µL EcoR1
* 20 µL H2Odest

## Gelelectrophoresis

The samples was mixed with loading dye and loaded the samples to a 1% Agarose-gel. It ran for 40 min by 100 V.

![](https://static.igem.org/mediawiki/2018/5/5c/T--Hamburg--180817_Testverdau.JPG)

## Minipreps of the overnight cultures from 180821 + Restriction and ligation and afterwards transformation

Miniprep was made by GeneJET Plasmid Miniprep Kit. Minipreps of 180712BK04 and 180802BK01/02.

Solving the DNA of the kit:

|  |  |  |  |
| --- | --- | --- | --- |
| Plate |  | Kit | Lotnumber |
| Plate 3 | 8E | Kit2018 | 180822NiF01 |
| Plate 6 | 19P | Kit2018 | 180822NiF02 |
| Plate 6 | 20K | Kit2018 | 180822NiF03 |
| Plate 6 | 10F | Kit2018 | 180822NiF04 |

Afterwards we did a heat shock transformation in DH5a.

## Restriction

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** |
|  | Part | pSB1C3-I13453 | pSB1C3-NOTMlcRE | pSB1C3-E0240 | pSB1C3-BBa\_B0032-cspD | pSB1C3-BBa\_B0032-mraZ | pSB1C3-AOX1 | pSB1C3 | OmpT | BBa\_J45503 |
|  | Lot# | 180822BK01 | 180822BK02 | 180817LD10 | 180531LP05 | 180703BK06 | 180726LP02 | 180814LP01 | 180806BK10 | 180625NiF12 |
|  | **ρ** | 115 ng/µL | 119 ng/µL | 163 ng/µL | 54 ng/µL | 250 ng/µL | 114 ng/µL | 101 ng/µL | 60 ng/µL | 45 ng/µL |
|  | Use |  |  |  |  |  |  |  |  |  |
|  | target m |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 0.87 µL | 0.84 µL | 0.61 µL | 1.87 µL | 0.40 µL | 0.88 µL | 0.99 µL | 1.66 µL | 2.24 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF |  |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |
| XbaI |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |
| SpeI | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |
| H2O | 6.63 µL | 6.66 µL | 6.89 µL | 5.63 µL | 7.10 µL | 6.62 µL | 7.01 µL | 6.34 µL | 5.76 µL |
|  | ad | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |

## Making overnight cultures (for the sulA characterization)

New lotnumbers:

|  |  |
| --- | --- |
| Lotnumber | Part |
| 180518BK08 | 32 sulA |
| 180518BK09 | 34 sulA |
| 180518BK12 | 31 sulA |

## Colony PCR of L7 and L9

Mastermix:

* 10 µL Dream Taq Buffer (10x)
* 2 µL dNTPs (10mM)
* 3 µL MgCl2
* 25 µL Taq DNA Polymerase
* 3 µL VF2
* 3 µL VR
* 8 µL H2Odest

|  |  |
| --- | --- |
| Tube | Part |
| 1-2 | L7 |
| 3-4 | L9 |

## Gelelectrophoresis

The samples was mixed with loading dye and loaded the samples to a 1% Agarose-gel. It ran for 40 min by 80 V. We made over night cultures of the samples.

![](https://static.igem.org/mediawiki/2018/2/2f/T--Hamburg--180820_cPCR_%28unvollständig_beschriftet%29.JPG)

## Sequencing

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Seq. ID | Lotnumber | Part | DNA-concentration [ng/µL] | length [bp] | Part length  [bp] | Primer |
| 98ED21 | 180817LD09 | pSB1C3-pBAD-34cbta-34cspD-34-mraz-34sulA | 191 | 1896 | 1136 | GGA\_cPCR\_fw |
| 98ED22 | 180817LD09 | pSB1C3-pBAD-34cbta-34cspD-34-mraz-34sulA | 191 | 1896 | 1136 | GGA\_cPCR\_r |
| 98ED23 | 180817LD03 | pSB1C3-33killR | 66 | 1896 | 250 | Vf2 |

## Miniprep of pSB1C3-I13453-B0032-cbtA

Miniprep was made by GeneJET Plasmid Miniprep Kit.

180821LP01 = pSB1C3-I13453-BBa-B0032-cbtA à Cryostock

* Mixed 400 µL of the cryostock + 600 µL 50% Glycerol and stored at -80°C.

180821LP02 = pSB1C3-I13453-BBa-B0032-cbtA (stored at -20°C)

## Making overnight cultures of the cryostocks

180712BK04 and 180802 BK01/02. We put the cultures into tubes with 7 mL LB-medium and 7 µL Camp. The cultures was stored over night by 37°C at 200 rpm.

## Ligation

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** |
| Product | | pSB1C3-I13453-pSB1C3-BBa\_B0032-cspD | pSB1C3-I13453-pSB1C3-BBa\_B0032-mraZ | pSB1C3-I13453-pSB1C3-AOX1 | pSB1C3-NOTMlcRE-pSB1C3-E0240 | pSB1C3-OmpT | pSB1C3-BBa\_J45503 |
|  | |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-NOTMlcRE | pSB1C3 | pSB1C3 |
| Tube | **1** | **1** | **1** | **2** | **7** | **7** |
| ρ | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |
| Länge | 2200 bp | 2200 bp | 2200 bp | 2500 bp | 2070 bp | 2070 bp |
| V | 4.40 µL | 3.01 µL | 1.55 µL | 2.21 µL | 1.71 µL | 4.08 µL |
| **Part 1** | Part 1 | pSB1C3-BBa\_B0032-cspD | pSB1C3-BBa\_B0032-mraZ | pSB1C3-AOX1 | pSB1C3-E0240 | OmpT | BBa\_J45503 |
| Tube | **4** | **5** | **6** | **3** | **8** | **9** |
| ρ | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |
| Länge | 280 bp | 510 bp | 1200 bp | 880 bp | 1000 bp | 300 bp |
| V | 5.60 µL | 6.99 µL | 8.45 µL | 7.79 µL | 8.29 µL | 5.92 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |
| V |  |  |  |  |  |  |
| T4 Buffer | | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 7.50 µL | 7.50 µL | 7.50 µL | 7.50 µL | 7.50 µL | 7.50 µL |
| ad | | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL |

## PCR of the transformation from 180822

Mastermix for PCR (30x):

* 60 µL Dream Taq buffer 10x
* 12 µL dNTPs
* 18 µL MgCl2
* 3 µL Dream Taq Polymerase
* 18 µL VR
* 18 µL VF2
* 471 µL H2Odest

|  |  |
| --- | --- |
| Tube | Part |
| 1-5 | L1 |
| 6-10 | L2 |
| 11-15 | L3 |
| 16-20 | L4 |
| 21-25 | L5 |

![](https://static.igem.org/mediawiki/2018/8/8b/T--Hamburg--180823_cPCR.JPG)

We didn’t make overnight cultures of anything, because nothing was successful.

## Golden Gate Assembly

Restriction of the PCR-products from 30.07.18 (+ accC from 02.08.18). Afterwards we made a 1% Agarose-gel.

![](https://static.igem.org/mediawiki/2018/0/03/T--Hamburg--180823_Restriktion_GGA.JPG)

The restriction products we cut out of the gel and cleaned them with the GeneJET Gelextraction Kit. The ligation ran by 16°C overnight.

## Characterization of sulA (OD-measure)

We measured the growth curve at 600 nm.

Measure of the overnight cultures:

|  |  |
| --- | --- |
| Cultures | OD |
| DH5a | 0.6 |
| 31-sulA | 1.05 |
| 32-sulA | 0.92 |
| 34-sulA | 0.97 |

We had to adjust the OD of each culture with the following formula.

Requested start-OD: 0.01

0.01/0.06 \* 35 mL = 0.583 mL of the overnight cultures in 35 mL LB-medium.

After preparing the cultures they were stored at 37°C, 170 rpm and measured every half hour.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Time [h] | DH5a | 31-sulA | 32-sulA | 34-sulA |
| 0:30 | 0.014 | 0.014 | 0.018 | 0.022 |
| 1:00 | 0.05 | 0.043 | 0.035 | 0.032 |
| 1:30 | 0.136 | 0.084 | 0.094 | 0.089 |
| 2:00 | 0.253 | 0.178 | 0.203 | 0.194 |
| 2:30 | 0.554 | 0.476 | 0.519 | 0.497 |
| 3:00 | 0.878 | 0.809 | 0.855 | 0.843 |
| 3:30 | 1.129 | 1.145 | 1.159 | 1.112 |

After 2 h we added Arabinose to the samples.

Calculation:

V = 30 mL

c = 60 µM

M = 150.13 g/mol

m = 1.8 x10-6 mol

m = c \* V \* M = 0.225 mg Arabinose to each culture.

Measure was canceled, because we had not important results. We had a permanent growth and had not added the Arabinose at the beginning.

## Repetition of sulA characterization

|  |  |  |  |
| --- | --- | --- | --- |
| Culture | OD 600 | Timepoint | Requested start-OD: 0.01 |
| DH5a | 3.28 | 1:32 pm | 0.108 |
| BBa-B0031-sulA | 3.456 | 1:34 pm | 0.126 |
| BBa-B0034-sulA | 4.192 | 1:42 pm | 0.11 |

We had to adjust the OD of each culture. We added 100 mL cultures in LB-medium (+ Camp in sulA) into 300 mL-flasks and shacked them by 170 rpm and 37°C. After 2 hours we added 5 ml 100%-L-Arabinose.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | Time [min] | DH5a | BBa-B0031-sulA | BBa-B0034-sulA |
| t0 | 0 | 0.108 | 0.126 | 0.11 |
| t1 | 60 | 0.222 | 0.324 | 0.284 |
| t2 | 90 | 0.408 | 0.592 | 0.532 |
| t3 | 120 | 0.82 | 1.156 | 1.096 |
| t4 | 180 | 1.852 | 3.138 | 3.262 |

After the measurement we microscoped the cultures and took some pictures. We had not noticed differences between DH5a and sulA.

## Heat-shock transformation

The transformation was made by the iGEM transformation protocol. The following Part was transformed: 180822NiF02, 180817LD02, 09, 10, 11, 12, 13, 14 and the products of the GGA ligation from 23.08.18 (3-Methyl-1-butanol). The parts were transformed in DH5a cells and plated on LB-Agar-Camp Plates. Afterwards we stored the plates over night by 37°C.

## Set up Cryostocks

|  |  |
| --- | --- |
| Part with the old lotnumber | New lotnumber |
| 180826LP01 | 180817LD02 |
| 180826LP02 | 180817LD09 |
| 180826LP03 | 180817LD10 |
| 180826LP04 | 180817LD11 |
| 180826LP05 | 180817LD12 |
| 180826LP06 | 180817LD14 |

We mixed 600 µL culture with 200  µL 50%-Glycerol and stored the samples at -80°C. 180817LD13 and the GGA-ligations had not worked.

## Set up overnight cultures for competent cells

We put the DH5a cells in 10 mL LB-medium and stored over night by 37°C and 300 rpm.

## Set up competent cells according to Zhang

We mixed two 250 mL LB-medium with each 1 mL DH5a-preculture. The cells incubated by 37°C and 160 rpm for about 3 h until an OD of 0.03. Afterwards the cultures chilled for 5 min by 4°C on ice. Then we put the samples into 50 mL falcon tubes and centrifugated them for 10min by 5000 rpm and 4°C. Resuspended the cells in 4 mL Ca/glycerol buffer.

## PCR of ompT, sulA, tesA, MlcRE

Mastermix for 5 samples:

* 50 µL 5x Phusion HF buffer
* 5 µL dNTPs
* 5 µL DNA (E.coli DH5a)
* 5 µL Phusion Polymerase
* 1 µL Fw-Primer
* 1 µL Pv-Primer
* ad 250 µL H2Odest

|  |  |  |  |
| --- | --- | --- | --- |
|  | Part | Fw-Primer | Rv-Primer |
| 1 | ompT | Pre-ompT-fz | OmpT-Suf-rz |
| 2 | sulA | Pre-sulA-fw | sulA-Suf-rv |
| 3 | tesA | Pre-tesA-f | tesA-Suf-r |
| 4 | MlcRE | Pre-MlcRE-fw | MlcRE-Suf-rv |

## Gelelectrophoresis

We made a 1%-Agarose gel. On tesA we did not see a band. Afterwards we made a Gel-extraction of the samples 1, 2 and 4 with the GeneJET Gel-extraction Kit.

![](https://static.igem.org/mediawiki/2018/d/dc/T--Hamburg--180827.JPG)

New lotnumbers:

|  |  |
| --- | --- |
| Lotnumber | Part |
| 180827LP01 | ompT |
| 180827LP02 | sulA |
| 180827LP03 | MlcRE |

## Heat shock transformation

After the gel extraction we did a heat shock transformation of 180827AK01 (pSB1C3\_K206000) in DH5a. The culture did not grow.

## Verification of the sequencing

Digest with EcoRI:

* 12 mL 10x NEBuffer 2.1
* 5 mL EcoRI
* 1 mg DNA
* Ad 20 mL H2O

|  |  |  |  |
| --- | --- | --- | --- |
|  | Lotnumber | Part | Volume [µL] |
| 1 | 180329DW03 | pSB1C3-MIcRe | 10 |
| 2 | 180329DW09 | pSB1C3-sulA | 7 |
| 3 | 180329DW21 | pSB1C3-BBa\_B0034-sulA | 10 |
| 4 | 180329DW28 | pSB1C3-E0840-oHybB | 7 |
| 5 | 180329DW41 | pSB1C3-K206000-32sulA | 10 |
| 6 | 180531LP05 | pSB1C3-cspD | 10 |

Incubated the samples for 60 min by 37°C and 20 min by 65°C.

## Gelelctrophoresis

We made a 1%-Agarose gel. Afterward we did a gel extraction with GeneJet Gel-extraction Kit.

## Transformation of 180827AK01

Repetition of the transformation of 180827AK01 into new competent cells from 27.08.18 (DH5a). Then incubated over night by 37°C.

New lotnumbers:

|  |  |
| --- | --- |
| Lotnumber | DNA-concentration [ng/µL] |
| 180329DW03 | 5.6 |
| 180329DW09 | 40 |
| 180329DW21 | 2.9 |

We discarded all of the samples.

## PCR of multiple Parts

PCR were prepapared as followed:

|  |  |  |  |
| --- | --- | --- | --- |
| Tube | Part | Primer-fw | Primer-rv |
| 1 | pre-31-sulA-suf | 31-sulA-Fw | SulA-Suf-Rev |
| 2 | pre-oHybB-suf | Pre-HybB-Fw | HybB-Suf-Rev |
| 3 | pre-34-sulA-suf | 34-sulA-Fw | SulA-Suf-Rev |
| 4 | pre-32-sulA-suf | 32-sulA-Fw | SulA-Suf-Rev |
| 5 | pre-32-cspD-suf | Pre\_cspD\_f | cspD\_Suf\_r |
| 6 | pre-32-LdhA-suf | 32-Idha-Fw | ldhA-Suf-Rev |
| 7 | pre-cspD-suf | Pre-cspD-f | cspD-Suf-Rev |

Mastermix for 10 approaches were prepared as shown:

|  |  |
| --- | --- |
| 50 µL | Q5 Mastermix |
| 5 µL | Template DNA (DH5α) |
| 35 µL | H2O |

This was aliquoted into 0.5 mL reaction tube to total volume of 9 µL. To each aliquot were added the specific primer pair, 0.5 µL forward and 0.5 µL reverse primer.

The PCR program was done as shown in the next table.

|  |  |  |
| --- | --- | --- |
| 1 | 98 °C | 30 s |
| 2 | 98 °C | 10 s |
| 3 | 63 °C | 30 s |
| 4 | 72 °C | 30 s |
| 5 | go to step 2 30 times | |
| 6 | 72 °C | 2 min |

For the analytic of the PCR products we used a 1 % agarose gel. 2 µL of the PCR product were loaded on the gel.

![](https://static.igem.org/mediawiki/2018/6/64/T--Hamburg--180829_PCR_%28nicht_erfolgreich%29_%281%29.JPG)

Because we used undiluted primers in this approach, we repeated the PCR with a new mastermix, the right primers and the same PCR program. New mastermix is shown in the following table.

|  |  |
| --- | --- |
| 250 µL | Q5 Mastermix |
| 25 µL | Template DNA (DH5α) |
| 175 µL | H2O |

This was aliquoted into 0.5 mL reaction tube to total volume of 4.5 µL. To each aliquot were added the specific primer pair, 2.5 µL forward and 2.5 µL reverse primer. Here we used a wrong ratio of primer to Q5 mastermix. So, we repeated this experiment.

## <https://static.igem.org/mediawiki/2018/7/75/T--Hamburg--180829_PCR_%28nicht_erfolgreich%29_%282%29.JPG>

## Miniprep of pSB1C3-K206000

For the miniprep of this construct we used the Mini Prep Kit from Thermo Scientific. Afterwards the concentration of the plasmid was measured with a nanodrop.

|  |  |  |
| --- | --- | --- |
| Lot number | Part | Concentration |
| 180829LD01 | pSB1C3\_K2060000 | 115.3 ng/µL |
| 180829LD02 | pSB1C3\_K2060000 | 49.8 ng/µL |

## Cryostock of pSB1C3\_K206000

600µL of the culture 180829LD03 was mixed with 200µL 50 % glycerol and stored at -80 °C.

## PCR repetition from 180829

Mastermix for 50 samples:

* 250 µL
* 25 µL of each primer
* 25 µL Template
* 175 µL H2Odest

|  |  |  |
| --- | --- | --- |
| Tube | Part | Length [bp] |
| 1 | Pre-31-sulA-suf | 530 |
| 2 | Pre-32-sulA-suf | 529 |
| 3 | Pre-34-sulA-suf | 528 |
| 4 | Pre-oHybB-suf | 173 |
| 5 | Pre-32-ldhA-suf | 1055 |
| 6 | Pre-cspD-suf | 222 |
| 7 | Pre-32-cspD-suf | 241 |

PCR program:

|  |  |
| --- | --- |
| 98°C | 2 min |
| 98°C | 40 s |
| 55°C | 20 s |
| 72°C | 1:20 min |
| Go to step 2 30x | |
| 72°C | 2 min |

## Gelelectrophoresis

We made a 1% agarose gel. It ran for 40 min by 80 V.

![](https://static.igem.org/mediawiki/2018/b/bd/T--Hamburg--180830_PCR1_3.JPG)
![](https://static.igem.org/mediawiki/2018/0/0a/T--Hamburg--180830-1.JPG)

## Restriction of multiple parts

               Part                                      Lot-No.

               pSB1C3-RNAG120-MlcrE 180822BK02
                pSB1C3-oHybB                                 180731NF08
                pSB1C3-E0240                                  180817LD10
                pSB1C3-E0840                                  180627LP02
 Reaction mix:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | pSB1C3-RNAG120-MlcRE | pSB1C3-oHybB | pSB1c3-E0240 | pSB1C3-E0840 |
| DNA | 1.69 µL | 1.48 µL | 1.23 µL | 2.17 µL |
| NE-Buffer 2.1 | 1 µL | 1 µL | 1 µL | 1 µL |
| SpeI | 0.5 µL | 0.5 µL | - | - |
| PstI | 0.5 µL | 0.5 µL | 0.5 µL | 0.5 µL |
| XbaI | - | - | 0.5 µL | 0.5 µL |
| FastAP | 0.5 µL | 0.5 µL | 0.5 µL | 0.5 µL |
| H2O | 5.81 µL | 6.02 µL | 6.27 µL | 5.33 µL |

Restriction ran for 1 h at 37 °C, followed by 20 min at 80 °C.

Separation of gfp-parts by gel electrophoresis with a 1 % agarose gel, followed by a gel extraction.

![](https://static.igem.org/mediawiki/2018/4/42/T--Hamburg--180903-1.JPG)

Resulting concentrations were:

               Part                                      c [ng/µL]

               pSB1C3-RNAG120-MlcrE 75.43
                pSB1C3-oHybB                                 259.27
                pSB1C3-E0240                                  1
                pSB1C3-E0840                                  2

The gfp parts showed very low concentrations and were discarded.

## Repeating the PCR from 30.08.18

PCR was performed as follows.

|  |  |
| --- | --- |
| 5xPhusion HF Buffer | 70 µL |
| dNTPs | 7 µL |
| DNA (*E. coli*) | 7µL |
| Phusion Polymerase | 3.5 µL |
| Fw primer | 1 µL |
| Rv prmer | 1 µL |
| ad 350 µL H2O | 248.5 µL |

The PCR programme was carried out as follows.

|  |  |  |
| --- | --- | --- |
| Step | Temperature | Time [s] |
| 1 | 98 °C | 180 |
| 2 | 98 °C | 10 |
| 3 | 55 °C | 30 |
| 4 | 72 °C | 120 |
| go to step 2 (30x) |  |  |
| 5 | 72 °C | 300 |

The following primer pairs were used.

|  |  |  |
| --- | --- | --- |
| 1 | 31sulA | (31-sulA-Fw, 31-sulA-Suf-Rv) |
| 2 | 32sulA | (32-sulA-Fw, 32-sulA-Suf-Rv) |
| 3 | 34sulA | (34-sulA-Fw, 34-sulA-Suf-Rv) |
| 4 | HybB | (pre-HybB-fw, pre-HybB-Suf-Rv) |
| 5 | 32IdhA | (32IdhA-fw, 32IdhA-Suf-Rv) |
| 6 | cspD | (pre-cspD-fw, pre-csp-Suf-Rv) |

The PCR results were analysed in an agarose gel (1%, 80 V, 45 min).

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 |
| 31sulA | 32sulA | 34sulA | HybB | 32IdhA | cspD |
| positive | positive | positive | positive | negative | positive |

## Restriction of the PCR from 30.08.18

The PCR products were restricted according to the following table. Afterwards, the DNA concentration was measured via nano drop.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | 31sulA | 32sulA | 34sulA | HybB | cspD |
| NEB2.1 [µL] | 2 | 2 | 2 | 2 | 2 |
| DNA [µL] | 4 | 4 | 4 | 4 | 4 |
| EcoRI [µL] |  |  |  | 0.5 | 0.5 |
| XbaI [µL] | 0.5 | 0.5 | 0.5 |  |  |
| SpeI [µL] |  |  |  | 0.5 |  |
| pstI [µL] | 0.5 | 0.5 | 0.5 |  | 0.5 |
| H2O [µL] | 13 | 13 | 13 | 13 | 13 |
| concentration [ng/µL] | 129.38 | 183.26 | 169.36 | 210.06 | 166.14 |

## Repeating the restriction of 3 and 4 of the day before

psB1C3-E0840 à 3 à

psB1C3-E0240 à 4 à

4 = 3.889 ng/µL = too little

3 = 2.206 ng/µL = too little

Samples were discarded.

Ligation of the samples of the restrictions 1 - 5 were incubated at 37 °C for 1 h.

Transformation of the products into DH5α cells.

## cPCR

Master mix:

* 16 µL Taq buffer
* 2 µL dNTPs
* 8 µL MgCl2
* 8 µL VR
* 8 µL VF2
* 1 µL Taq polymerase
* 125.4 µL H2Odest

|  |  |
| --- | --- |
| Tube | Part |
| 1 | pSB1C3-BBa\_K20600-32-sulA |
| 2-6 | pSB1C3-B0840-oHybB |
| 7 | pSB1C3-cspD |

PCR program:

|  |  |
| --- | --- |
| 95°C | 10 min |
| 95°C | 30 s |
| 65°C | 30 s |
| 72°C | 1:10 min |
| Go to step 2 30x | |
| 72°C | 5 min |

## Gelelectrophoresis

We made 1% agarose gel. It ran for 45 min by 80 V.

![](https://static.igem.org/mediawiki/2018/5/52/T--Hamburg--180905-1.JPG)
![](https://static.igem.org/mediawiki/2018/0/0f/T--Hamburg--180905-2.JPG)

## PCR Purification

The purification was made with GeneJET PCR purification Kit by Thermo Fischer.

|  |  |  |
| --- | --- | --- |
| Lot number | Part | DNA-concentration [ng/µL] |
| 180905AW01 | 31-sulA | 28 |
| 180905AW02 | 32-sulA | 40 |
| 180905AW03 | 34-sulA | 43 |
| 180905AW04 | HybB | 69 |
| 180905AW05 | cspD | 48 |

## PCR of AOX

Master mix:

* 25 µL Q5-Mastermix
* 5 µL of each primer
* 5 µL template
* 50 µL H2Odest

|  |  |  |
| --- | --- | --- |
| Part | Fw primer | Rv primer |
| 34-AOX | 34-AOX-f | Syn-r |

PCR program:

|  |  |
| --- | --- |
| 98°C | 2 min |
| 98°C | 10 s |
| 55°C | 20 s |
| 72°C | 1:45 min |
| Go to step 2 30x | |
| 72°C | 2 min |

## Gelelectrophoresis

We made a 1% agarose gel.

![](https://static.igem.org/mediawiki/2018/c/c4/T--Hamburg--180906_PCR_AOX1.JPG)

## Restriction

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **9** | **10** | **11** | **12** | **13** | **14** | **15** |
|  | Part | MlcRE | sulA | 31sulA | psB1C3-E0840 | oHybB | pSB1C3-BBa\_K206000 | pSB1C3 | 32sulA | 34sulA | cspD | pSB1C3-BBa\_B0032 | cspD | psB1C3-E0840 | pSB1C3-NOT-MlcRE |
|  | Lot# | 180827LP03 | 180827LP02 | 180905AW01 | 180315NB01 | aus PCR | 180829LD01 | 180814LP01 | 180905AW02 | 180905AW03 | aus PCR | 180514NB07 | aus PCR | 180315NB01 | 180822BK02 |
|  | **ρ** | 63 ng/µL | 71 ng/µL | 28 ng/µL | 128 ng/µL | 69 ng/µL | 115 ng/µL | 101 ng/µL | 40 ng/µL | 43 ng/µL | 48 ng/µL | 81 ng/µL | 48 ng/µL | 128 ng/µL | 118 ng/µL |
|  | Use | single insert | single insert | assembly insert 2 | vector | single insert | vector | vector | assembly insert 2 | assembly insert 2 | single insert | vector | assembly insert 2 | assembly insert 2 | vector |
|  | target m | 100 ng | 100 ng |  | 2000 ng | 100 ng | 2000 ng | 2000 ng | 100 ng | 100 ng | 100 ng | 2000 ng | 100 ng | 100 ng | 2000 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 1.60 µL | 1.40 µL | 3.57 µL | 7.00 µL | 1.45 µL | 5.00 µL | 5.00 µL | 2.50 µL | 2.33 µL | 2.08 µL | 5.00 µL | 2.08 µL | 0.78 µL | 7.00 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF | 0.20 µL | 0.20 µL |  | 0.40 µL | 0.20 µL |  | 0.20 µL |  |  | 0.20 µL | 0.20 µL |  |  |  |
| XbaI |  |  | 0.20 µL | 0.40 µL |  |  |  | 0.20 µL | 0.20 µL |  | 0.20 µL | 0.20 µL | 0.20 µL |  |
| SpeI |  |  |  |  |  | 0.20 µL |  |  |  |  |  |  |  | 0.20 µL |
| PstI | 0.20 µL | 0.20 µL | 0.20 µL |  | 0.20 µL | 0.20 µL | 0.20 µL | 0.20 µL | 0.20 µL | 0.20 µL |  | 0.20 µL | 0.20 µL | 0.20 µL |
| FastAP |  |  |  | 0.50 µL |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL |  |  | 0.50 µL |
| H2O | 7.00 µL | 7.20 µL | 5.03 µL | 0.70 µL | 7.15 µL | 3.10 µL | 3.10 µL | 6.10 µL | 6.27 µL | 6.52 µL | 3.10 µL | 6.52 µL | 7.82 µL | 1.10 µL |
|  | ad | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt | 252 bp | 556 bp | 565 bp | 2948 bp | 224 bp | 2200 bp | 2070 bp | 565 bp | 565 bp | 245 bp | 2083 bp | 245 bp | 904 bp | 2697 bp |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 10 ng/µL | 10 ng/µL | 10 ng/µL | 90 ng/µL | 10 ng/µL | 58 ng/µL | 51 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 40 ng/µL | 10 ng/µL | 10 ng/µL | 83 ng/µL |

## Ligation

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L5** | **L6** | **L7** | **L9** |
| Product | | pSB1C3-MlcRE | pSB1C3-sulA | pSB1C3-BBa\_K206000-31sulA | pSB1C3-BBa\_K206000-32sulA | pSB1C3-BBa\_K206000-34sulA | pSB1C3-BBa\_B0032-cspD |
|  | |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3 | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_B0032 |
| Tube | **7** | **7** | **6** | **6** | **6** | **12** |
| ρ | 51 ng/µL | 51 ng/µL | 58 ng/µL | 58 ng/µL | 58 ng/µL | 40 ng/µL |
| Länge | 2070 bp | 2070 bp | 2200 bp | 2200 bp | 2200 bp | 2083 bp |
| V | 0.89 µL | 0.54 µL | 0.49 µL | 0.49 µL | 0.49 µL | 1.14 µL |
| **Part 1** | Part 1 | MlcRE | sulA | 31sulA | 32sulA | 34sulA | cspD |
| Tube | **1** | **2** | **3** | **9** | **10** | **13** |
| ρ | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |
| Länge | 252 bp | 556 bp | 565 bp | 565 bp | 565 bp | 245 bp |
| V | 5.49 µL | 7.29 µL | 7.20 µL | 7.20 µL | 7.20 µL | 5.40 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |
| V |  |  |  |  |  |  |
| T4 Buffer | | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 2.12 µL | 0.68 µL | 0.82 µL | 0.82 µL | 0.82 µL | 1.95 µL |
| ad | | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |
| Länge gesamter Part |  | 252 bp | 556 bp | 695 bp | 695 bp | 695 bp | 258 bp |
|  |  | 597 bp | 901 bp | 1040 bp | 1040 bp | 1040 bp | 603 bp |

## Repetition of the PCR to create BB-34-ompA-Aox1

Mastermix:

* 10 µL Q5-Mastermix
* 1 µL xba-34-AOX-fw
* 1 µL syn-rev
* 0.2 µL Template (BBPre-ompA-NnAOX1a-BBsuf)

PCR-Program:

|  |  |
| --- | --- |
| 98°C | 2 min |
| 98°C | 10 s |
| 55°C | 20 s |
| 72°C | 1:45 min |
| Go to step 2  30x | |
| 72°C | 2 min |

## Gelelectrophoresis

We made a 1%-Agarose-gel to proof the length of the parts. It ran for 45 min by 80 V.

![](https://static.igem.org/mediawiki/2018/c/c4/T--Hamburg--180906_PCR_AOX1.JPG)

We cleaned up the PCR-products with GeneJET PCR Purification Kit and eluted in 30 µL H2Odest. Afterwards we measured the DNA-concentration with a Nano Drop.

|  |  |
| --- | --- |
| Lotnumber | DNA-concentration [ng/µL] |
| 180906LP01 | 84 |

## Restriction

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** |
|  | Part | pSB1C3-BBa\_K206000 | pSB1C3 | pSB1C3-BBa\_I13504 | 34-AOX1 | pSB1C3-BBa\_E0840 | pSB1C3-BBa\_E0840 | pSB1C3\_RNAG120\_MlcRE | pSB1C3\_oHybB |
|  | Lot# | 180829LD01 | 180906LP02 | 180208BK04 | 180906LP01 | 180627LP02 | 180627LP02 | 180822BK02 | 180731NF08 |
|  | **ρ** | 115 ng/µL | 138 ng/µL | 141 ng/µL | 84 ng/µL | 91 ng/µL | 91 ng/µL | 119 ng/µL | 135 ng/µL |
|  | Use | vector | vector | vector | single insert | vector | single insert |  |  |
|  | target m | 2000 ng | 2000 ng | 2000 ng | 100 ng | 2000 ng | 100 ng | 200 ng | 200 ng |
|  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 16.60 µL | 14.49 µL | 5.00 µL | 1.19 µL | 3.00 µL | 1.10 µL | 1.69 µL | 1.48 µL |
| NEBuffer 2.1 | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF |  |  |  |  | 0.20 µL | 0.20 µL |  |  |
| XbaI |  | 0.40 µL |  | 0.20 µL | 0.20 µL |  |  |  |
| SpeI | 0.40 µL |  | 0.20 µL |  |  |  | 0.50 µL | 0.50 µL |
| PstI | 0.40 µL | 0.40 µL | 0.20 µL | 0.20 µL |  | 0.20 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL |  | 0.50 µL | 0.50 µL |
| H2O | 0.10 µL | 2.21 µL | 3.10 µL | 7.41 µL | 5.10 µL | 7.50 µL | 5.81 µL | 6.02 µL |
|  | ad | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt | 2200 bp | 2070 bp | 2200 bp | 1200 bp | 2948 bp | 878 bp | 2697 bp | 2326 bp |
|  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 96 ng/µL | 100 ng/µL | 71 ng/µL | 10 ng/µL | 27 ng/µL | 10 ng/µL | 20 ng/µL | 20 ng/µL |
|  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  | "Tube 1" | "Tube 2" |
|  |  |  |  |  |  |  |  | vom 4.9. | |

## Ligation

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** |
| Product | | pSB1C3-BBa\_K206000-34-AOX1 | pSB1C3-34-AOX1 | pSB1C3-BBa\_I13504-34-AOX1 | pSB1C3\_RNAG120\_MlcRE-pSB1C3-BBa\_E0840 | pSB1C3\_oHybB-pSB1C3-BBa\_E0840 |
|  | |  |  |  |  |  |
| **Vector** | Vector | pSB1C3-BBa\_K206000 | pSB1C3 | pSB1C3-BBa\_I13504 | pSB1C3\_RNAG120\_MlcRE | pSB1C3\_oHybB |
| Tube | **1** | **2** | **3** | **7** | **8** |
| ρ | 96 ng/µL | 100 ng/µL | 71 ng/µL | 20 ng/µL | 20 ng/µL |
| Länge | 2200 bp | 2070 bp | 2200 bp | 2697 bp | 2326 bp |
| V | 0.16 µL | 0.15 µL | 0.22 µL | 0.50 µL | 0.50 µL |
| **Part 1** | Part 1 | 34-AOX1 | 34-AOX1 | 34-AOX1 | pSB1C3-BBa\_E0840 | pSB1C3-BBa\_E0840 |
| Tube | **4** | **4** | **4** | **6** | **6** |
| ρ | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |
| Länge | 1200 bp | 1200 bp | 1200 bp | 878 bp | 878 bp |
| V | 8.45 µL | 8.53 µL | 8.45 µL | 5.00 µL | 5.00 µL |
| **Part 2** | Part 2 |  |  |  |  |  |
| Tube |  |  |  |  |  |
| ρ |  |  |  |  |  |
| Länge |  |  |  |  |  |
| V |  |  |  |  |  |
| T4 Buffer | | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 8.89 µL | 8.82 µL | 8.83 µL | 3.00 µL | 3.00 µL |
| ad | | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |
| Länge gesamter Part |  | 1330 bp | 1200 bp | 1330 bp | 1505 bp | 1134 bp |
| plus VR VF2 |  | 1675 bp | 1545 bp | 1675 bp | 1850 bp | 1479 bp |

## PCR of linear pSB1C3

Mastermix:

* 10 µL HF buffer 5x
* 1 µL dNTPs
* 35 µL SB-prep-3P-1
* 35 µL SB-prep-2Ea
* 5 µL Template DNA (180208BK01 = pSB1C3-I20270)
* 5 µL Phusion polymerase
* 50 µL H2Odest

PCR-program

|  |  |
| --- | --- |
| 98°C | 2 min |
| 98°C | 30 s |
| 55°C | 30 s |
| 72°C | 3 min |
| Go to step 2 35x | |
| 72°C | 10 min |

## Gelelectrophoresis

We made a 1%-Agarose-gel to proof the length. It ran for 45 min by 80 V.

![](https://static.igem.org/mediawiki/2018/c/cd/T--Hamburg--180906_PCR_linear_pSB1C3.JPG)

The part was cleaned up with GeneJET PCR Purification Kit and eluted in 30 µL H2Odest. The concentration was measured with a Nano Drop.

|  |  |
| --- | --- |
| Lotnumber | DNA-concentration [ng/µL] |
| 180906LP02 | 138 |

## Minipreps and Cryostocks of the cPCR from 180905

We did the plasmid prep with the GeneJET Plasmid Purification Kit. Afterwards we measured the DNA-concentration with a Nano Drop.

|  |  |
| --- | --- |
| Lotnumber | DNA-concentration [ng/µL] |
| 180906NB03 | 191 |
| 180906NB04 | 170 |

We mixed the Cryostocks with 500 µL 50% Glycerol and 500 µL cellsuspension and stored at -20°C.

## Heat shock transformation of the ligations from 180905 and 180906

We transformed the parts 180906LP03-13. The cells chilled 5 min on ice at 4°C. After that we added 10-20 µL DNA to the cells. Afterwards the cells had to incubate for 30 min on ice. Then we made a heat shock for 45 s at 42°C and added 800 µL LB-medium. After that the cells incubate for 30 min at 37°C and 700 rpm. 300 µL of the samples were plated and stored overnight at 37°C.

After the transformation we set up overnight cultures for miniprep.

|  |  |
| --- | --- |
| Lotnumber | Part |
| 180208AK02 | pSB1C3-E0840 |
| 180208AK03 | pSB1C3-I13504 |

## cPCR oft the ligation form 06.09.18

On all plates except from 180906LP05 and 180906LP04 colonies are grown. Plates 180906LP05 and 180906LP04 were further incubated at 37 °C.

|  |  |  |
| --- | --- | --- |
| PCR preparation | 1x | 47x |
| 10x Taq Buffer | 2 µL | 94 µL |
| dNTPs 10 mM | 0.4 µL | 18 µL |
| 50 mM MgCl2 | 0.6 µL | 28.2 µL |
| fw primer | 0.6 µL | 28.2 µL |
| rv primer | 0.6 µL | 28.2 µL |
| Taq polymerase | 0.1 µL | 4.7 µL |
| H2O ad 20 µL | 14.5 µL | 681.5 µL |

The PCR programme was carried out as follows.

|  |  |  |
| --- | --- | --- |
| Step | Temperature | Time [s] |
| 1 | 95 °C | 600 |
| 2 | 95 °C | 30 |
| 3 | 55 °C | 30 |
| 4 | 72 °C | 120 |
| go to step 2 (30x) |  |  |
| 5 | 72 °C | 300 |

## Minipreparation of ONC from 06.09.18

A minipreparation of 180907NiF03 and 180907NiF04 was performed using GeneJET Plasmid Miniprep Kit #K0503 by Thermo Scientific following protocol A. Afterwards, the concentration of DNA was measured via nano drop.

|  |  |
| --- | --- |
| Minipreparation | Concentration (ng/µL) |
| 180907NiF03 | 83.791 |
| 180907NiF04 | 85.965 |

180907-2.png

Control of the gel electrophoresis is listed in the following table.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| LOT number | Part | Number | Length | Result |
| 180906LP03 | pSB1C3-K206000-34-AOX1 | 1-4 | 1675 bp | positive |
| 180906LP06 | pSB1C3-RNAG120-MlcRE-RE-BBa\_E0840 | 5-8 | 1850 bp | negative |
| 180906LP07 | pSB1C3-oHybB-BBa\_E0840 | 9-12 | 1479 bp | positive |
| 180906LP08 | pSB1C3-MlcRE | 13-16 | 597 bp | positive |
| 180906LP09 | pSB1C3-SulA | 17-20 | 901 bp | positive |
| 180906LP10 | pSB1C3-BBa\_K02600-31-sulA | 21-24 | 1040 bp | negative |
| 180906LP11 | pSB1C3-BBa\_K02600-32-sulA | 25-28 | 1040 bp | negative |
| 180906LP12 | pSB1C3-BBa\_K02600-34-sulA | 29-32 | 1040 bp | negative |
| 180906LP13 | pSB1C3-BBA\_B0032-cspD | 33-36 | 603 bp | negative |

ONCs were prepared of the parts showing a positive result.

|  |  |
| --- | --- |
| LOT number | Part |
| 180907LP01 | psB1C3-K206000-34-AOX1 |
| 180907LP02 | psB1C3-oHybB-E0840 |
| 180907LP03 | psB1C3-MlcRE |
| 180907LP04 | psB1C3-sulA |

## Cryostocks of ONCs from 06.09.18

To 600 µL of each culture 200 µL glycerol (50%) were added. These samples were stored at -80 °C.

|  |  |
| --- | --- |
| LOT number | Part |
| 180908NF01 | psB1C3-K206000-34-AOX1 |
| 180908NF 02 | psB1C3-oHybB-E0840 |
| 180908NF 03 | psB1C3-MlcRE |
| 180908NF 04 | psB1C3-sulA |

## Minipreparation of ONC from 07.09.18

A minipreparation of the ONCs were performed using GeneJET Plasmid Miniprep Kit #K0503 by Thermo Scientific following protocol A. Afterwards, the concentration of DNA was measured via nano drop.

|  |  |  |
| --- | --- | --- |
| LOT number | Part | Concentration [ng/µL] |
| 180908NF05 | psB1C3-K206000-34-AOX | 64.700 |
| 180908NF06 | psB1C3-oHybB-E0840 | 65.967 |
| 180908NF07 | psB1C3-MlcRE | 28.584 |
| 180908NF08 | psB1C3-sulA | 53.158 |

A new ONC of 180908NF07 was prepared since its concentration was below 50 ng/µL.

## Restriction and ligation

The restriction was performed according to the following table. The preparations were incubated at first for 1 h at 37 °C and afterwards at 80 °C for 20 min.

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| Part | pSB1C3\_RNAG120\_MlcRE | pSB1C3-BBa-E0840 | pSB1C3-BBa\_K206000 | 31sulA | 32sulA | 34sulA | cspD | pSB1C3-BBa\_B0032 | cspD | 34-AOX1 | pSB1C3-BBa\_I13453 | pSB1C3 |
| Lot# | 180822BK02 | 180907NiF03 | 180829LD01 | 180905AW01 | 180905AW02 | 180905AW03 | 180905AW05 | 180514NB07 | 180905AW05 | 180906LP01 | 180822BK01 | 180906LP02 |
| ρ | 119 ng/µL | 92 ng/µL | 115 ng/µL | 28 ng/µL | 40 ng/µL | 43 ng/µL | 48 ng/µL | 81 ng/µL | 48 ng/µL | 84 ng/µL | 115 ng/µL | 138 ng/µL |
| Use | vector | single insert | vector | single insert | single insert | single insert | single insert | vector | single insert | single insert | vector | single insert |
| target m | 2000 ng | 100 ng | 2000 ng | 100 ng | 100 ng | 100 ng |  | 2000 ng | 100 ng | 100 ng | 2000 ng | 100 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |
| DNA | 7.00 µL | 7.00 µL | 16.00 µL | 3.57 µL | 2.50 µL | 2.33 µL | 2.08 µL | 7.00 µL | 2.08 µL | 1.19 µL | 17.34 µL | 0.72 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 3.00 µL | 1.00 µL |
| EcoRI-HF |  |  |  |  |  |  |  |  | 0.50 µL |  |  |  |
| XbaI |  | 0.50 µL |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  | 0.50 µL |
| SpeI | 0.50 µL |  | 0.50 µL |  |  |  |  | 0.50 µL |  |  | 0.50 µL |  |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  | 0.50 µL |  |  | 0.50 µL |  |
| H2O | 0.50 µL | 0.50 µL | 0.50 µL | 4.43 µL | 5.50 µL | 5.67 µL | 5.92 µL | 0.50 µL | 5.92 µL | 6.81 µL | 8.16 µL | 7.28 µL |
| ad | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 30.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |
| Länge Produkt | 2697 bp | 878 bp | 2200 bp | 565 bp | 565 bp | 565 bp | 245 bp | 2083 bp | 245 bp | 1200 bp | 2200 bp | 2070 bp |
|  |  |  |  |  |  |  |  |  |  |  |  |  |
| ρ final | 83 ng/µL | 64 ng/µL | 92 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 56 ng/µL | 10 ng/µL | 10 ng/µL | 67 ng/µL | 10 ng/µL |

After the gel extraction DNA concentrations were determined. Restriction 1 was not successful.

|  |  |
| --- | --- |
| Restriction | Concentration [ng/µL] |
| 2 | 3.8 |
| 3 | 8.3 |
| 8 | 5.0 |

The ligation was performed overnight according to the following table.

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** |
| Product | | pSB1C3-34-AOX1 | pSB1C3-BBa\_K206000-34-AOX1 | pSB1C3\_RNAG120\_MlcRE-pSB1C3-BBa-E0840 | pSB1C3-BBa\_K206000-31sulA | pSB1C3-BBa\_K206000-32sulA | pSB1C3-BBa\_K206000-34sulA | pSB1C3-BBa\_B0032-cspD | pSB1C3-cspD |
|  | |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3\_RNAG120\_MlcRE | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_B0032 | pSB1C3 |
| Tube | **12** | **3** | **1** | **3** | **3** | **3** | **8** | **12** |
| ρ | 10 ng/µL | 8 ng/µL | 83 ng/µL | 8 ng/µL | 8 ng/µL | 8 ng/µL | 5 ng/µL | 10 ng/µL |
| Länge | 2070 bp | 2200 bp | 2697 bp | 2200 bp | 2200 bp | 2200 bp | 2083 bp | 2070 bp |
| V | 1.47 µL | 1.87 µL | 0.28 µL | 3.38 µL | 3.38 µL | 3.38 µL | 9.19 µL | 4.58 µL |
| **Part 1** | Part 1 | 34-AOX1 | 34-AOX1 | pSB1C3-BBa-E0840 | 31sulA | 32sulA | 34sulA | cspD | cspD |
| Tube | **10** | **10** | **2** | **4** | **5** | **6** | **7** | **9** |
| ρ | 10 ng/µL | 10 ng/µL | 64 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |
| Länge | 1200 bp | 1200 bp | 878 bp | 565 bp | 565 bp | 565 bp | 245 bp | 245 bp |
| V | 8.53 µL | 8.45 µL | 1.19 µL | 7.20 µL | 7.20 µL | 7.20 µL | 5.40 µL | 5.42 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |
| T4 Buffer | | 2.00 µL | 2.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 7.00 µL | 7.00 µL | 7.02 µL | 7.00 µL | 7.00 µL | 7.00 µL | 2.90 µL | 7.50 µL |
| ad | | 20.00 µL | 20.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL |

## PCR for GG Assembly (+BjaIT) + RNAG120 + pGGA

PCR: 25 µL Q5 MM, 20 µL H2O, 2.5 mL primer each, 0.25 µL template

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Part | fw primer | rv primer | Template | LOT number | Conc [ng/µL] | pred. length |
| Bsa\_HlyB | BsaI\_HlyB\_f | BsaI\_HlyB\_r | BBa\_K554013 | 18090901 | 184 | 3800 |
| Bsa\_TolC | BsaI\_TolC\_f | BsaI\_TolC\_r | BBa\_K554013 | 18090902 | 138 | 1300 |
| Bsa\_OmpT | BsaI\_OmpT\_f | BsaI\_OmpT\_r | 180827LP01 | 18090903 | 177 | 1000 |
| Bsa\_pBAD | BsaI-dopAP\_f | BsaI\_pBAD\_r | 180829LD01 | 18090904 | 143 | 200 |
| Bsa\_NOTMlcRE | BsaI-dopAP\_f | BsaI\_RnaG\_r | 180822BK02 | 18090905 | 101 | 540 |
| BjalT-OmpTSite-FLAG | BBPre\_Syn\_f | BBSuf\_Syn\_r | IDT(76546522) | 18090906 | 94 | 300 |
| RnaG120 | RnaG120\_f | RnaG120\_r | 180822BK02 | 18090907 | 76 | 120 |
| pGGA | pGGA\_Prep\_f | pGGA\_Prep\_r | pGGA(NEBKit) | 18090908 | 53 | 2170 |
| RnaG120+Terminator | BBPre\_Syn\_f | RnaG120\_r | 180822BK02 | 18090909 | 77 | 314 |

1% agarose gel à all successful

PCR purification with thermos-kit à at -20 °C

GG Assembly after 180909 GGA

Transformation following the protocol by Zhang *et al.*

remaining ligation approaches (10 µL) were stored at -20 °C

180909DW01 psB1C3\_BBa\_B0034\_AOX1

180909DW02 psB1C3\_BBa\_K206000\_BBa\_B0034\_AOX1

180909DW03 psB1C3\_BBa\_K206000\_BBa\_B0031\_SulA

180909DW04 psB1C3\_BBa\_K206000\_BBa\_B0032\_SulA

180909DW05 psB1C3\_BBa\_K206000\_BBa\_B0034\_SulA

180909DW06 psB1C3\_BBa\_B0032-cspD

180909DW07 pGGA\_pBAD\_HlyB-HlyD-TolC-OmpT

180909DW08pGGA\_NotMlcRE-HlyB-HlyD-TolC-OmpT

## GG Assembly

Procedure for the GGA:

1. PCR with Q5, with E. coli as a template or the existing parts
2. GGA where pGGA (Vector with 2174 bp), Assembly Mix und Buffer, DNA are used (fill with water). The DNA volume of the 3 to 7 inserts depends on the part’s length (in bp) and its density (in ng/µL)
3. Transformation, cPCR, mini, cryos, sequencing, characterization

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| **Overview** |  | Part 1 | Part 2 | Part 3 | Part 4 | Part 5 | Part 6 |
| Growth Inhbition | pBAD | cbtA | cspD | mraZ | sulA |  |  |
| Myristic Acid | pBAD | accA | accB | accC | accD | tesA |  |
| 3MB | pBAD | ADH2 | kivD | LeuA | LeuB | LeuC | LeuD |
| Toxin Synthesis | pBAD | BjaIT | HlyA |  |  |  |  |
| Excretion System | pBAD | HlyB | HlyD | TolC | OmpT |  |  |
|  |  |  |  |  |  |  |  |
| **Calculation** | length (bp) | density (ng/µL) | volume (µL) | Excretion |  |  |  |
| Promotor | 161 | 143 | 0.2 | pBAD |  |  |  |
| Part 1 | 3872 | 184 | 1.45194192 | HlyB+HlyD |  |  |  |
| Part 2 | 1296 | 138 | 0.64797408 | TolC |  |  |  |
| Part 3 | 975 | 177 | 0.38006954 | ompT |  |  |  |
| Buffer |  |  | 2 |  |  |  |  |
| Mix |  |  | 1 |  |  |  |  |
| pGGA |  |  | 1 |  |  |  |  |
| H2O |  |  | 13.3200145 |  |  |  |  |
|  |  |  |  |  |  |  |  |
| **Calculation** | length (bp) | density (ng/µL) | volume (µL) | Excretion |  |  |  |
| Promotor | 161 | 101 | 0.2 | NotMlcRE |  |  |  |
| Part 1 | 3872 | 184 | 1.45194192 | HlyB+HlyD |  |  |  |
| Part 2 | 1296 | 138 | 0.64797408 | TolC |  |  |  |
| Part 3 | 975 | 177 | 0.38006954 | ompT |  |  |  |
| Buffer |  |  | 2 |  |  |  |  |
| Mix |  |  | 1 |  |  |  |  |
| pGGA |  |  | 1 |  |  |  |  |
| H2O |  |  | 13.3200145 |  |  |  |  |

Assembly protocol:

(37 °C, 1 min à 16 °C, 1min) 30 times

55 °C à 5 min

## cPCR of the ligation from 08.09.18 and 09.09.18

Colonies have grown on all plates.

|  |  |  |
| --- | --- | --- |
| PCR preparation | 1x | 26x |
| 10x Taq Buffer | 2 µL | 52 µL |
| dNTPs 10 mM | 0.4 µL | 10.4 µL |
| 50 mM MgCl2 | 0.6 µL | 15.6 µL |
| fw primer | 0.6 µL | 15.6 µL |
| rv primer | 0.6 µL | 15.6 µL |
| Taq polymerase | 0.1 µL | 2.6 µL |
| H2O ad 20 µL | 14.5 µL | 377 µL |

The PCR programme was carried out as follows.

|  |  |  |
| --- | --- | --- |
| Step | Temperature | Time [s] |
| 1 | 95 °C | 600 |
| 2 | 95 °C | 30 |
| 3 | 55 °C | 30 |
| 4 | 72 °C | 120 |
| go to step 2 (30x) |  |  |
| 5 | 72 °C | 300 |

Control of the cPCR is shown in the following table and the following image.

![](https://static.igem.org/mediawiki/2018/7/76/T--Hamburg--180910-1.JPG)

|  |  |  |  |
| --- | --- | --- | --- |
| Part | Number | Length | Gel |
| pSB1C3-BBa-K206000-BBa-B0032-sulA | 1-3 | 1040 bp | negative |
| pSB1C3-BBa-K206000-BBa-B0034-sulA | 4-6 | 1040 bp | negative |
| pSB1C3-BBa\_B0032-cspD | 7-9 | 603 bp | negative |
| pSB1C3-BBa\_K206000-BBa\_B0031-sulA | 10-12 | 1040 bp | positive |
| pSB1C3-BBa\_B0034-AOX1 | 13-15 |  | negative |
| pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | 16-18 |  | negative |
| pGGA-pBAD-HlyB-HlyD-TolC-OmpT | 19-21 | 6304 bp | negative |
| pSB1C3-BBa-K206000-BBa-B0034-AOX1 | 22-24 | 1675 bp | positive |

ONCs of the numbers 10 and 23 as well as from cryostock 180518BK06 were prepared.

The following samples were sent for sequencing.

|  |  |
| --- | --- |
| LIGHTrun tube | lab record Seq. ID 98ED24 | 180906NB04 |
| LIGHTrun tube | lab record Seq. ID 98ED24 | 180906NB03 |
| LIGHTrun tube | lab record Seq. ID 98ED24 | 180906LP02 |
| LIGHTrun tube | lab record Seq. ID 98ED24 | 180906NB05 |
| LIGHTrun tube | lab record Seq. ID 98ED24 | 180906NB06 |

## Colony PCR of GGA (from the 9th of September)

A cPCR of two cell cultures (DH5α) with both: a) pGGA-NotMIcRE-HlyB-HlyD-TolC-OmpT and b) pGGA-pBAD-HlyB-HlyD-TolC-OmpT (from the 9th of September) were prepared according to the table below to verify their presence. Due to the extreme length of the constructs, the insert was split in two using two primer pairs per construct.

PCR-mixture, for 9 colonies per plate and 2 PCR-preparations per colony (two different primer pairs)

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | | 1x | Mastermix (38x) (2 preparations buffer) | MM primer pair 1 | MM primer pair 2 |
| 10x DreamTaq Buffer | | 2 µL | 76 µL |  |  |
| dNTPs 10 mM | | 0.4 µL | 15.2 µL |  |  |
| 50 mM MgCl2 | | 1.2 µL | 45.6 µL |  |  |
| Primer 1 | GGA\_cPCR\_fw | 0.6 µL per primer (1 primer pair) | / | 11.4 µL | / |
| BSAI\_HlyB=rev | / | 11.4 µL | / |
| Primer 2 | BSAI\_TolC\_fw | / | / | 11.4 µL |
| GGA\_cPCR\_rev | / | / | 11.4 µL |
| DreamTaq Polymerase | | 0.1 µL | / | 1.9 µL | 1.9 µL |
| H2O ad final volume (1x: 20 µL) | | 15.1 µL | 573.8 µL | \*      ↓  380 µL  ↓  18 x 20 µL | \*      ↓  380 µL  ↓  18 x 20 µL |

= 36 PCR mixtures in total

\* (an arrow pointed from the 573.8 µL onto the marked fields saying “split between 2 master mixes (á 355.3 µL)”)

- 9 colonies have been picked per plate. Each colony has been dipped into the PCR mixture containing the first primer pair, and then into the mixture containing the second primer pair. The tips were then transferred into 1 mL of LB (+ CAMP) and incubated for 10 minutes at 37 °C.

(- The PCR program is described on the next page)

## Miniprep and Cryostocks of the ONCs (from the 10th of September)

→ Cryostocks of positive colonies (cPCR)

pSB1C3-K206000-31sulA → 1809//LP01

pSB1C3-K206000-34AOX1 → 1809//LP02

→ Minipreps: 1809//LP03, LP04

cPCR - program

|  |  |  |
| --- | --- | --- |
| Step | Temperature | Time [s] |
| 1 | 95 °C | 600 |
| 2 | 95 °C | 30 |
| 3 | 55 °C | 30 |
| 4 | 72 °C | 180 |
| go to step 2 (30x) |  |  |
| 5 | 72 °C | 600 |

Following lengths were expected:

GGA-NOTMIcRE-HlyB-HlyD-TolC-OmpT

               PCR-mixture of primer pair 1: 2700 bp → wrong, because BSAHlyB\_rev starts behind HlyD

               PCR-mixture of primer pair 2: 2500-2600 bp

→ The same lengths were expected for GGA-pBAD-HlyB-HlyD-TolC-OmpT.

→ An ONC of pGGA-NotMIcRE-HlyB-HlyD-TolC -OmpT, colony 3: in LB + Camp, 37 °C, overnight was prepared.

## Restriction/Ligation

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Part | pSB1C3\_RNAG120\_BBa-E0840 | | pSB1C3-BBa-E0840 | BBa\_J45503 | pSB1C3-oHybB | 34-AOX1 |
| Lot # | 180822BK02 | 180907NF03 | 180907NF03 | 180625NF12 | 180911LP03 | 180906LP01 |
| C | 119 ng/μL | 92 ng/μL | 92 ng/ μL | 45 ng/ μL | 69 ng/ μL | 84 ng/ μL |
| Use | vector | vector | vector | single insert | vector | single insert |
| Target m | 2000 ng | | 2000 ng | 100 ng | 2000 ng | 100 ng |
| DNA | 5.70 μL | 21.81 μL | 17.30 μL | 2.24 μL | 25.00 μL | 4.80 μL |
| NEBuffer 2.1 | 1.00 μL | 3.00 μL | 3.00 μL | 1.00 μL | 3.00 μL | 4.00 μL |
| EcoRI-HF | - | - | 0.5 μL | 0.5 μL | - | - |
| XbaI | - | 0.5 μL | 0.5 μL |  | - | 0.5 μL |
| SpeI | 0.5 μL | - | - | 0.5 μL | 0.5 μL |  |
| PstI | 0.5 μL | 0.5 μL | - | - | 0.5 μL | 0.5 μL |
| FastAP | 0.5 μL | 0.5 μL | 0.5 μL |  | 0.5 μL |  |
| H2O | 1.80 μL | 3.69 μL | 8.20 μL | 5.76 μL | 0.5 μL | 30.20 μL |
| CFinal | 68 ng/ μL | 67 ng/ μL | 10 ng/ μL | 10 ng/ μL | 10 ng/ μL | 10 ng/ μL |

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Part | pSB1C3\_K20600 | | | 31SulA | 32SulA | 34SulA | cspD | pSB1C3-BBa\_B0032 |
| Lot # | 180829LD01 | | 180905AW01 | | 180905AW02 | 180905AW03 | 180905AW05 | 180514NB07 |
| C | 115 ng/μL | | 28 ng/μL | | 40 ng/ μL | 43 ng/ μL | 48 ng/ μL | 81 ng/ μL |
| Use | vector | | single insert | | single insert | single insert | single insert | vector |
| Target m | 2000 ng | 100 ng | | | 100 ng | 100 ng | 100 ng | 2000 ng |
| DNA | 4.88 μL | | 3.57 μL | | 2.5 μL | 2.33 μL | 2.08 μL | 6.10 μL |
| NEBuffer 2.1 | 1.00 μL | | 1.00 μL | | 1.00 μL | 1.00 μL | 1.00 μL | 1.00 μL |
| EcoRI-HF | - | | - | | - | - | - | - |
| XbaI | - | | 0.5 μL | | 0.5 μL | 0.5 μL | 0.5 μL | - |
| SpeI | 0.5 μL | | - | | - | - | - | 0.5 μL |
| PstI | 0.5 μL | | 0.5 μL | | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL |
| FastAP | 0.5 μL | | - | | - |  | - | 0.5 μL |
| H2O | 2.62 μL | | 4.43 μL | | 5.5 μL | 567 μL | 5.92 μL | 1.40 μL |
| CFinal | 6 ng/ μL | | 10 ng/ μL | | 10 ng/ μL | 10 ng/ μL | 10 ng/ μL | 10 ng/ μL |
|  |  |  |  |  |  |  |  |  |

|  |  |  |
| --- | --- | --- |
| Part | pSB1C3 | pSB1C3-I13453 |
| Lot # | 180906LP02 | 180822BK01 |
| C | 138 ng/μL | 115 ng/μL |
| Use | vector | vector |
| Target m | 2000 ng | 2000 ng |
| DNA | 14.49 μL | 16.12 μL |
| NEBuffer 2.1 | 2.00 μL | 2.00 μL |
| EcoRI-HF | - | - |
| XbaI | 0.5 μL | - |
| SpeI | - | 0.5 μL |
| PstI | 0.5 μL | 0.5 μL |
| FastAP | 0.5 μL | 0.5 μL |
| H2O | 2.01 μL | 0.38 μL |
| CFinal | 100 ng/ μL | 7 ng/ μL |

Ligations

|  |  |  |  |
| --- | --- | --- | --- |
| Tube product | pSB1C3\_RNAG120\_MIcRE\_BBa-E0840 | pSB1C3-BBa-E0840\_BBa\_J45503 | pSB1C3\_oHybB-34-AOX1 |
| vector | pSB1C3\_RNAG120\_MIcRe (68 ng/ μL, 0.35 μL) | pSB1C3-BBa-E0840 (10 ng/ μL, 4.29 μL) | pSB1C3\_oHybB (10 ng/ μL, 1.55 μL) |
| Part 1 | BBa-E0840 (67 ng/ μL, 1.15 μL) | BBa\_J45503 (10 ng/ μL, 5.71 μL) | 41-AOX1 (10 ng/ μL, 8.38 μL) |
| Part 2 | - | - |  |
| T4 Buffer | 1.00 μL | 2.00 μL | 2.00 μL |
| T4 Ligase | 0.5 μL | 0.5 μL | 0.5 μL |
| H2O | 7.00 μL | 7.5 μL | 7.57 μL |
| ad | 10.0 μL | 20.00 μL | 20.00 μL |

|  |  |  |  |
| --- | --- | --- | --- |
| Tube product | pSB1C3\_mraz | pSB1C3-BBa- K20600-32SulA | pSB1C3-BBa- K20600-34SulA |
| vector | pSB1C3 (74 ng/ μL, 0.61 μL) | pSB1C3-BBa-k20600 (6 ng/ μL, 4.25 μL) | pSB1C3-BBa-k20600 (6 ng/ μL, 4.25 μL) |
| Part 1 | mraz (10 ng/ μL, 10.0 μL) | 32-SulA (10 ng/ μL, 7.20 μL) | 34-SulA (10 ng/ μL, 7.20 μL) |
| Part 2 | - | - |  |
| T4 Buffer | 2.00 μL | 2.00 μL | 2.00 μL |
| T4 Ligase | 0.5 μL | 0.5 μL | 0.5 μL |
| H2O | 6.89 μL | 7.5 μL | 5.63 μL |
| ad | 20.0 μL | 20.00 μL | 20.00 μL |

|  |  |  |  |
| --- | --- | --- | --- |
| Tube product | pSB1C3\_BBa-80032-cspD | pSB1C3-34-AOX1 | pSB1C3-I13453-34-AOX1 |
| vector | pSB1C3\_BBa\_80023 (8 ng/ μL, 5.74 μL) | pSB1C3 (100 ng/ μL, 0.15 μL) | pSB1C3-I13453 (7 ng/ μL, 42.21 μL) |
| Part 1 | cspD (10 ng/ μL, 5.40 μL) | 34-AOX1 (10 ng/ μL, 8.46 μL) | 34-AOX1 (10 ng/ μL, 8.38 μL) |
| Part 2 | - | - | - |
| T4 Buffer | 2.00 μL | 2.00 μL | 2.00 μL |
| T4 Ligase | 0.5 μL | 0.5 μL | 0.5 μL |
| H2O | 6.35 μL | 6.89 μL | 6.90 μL |
| ad | 20.0 μL | 20.00 μL | 20.00 μL |

|  |  |
| --- | --- |
| Tube product | pSB1C3\_oHybB-34-AOX1 |
| vector | pSB1C3\_oHybB (10 ng/ μL, 1.55 μL) |
| Part 1 | 34-AOX1 (10 ng/ μL, 8.38 μL) |
| Part 2 | - |
| T4 Buffer | 2.00 μL |
| T4 Ligase | 0.5 μL |
| H2O | 7.57 μL |
| ad | 20.0 μL |

* Gelextraction using GeneJetKit of Restrictions 3, 5, 9, 14, 16
* Problems: 1 could not be detected on the gel, 2 did not show the right length on the gel

## Miniprep + Cryostock

* Of the positive cPCR colony (11.9.)

180912LP01: pGGA-Not-MIcRE-HyB-HyD-TolC-OmpT

* Dissolve in 1:1 Glycerol (50 %), store at -80 °C in DH5α

180912LP02: pGGA-Not-MIcRE-HyB-HyD-TolC-OmpT

* Miniprep: c = 129.25 ng/μL, stored at -20 °C
* Heat shock transformation according to protocol on p. 161

## Colony PCR of the transformation performed on the 12th of September

Mastermix

|  |  |  |
| --- | --- | --- |
| Substance | 1x (volume in µl) | 40x (volume in µl) |
| 10x DreamTaq Buffer | 2 | 80 |
| dNTPs 10mM | 0.4 | 16 |
| MgCl2 50mM | 1.2 | 48 |
| Fw-Primer VF2 | 0.6 | 24 |
| Rv-Primer VR | 0.6 | 24 |
| Taq-Polymerase | 0.1 | 4 |
| H2O ad20µl | 15.1 | 604 |

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Programm (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 95 | 180 |
| 95 | 30 |
| 55 | 30 |
| 72 | 120 |
| 72 | 600 |

180913 cPCR.PNG

The colonies of following bands were used to set up ONCs.

|  |  |  |
| --- | --- | --- |
| Band | Part | Length (bp) |
| 6 | pSB1C3-BBa\_E0840-BBa\_J45503 | 1571 |
| 10 | pSB1C3-mraz | 758 |
| 18 | pSB1C3-BBa\_K206000-BBa\_B0032-sulA | 995 |
| 31 | pSB1C3-BBa\_B0032-cspD | 558 |

## Set up of minipreps and cryostocks of the ONCs performed on the 13th of September

The minipreps were set up using the “GeneJet Plasmid Miniprep Kit” from Thermo Fisher.

## PCR of linear pSB1C3

Mastermix

|  |  |  |
| --- | --- | --- |
| Substance | 1x (volume in µl) | 3x (volume in µl) |
| 5x HF Buffer | 10 | 30 |
| DNA-Template | 0.5 | 1.5 |
| F-Primer | 0.35 | 1.05 |
| R-Primer | 0.35 | 1.05 |
| Phusion-Polymerase | 0.5 | 1.5 |
| H2O ad 50µl | 37.3 | 111.9 |

PCR-Programm (35 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 98 | 120 |
| 98 | 30 |
| 55 | 30 |
| 72 | 180 |
| 72 | 600 |

The 3 set ups were purified with the “GeneJet PCR-Purification Kit” of Thermo Fisher and eluted in 100 µl ddH2O.

C = 89 ng/µl

## Restriction and ligation

Restriction

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Part | pSB1C3-NOT-MlcRE | pSB1C3-BBa\_Eo840 | pSB1C3-oHybB | pSB1C3-K206000 | pSB1C3 | BBa\_B0034-AOX1 | BBa\_B0034-sulA |
| DNA | 38.46 µl | 30.77 µl | 22 µl | 34.48 µl | 22.47 µl | 2.4 µl | 2.33 µl |
| NEBBuffer 2.1 | 5 µl | 4 µl | 4 µl | 4 µl | 3 µl | 2 µl | 1 µl |
| EcoRI-HF | - | - | - | - | - | - | - |
| XbaI | - | 0.5 µl | - | - | 0.5 µl | 0.5 µl | 0.5 µl |
| SpeI | 0.5 µl | - | 0.5 µl | 0.5 µl | - | - | - |
| PstI | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl |
| FastAP | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl | - | - |
| ddH2O | 5.04 µl | 3.73 µl | 5.51 µl | - | 3.03 µl | 14.6 µl | 5.67 µl |
| ad | 50 µl | 40 µl | 40 µl | 40 µl | 30 µl | 20 µl | 10 µl |

The samples were incubated at 37°C for 1 h and following 20 min at 80°C. Samples 1-4 were applied on a 1% agarose gel and extracted through the “GeneJet Gel Extraction Kit” of Thermo Fisher.

![](https://static.igem.org/mediawiki/2018/c/c6/T--Hamburg--180914_Restriktion.JPG)

Ligation

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Product | pSB1C3-NOT-MlcRE-BBa\_E0840 | pSB1C3-oHybB-BBa\_B0034-AOX1 | pSB1C3-BBa\_K206000-BBa\_B0034-sulA | pSB1C3-BBa\_B0034-AOX1 |
| Vector | 1.46 µl | 0.44 µl | 1.82 µl | 0.24 µl |
| Part | 8.5 µl | 8.25 µl | 10 µl | 8.35 µl |
| T4 Buffer | 2 µl | 2 µl | 2 µl | 2 µl |
| T4 Ligase | 0.5 µl | 0.5 µl | 0.5 µl | 0.5 µl |
| ddH2O | 7.54 µl | 8.81 µl | 5.68 µl | 8.91 µl |
| ad | 20 µl | 20 µl | 20 µl | 20 µl |

The samples were incubated at 16°C overnight.

## Set up of ONCs

The pSB1C3-oHybB cryostock was used to set up an ONC.

## Template preparation for GGA by PCR

All PCR Reactions were prepared as written below.

|  |  |
| --- | --- |
| 8 µL | H2O |
| 0.2 µL | Template |
| 1 µL | Fw-Primer |
| 1 µL | Rv-Primer |
| 10 µL | Q5 High-Fidelity 2X Master Mix |

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Tube | Part | Fw-Primer | Rv-Primer | Predicted length (bp) | Template |
| 1 | BsaI-pBAD | BsaI-pBAD-f | BsaI-pBAD-r | 186 | 180909BK04 |
| 2 | BsaI-NOTMlcRE | BsaI-NOTMlcRE-f | BsaI-NOTMlcRE-r | 549 | 180909BK05 |
| 3 | BsaI-HlyB | BsaI-HlyB-f | BsaI-HlyB-r | 3895 | 180909BK01 |
| 4 | BsaI-TolC | BsaI-TolC-f | BsaI-TolC-r | 1322 | 180909BK02 |
| 5 | BsaI-OmpT | BsaI-OmpT-f | BsaI-OmpT-r | 1001 | 180909BK03 |
| 6 | BsaI-ADH2 | BsaI-ADH2-f | BsaI-ADH2-r | 1100 | 180726LP03 |
| 7 | BsaI-kivD | BsaI-kivD-f | BsaI-kivD-r | 1686 | 180726LP01 |
| 8 | BsaI-LeuA | BsaI-LeuA-f | BsaI-LeuA-r | 1033 | e. coli |
| 9 | BsaI-LeuB | BsaI-LeuB-f | BsaI-LeuB-r | 1115 | e. coli |
| 10 | BsaI-LeuC | BsaI-LeuC-f | BsaI-LeuC-r | 1441 | e. coli |
| 11 | BsaI-LeuD | BsaI-LeuD-f | BsaI-LeuD-r | 664 | e. coli |
| 12 | BsaI-accA | BsaI-accA-f | BsaI-accA-r | 1017 | e. coli |
| 13 | BsaI-accB | BsaI-accB-f | BsaI-accB-r | 515 | 180703BK05 |
| 14 | BsaI-accC | BsaI-accC-f | BsaI-accC-r | 1384 | 180726LP04 |
| 15 | BsaI-accD | BsaI-accD-f | BsaI-accD-r | 951 | IDT syn |
| 16 | BsaI-tesA | BsaI-tesA-f | BsaI-tesA-r | 705 | e. coli |

PCR was performed using following Cycler-Protocol

|  |  |  |  |
| --- | --- | --- | --- |
| Temperature (°C) | Time | Time (for e. coli templates) | Repeats |
| 98 | 0:20 | 2:00 | 1 |
| 98 | 0:10 | 0:10 | 30 |
| 60 | 0:20 | 0:20 | 30 |
| 72 | 1:00 | 1:00 | 30 |
| 72 | 2:00 | 2:00 | 1 |
| 4 | ∞ | ∞ | 1 |

A gel electrophoresis was performed using a 1%-Agarose-Gel.

![](https://static.igem.org/mediawiki/2018/4/40/T--Hamburg--180915_PCR_GGA.JPG)

Only BsaI-ADH2 was successful.

## Restriction and ligation of basic parts

Restriction was set up according to following table, performed at 37 °C for an hour and heat inactivated at 80 °C for 20 minutes.

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** | **4** | **5** | **6** |
|  | Part | accA | accB | icsA | RnaG120 | BjaIT-OmpTSite-FLAG | pSB1C3 |
|  | Lot# | 180622NB05 | 180622NB06 | 180909BK07 | 180909BK09 | 180909BK08 | 180914LP01 |
|  | **ρ** | 49 ng/µL | 52 ng/µL | 76 ng/µL | 77 ng/µL | 94 ng/µL | 89 ng/µL |
|  | Use |  |  |  |  |  |  |
|  | target m |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |
| Volumes | DNA | 2.04 µL | 1.92 µL | 1.32 µL | 1.30 µL | 1.06 µL | 3.00 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| XbaI |  |  |  |  |  |  |
| SpeI |  |  |  |  |  |  |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP |  |  |  |  |  |  |
| H2O | 5.96 µL | 6.08 µL | 6.68 µL | 6.70 µL | 6.94 µL | 5.00 µL |
|  | ad | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |
|  | Länge Produkt |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |
|  | ρ final | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 27 ng/µL |

Ligation was set up according to following table and performed at 37 °C for an hour.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** |
| Product | | pSB1C3-accA | pSB1C3-accB | pSB1C3-icsA | pSB1C3-RnaG120 | pSB1C3-BjaIT-OmpTSite-FLAG |
|  | |  |  |  |  |  |
| **Vector** | Vector | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 |
| Tube | **6** | **6** | **6** | **6** | **6** |
| ρ | 27 ng/µL | 27 ng/µL | 27 ng/µL | 27 ng/µL | 27 ng/µL |
| Länge | 2046 bp | 2046 bp | 2046 bp | 2046 bp | 2046 bp |
| V | 0.64 µL | 1.09 µL | 2.36 µL | 1.48 µL | 1.52 µL |
| **Part 1** | Part 1 | accA | accB | icsA | RnaG120 | BjaIT-OmpTSite-FLAG |
| Tube | **1** | **2** | **3** | **4** | **5** |
| ρ | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |
| Länge | 1000 bp | 500 bp | 120 bp | 314 bp | 300 bp |
| V | 7.30 µL | 7.10 µL | 3.70 µL | 6.05 µL | 5.95 µL |
| **Part 2** | Part 2 |  |  |  |  |  |
| Tube |  |  |  |  |  |
| ρ |  |  |  |  |  |
| Länge |  |  |  |  |  |
| V |  |  |  |  |  |
| T4 Buffer | | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 0.56 µL | 0.32 µL | 2.44 µL | 0.97 µL | 1.04 µL |
| ad | | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |

## Transformation of the ligations perfomed on the 14th and 15th of September

19. 150 µl Competent cell aliquots were thawed on ice for 15-30 min.
20. 10 µl DNA were added and mixed
21. Cells were incubated for 35 min on ice
22. Heat-shock: 42°C for 45 sec, then put back on ice for 2-5 min
23. Pre-warm 850 µl LB-Medium
24. Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
25. Cetrifuge 4500 rpm 2 min to collect cells.
26. Put cells on appropriate plates or medium
27. Incubation at 37°C overnight.

## Colony PCR of the transformation performed on the 9th of September

Mastermix

|  |  |  |
| --- | --- | --- |
| Substance | 1x (volume in µl) | 20x (volume in µl) |
| 10x DreamTaq Buffer | 2 | 40 |
| dNTPs 10mM | 0.4 | 8 |
| MgCl2 50mM | 0.6 | 12 |
| Fw-Primer VF2 | 0.6 | 12 |
| Rv-Primer VR | 0.6 | 12 |
| Taq-Polymerase | 0.1 | 2 |
| ddH2O ad 20µl | 15.7 | 314 |

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Programm (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 95 | 180 |
| 95 | 30 |
| 55 | 30 |
| 72 | 360 |
| 72 | 600 |

![](https://static.igem.org/mediawiki/2018/b/bb/T--Hamburg--180916_cPCR_GGA.JPG)

Two ONCs of the part pSB1C3-NOT-MlcRE-HlyB-HlyD-TolC-OmpT were set up.

## Set up of Minipreps and cryostocks of the ONCs set up on the 16th of September

The ONCs were purified with the “GeneJet Plasmid Miniprep Kit” of Thermo Fisher.

## Colony PCR of the transformation performed on the 16th of September

MasterMix

|  |  |  |
| --- | --- | --- |
| Substance | 1x (volume in µl) | 40x (volume in µl) |
| 10x DreamTaq Buffer | 2 | 80 |
| dNTPs 10mM | 0.4 | 16 |
| MgCl2 50mM | 0.6 | 24 |
| Fw-Primer VF2 | 0.6 | 24 |
| Rv-Primer VR | 0.6 | 24 |
| Taq-Polymerase | 0.1 | 4 |
| ddH2O ad 20µl | 15.7 | 628 |

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Program (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 95 | 180 |
| 95 | 30 |
| 55 | 30 |
| 72 | 360 |
| 72 | 600 |

![](https://static.igem.org/mediawiki/2018/1/1d/T--Hamburg--180917_cPCR.JPG)

The colonies of following bands were used to set up ONCs.

|  |  |
| --- | --- |
| Band | Part |
| 3 | pSB1C3-accA |
| 8 | pSB1C3-accB |
| 11 | pSB1C3-icsA |
| 16 | pSB1C3-RnaG120 |
| 19 | pSB1C3-BjaIT-OmpTSite-FLAG |
| 25 | pSB1C3-oHybB-BBa\_B0034-AOX1 |

## Preparation of competent cells

1. Grow the cells at 37 till OD=0.2-0.4
2. Chill the culture on ice for 5 min
3. Collect cells by centrifugation. 6000rpm, 10 min, 4°C
4. Gently resuspend the cells from 500 ml LB in 40 ml Ca/glycerol buffer on ice.
5. Incubate the cells on ice for 5-30 min
6. Repeat the steps 3 and 4
7. Incubate the resuspended cells in Ca/glycerol buffer on ice for 30 min
8. Collect cells by centrifugation. 6000rpm, 10 min, 4°C
9. Resuspend cells from 500 ml LB in 6 ml Ca/glycerol buffer
10. Aliquot 150 µl/tube
11. Freeze in liquid-N2 and store at -80°C

## Test-digest for sequencing

Following parts were digested:

pSB1C3-K206000-BBa\_B0031-sulA

pSB1C3-J45503-E0840

pSB1C3-mraz

pSB1C3-K206000-BBa\_B0032-sulA

pSB1C3-BBa\_B0032-cspD

Mastermix

|  |  |
| --- | --- |
| Substance | 1x (volume in µl) |
| DNA-Template | 5 |
| EcoRI | 0.5 |
| FastAP | 0.5 |
| NEBBuffer 2.1 | 1 |
| ddH2O | 3 |

All probes were applied to a 1% agarose gel.

![](https://static.igem.org/mediawiki/2018/c/cb/T--Hamburg--180917_Restriktionsverdau.JPG)

All probes seemed fine and were sent in for sequencing.

## Set up of Minipreps and cryostocks of the ONCs set up on the 17th of September

The ONCs were purified with the “GeneJet Plasmid Miniprep Kit” of Thermo Fisher.

## Colony PCR of the transformed pSB1C3-NOT-MlcRE

MasterMix

|  |  |
| --- | --- |
| Substance | 1x (volume in µl) |
| 10x DreamTaq Buffer | 2 |
| dNTPs 10mM | 0.4 |
| MgCl2 50mM | 0.6 |
| Fw-Primer VF2 | 0.6 |
| Rv-Primer VR | 0.6 |
| Taq-Polymerase | 0.1 |
| ddH2O ad 20µl | 15.7 |

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Program (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 95 | 180 |
| 95 | 30 |
| 55 | 30 |
| 72 | 150 |
| 72 | 180 |

![](https://static.igem.org/mediawiki/2018/0/0e/T--Hamburg--Img_2018.09.18_16_04_20.JPG)

The colony of band 2 was used to set up an ONC.

## Interlab study Marburg – set up of media

12. 500 ml LB + V2 salt
    1. 450 ml autoclaved LB-Media plus 50 ml V2 salt (204 mM NaCl, 4.2 mM KCl, 23.14 mM MgCl2)
13. 500 ml LB + V2 salt + 2 µg/ml Chloramphenicol
14. 500 ml LB + V2 salt + agarose (1%)
15. Electroporation buffer:
    2. 200 ml 2.5 x electroporation buffer (680 mM sucrose, 7mM K2HPO4 pH7)
    3. 300 ml ddH2O

## Repitition of the PCR of sulA from 180904

Mastermix:

* 10 µL 5x Phusion buffer
* 1 µL dNTPs
* 1 µL DNA (E. coli)
* 5 µL Phusion polymerase
* 1 µL Primer 34-sulA-Fw
* 1 µL Primer sulA-suf-rv
* 5 µL H2O (ad. 50 µL)

PCR program:

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 98 | 180 |
| 98 | 10 |
| 55 | 30 |
| 72 | 60 |
| 72 | 300 |

## Gelelectrophoresis

We made a 1% agarose gel. It ran for 45 min at 80 V.

![](https://drive.google.com/file/d/1ViL-OZxjautsQkGGPTJcColZy_CAgBbn/view)

There was no band in the gel, so the PCR of 34sulA were repeated.

## Repetition of the PCR of 34sulA

Mastermix for sulA:

* 10 µL 5x Phusion buffer
* 1 µL dNTPs
* 1 µL DNA (E. coli)
* 5 µL Phusion polymerase
* 1 µL Primer 34-sulA-Fw
* 1 µL Primer sulA-suf-rv
* 34 µL H2O (ad. 50 µL)
* 5 µL DMSO

Mastermix for AOX1:

* 10 µL 5x Phusion buffer
* 1 µL dNTPs
* 1 µL DNA (BB-Pre-ompA-Aox1a-BB-suf)
* 1 µL Primer xba-34-Aox1-f
* 1 µL Primer suf-Syn-r
* 5 µL DMSO
* 34 µL H2O
* 5 µL Phusion polymerase

PCR program for sulA:

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 98 | 180 |
| 98 | 10 |
| 55 | 30 |
| 72 | 60 |
| 72 | 300 |

PCR program for Aox1:

|  |  |
| --- | --- |
| 98°C | 2 min |
| 98°C | 10 s |
| 55°C | 20 s |
| 72°C | 30 s |
| Go to step 2 30x | |
| 72°C | 2 min |

## Gelelectrophoresis

We made a 1% agarose gel. It ran for 45 min at 80 V.

![](https://drive.google.com/file/d/17U_08m8fm4v40C0NERNyeeW4N7jFZEV5/view)

## MiniPreps

The miniprep was made with the GeneJET Plasmid Miniprep Kit by Thermo Fischer.

|  |  |  |
| --- | --- | --- |
| Part | Lot number | DNA-concentration (ng/µL) |
| pSB1C3-I13500 | 180919AK01 | 151.75 |

## Test digestion of the sequencing parts

|  |  |  |
| --- | --- | --- |
| Tube | Part | Sequencing ID |
| 1 | 180918AK09 | 47GJ15 |
| 2 | 180918AK10 | 47GJ16 |
| 3 | 180918AK11 | 47GJ18 |
| 4 | 180918AK12 | 47GJ19 |
| 5 | 180918AK13 | 47GJ20 |
| 6 | 180918AK14 | 47GJ22 |
| 7 | 180917K03 | - |
| 8 | 180917K04 | - |
| 9 | 180726LP01 | 47GJ23 |
| 10 | 180726LP02 | empty |
| 11 | 180726LP03 | 47GJ27 |
| 12 | 180726LP04 | - |
| 13 | 180803NB02 | 47GJ29 |
| 14 | 180914AW01 | 47GJ31 |
| 15 | 180914AW02 | 47GJ32 |

Master mix for 16 samples:

* 2 µL DNA
* 2 µL EcoR1
* 2 µL FastAP
* 16 µL NEB 2.1
* 96 µL H2O

Incubated the samples for 1 h at 37°C.

## Gelelectrophoresis

We made a 1% agarose gel. It ran for 45 min at 100 V.

![](https://drive.google.com/file/d/1OosOhqS4KvP8sp6BtcsaPELP5FX2Znu7/view)

## Set up overnight cultures

Set up overnight cultures with LB-medium and added Camp.

|  |  |
| --- | --- |
| Lot number | Part |
| 180727DK02 | pSB1C3-OmpA-Aox1 |
| 180917AK01 | pGGA-NOTMlcRE-HlyB-HlyD-TolC-OmpT |
| 180917AK02 | pGGA-NOTMlcRE-HlyB-HlyD-TolC-OmpT |

## Proofed the sequences 98ED29 – 98ED34 from 180919

Mastermix for AOX1:

* 10 µL 5x Phusion buffer
* 1 µL dNTPs
* 1 µL DNA (BB-Pre-ompA-Aox1a-BB-suf)
* 1 µL Primer xba-34-Aox1-f
* 1 µL Primer suf-Syn-r
* 5 µL DMSO
* 34 µL H2O
* 5 µL Phusion polymerase

PCR program: 180827iGEM

## Miniprep of 180727DK02 and 180917AK02

The miniprep was made with GeneJET Miniprep Plasmid Kit by Thermo Fischer. Minipreps stored at -20°C.

|  |  |  |  |
| --- | --- | --- | --- |
| Old Lot number | New Lot number | Part | DNA-concentration (ng/µL) |
| 180727DK02 | 180920OM01 | pSB1C3-ompA-Aox1 | 513.055 |
| 180917AK02 | 180920OM02 | pGGA-NOTMlcRE-HlyB-HlyD-TolC-OmpT | 770.954 |

## Set up overnight cultures for characterizations

The overnight cultures were mixed with LB-medium and Camp.

|  |  |
| --- | --- |
| Lot number | Part |
| 180803NB01 | BjalT |
| 180908NF01 | AOX1 |
| Plate Retrafo | BBa\_I13500 |

## Set up over night cultures for minipreps

Set up over night cultures for minipreps, because there were empty. Mixed with 6 mL LB-medium and 6 µL Camp. Incubated over night at 37°C.

|  |  |
| --- | --- |
| Lot number | Part |
| 180826LP03 | pSB1C3-E240 |
| 180518BKK06 | pSB1C3-oHybB |
| 180712BK04 | pSB1C3-NOTMlcRE |

## Restriction

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** |
|  | Part | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-E0240 | BBa\_J45503 | pSB1C3-oHybB | 34-AOX1 | pSB1C3-I13453 | BBa\_B0032\_cbtA |
|  | Lot# | 180914AW05 | 180817LD10 | 180817LD10 | 180625NiF12 | 180915BK01 | 180906LP01 | 180914AW06 | 180731NF04 |
|  | **ρ** | 118 ng/µL | 200 ng/µL | 200 ng/µL | 45 ng/µL | 170 ng/µL | 84 ng/µL | 115 ng/µL | 173 ng/µL |
|  | Use | vector | vector | vector | single insert | vector | single insert | vector | vector |
|  | target m | 2000 ng | 2000 ng | 2000 ng | 100 ng | 2000 ng | 100 ng | 2000 ng | 2000 ng |
|  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 6.77 µL | 10.00 µL | 6.40 µL | 2.24 µL | 18.00 µL | 4.80 µL | 16.12 µL | 11.56 µL |
| NEBuffer 2.1 | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 3.00 µL | 4.00 µL | 2.00 µL | 2.00 µL |
| EcoRI-HF |  |  | 0.50 µL | 0.50 µL |  |  |  |  |
| XbaI |  | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  | 0.50 µL |
| SpeI | 0.50 µL |  |  | 0.50 µL | 0.50 µL |  | 0.50 µL |  |
| PstI | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL |  | 0.50 µL | 0.50 µL |
| H2O | 1.80 µL | 6.50 µL | 1.10 µL | 5.76 µL | 7.50 µL | 30.20 µL | 0.38 µL | 4.94 µL |
|  | ad | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 30.00 µL | 40.00 µL | 20.00 µL | 20.00 µL |
|  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt |  |  |  |  | 2200 bp |  | 2200 | 394 |
|  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 10 ng/µL | 14 ng/µL | 10 ng/µL | 10 ng/µL | 102 ng/µL | 10 ng/µL | 17 ng/µL | 8 ng/µL |

## Ligation

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** |
| Product | | pSB1C3\_RNAG120\_MlcRE-pSB1C3-E0240 | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-BBa\_B0032\_cbtA |
|  | |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-I13453 | pSB1C3-I13453 |
| Tube | **1** | **7** | **7** |
| ρ | 10 ng/µL | 17 ng/µL | 17 ng/µL |
| Länge | 2697 bp | 2200 bp | 2200 bp |
| V | 4.70 µL | 0.91 µL | 2.11 µL |
| **Part 1** | Part 1 | pSB1C3-E0240 | 34-AOX1 | BBa\_B0032\_cbtA |
| Tube | **2** | **6** | **8** |
| ρ | 14 ng/µL | 10 ng/µL | 8 ng/µL |
| Länge | 878 bp | 1200 bp | 394 bp |
| V | 10.92 µL | 8.38 µL | 8.02 µL |
| **Part 2** | Part 2 |  |  |  |
| Tube |  |  |  |
| ρ |  |  |  |
| Länge |  |  |  |
| V |  |  |  |
| T4 Buffer | | 2.00 µL | 2.00 µL | 2.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 1.88 µL | 8.21 µL | 7.37 µL |
| ad | | 20.00 µL | 20.00 µL | 20.00 µL |

## Gelelectrophoresis of the PCR and the restricion

We made a 1% agarose gel. It ran for 45 min at 100 V.

![](https://drive.google.com/file/d/1c3DtaFo1_jKUN1zlURVUN4ODivDk43Ic/view)

## Heat shock transformation

The cells chilled 5 min on ice at 4°C. After that we added 10-20 µL DNA to the cells. Afterwards the cells had to incubate for 30 min on ice. Then we made a heat shock for 45 s at 42°C and added 800 µL LB-medium. After that the cells incubate for 30 min at 37°C and 700 rpm. 300 µL of the samples were plated and stored overnight at 37°C.

## PCR for the GGA-parts and the basic parts

The master mix wasn’t noted, also the PCR program was deleted.

## Gelelectrophoresis

We made a 1% agarose gel. It ran for 1 h at 100 V.

![](https://drive.google.com/file/d/1LZ1zd37PKSwENEZnZMEBg-IOKtcJdCWD/view)

## Minipreps of the over night cultures from 180920

|  |  |  |
| --- | --- | --- |
| Old Lot number | New Lot number | DNA-concentration (ng/µL) |
| 1802826LP03 | 190921OM01 | 123 |
| 180712BK04 | 190921OM02 | 113 |
| 180518BK06 | 190921OM03 | 186 |
| pSB1C3-I13500 | 190921OM04 | 112 |

## Charcterization of sulA

Inculated the following cultures with 200 mL LB medium and Camp.

* DH5α + Aabinose (10 µM)
* pSB1C3-K206000-34ldhA + Arabinose (10 µM)
* pSB1C3-K206000-34ldhA (without Arabinose)

The start-OD should be for all cultures 0.1.

OD after 7 h:

|  |  |
| --- | --- |
| Part | OD |
| DH5α | 5.36 |
| ldhA + Arabinose | 4.784 |
| ldhA without Arabinose | 5.776 |

50 mL of the cultures were put in falcon tubes and incubated for 20 min at 8°C and 5000 rpm. Then stored on ice at 4°C. The supernatant was discarded, and the pellets stored at -80°C.

## Preparing of polyacrylamid gel

|  |  |  |
| --- | --- | --- |
|  | seperation gel | gathering gel |
| acryl amide | 6.75 mL | 750 μL |
| seperation gel buffer | 4.5 mL | - |
| gathering gel buffer | - | 1.7 mL |
| H2O | 6.75 mL | 4.25 mL |
| 10 % APS | 60 μL | 20 μL |
| TEMED | 13.5 μL | 12 μL |

Added to the samples from 180921 with 100 µL SDS buffer with β-Mercaptoethanol. Incubated for 10 min at 95°C and stored at -20°C.

## Preparing SDS buffer 10x

* 3 g Tris
* 1 g Glycin
* 10 g SDS
* 1 L H2O
* Adjusted the pH to 8.4 with HCl (1 M)

## PCR of several parts

Mastermix for BBa\_B0031-sulA and BBa\_B0034-sulA

|  |  |
| --- | --- |
| Substance | 2x (volume in µl) |
| 5x Phusion HF Buffer | 20 |
| dNTPs 10mM | 2 |
| F-Primer | 0.7 |
| R-Primer | 0.7 |
| Phusion-Polymerase | 4 |
| DNA-Template | 2 |
| ddH2O ad 100µl | 70.6 |

PCR-Program (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 98 | 180 |
| 98 | 10 |
| 55 | 30 |
| 72 | 60 |
| 72 | 300 |

Mastermix for pSB1C3

|  |  |
| --- | --- |
| Substance | 1x (volume in µl) |
| 5x Phusion HF Buffer | 10 |
| dNTPs 10mM | 1 |
| F-Primer | 0.35 |
| R-Primer | 0.35 |
| Phusion-Polymerase | 0.5 |
| DNA-Template | 0.5 |
| ddH2O ad 50µl | 37.3 |

PCR-Program (35 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 98 | 120 |
| 98 | 30 |
| 55 | 30 |
| 72 | 180 |
| 72 | 600 |

PCR-Products were applied to a 1% agarose gel. PSB1C3 was cut out and purified by using the “GeneJet Gelextraction Kit” by Thermo Fisher.

## <https://static.igem.org/mediawiki/2018/2/24/T--Hamburg--180924_PCR.JPG>

## Restriction, ligation and transformation

Restriction

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Tube** | **1** | **2** | **5** | **6** | **9** | **12** | **16** | **17** |
| **Part** | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-oHybB | 34-AOX1 | pSB1C3-BBa\_K206000 | 34sulA | pSB1C3-I13453 | pSB1C3-BBa\_B0032\_cbtA |
| **Lot#** | 180921OM02 | 180921OM01 | 180921OM03 | 180920LP01 | 180829LD02 | 180905AW03 | 180914AW06 | 180731NF04 |
| **ρ** | 113 ng/µL | 123 | 186 ng/µL | 575 ng/µL | 50 ng/µL | 43 ng/µL | 67 ng/µL | 173.5 |
| Use | vector | vector | vector | single insert | vector | single insert | vector | vector |
| target m | 2000 ng | 2000 ng | 2000 ng | 100 ng | 2000 ng | 100 ng | 2000 ng | 2000 ng |
|  |  |  |  |  |  |  |  |  |
| DNA | 17.70 µL | 16.26 µL | 10.75 µL | 0.17 µL | 40.00 µL | 2.33 µL | 29.85 µL | 11.53 µL |
| NEBuffer 2.1 | 3.00 µL | 2.00 µL | 2.00 µL | 4.00 µL | 5.00 µL | 1.00 µL | 4.00 µL | 2.00 µL |
| EcoRI-HF |  |  |  |  |  |  |  |  |
| XbaI |  | 0.50 µL |  | 0.50 µL |  | 0.50 µL |  | 0.50 µL |
| SpeI | 0.50 µL |  | 0.50 µL |  | 0.50 µL |  | 0.50 µL |  |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL |  | 0.50 µL | 0.50 µL |
| H2O | 7.80 µL | 0.24 µL | 5.75 µL | 34.83 µL | 3.50 µL | 5.67 µL | 4.65 µL | 4.97 µL |
| ad | 30.00 µL | 20.00 µL | 20.00 µL | 40.00 µL | 50.00 µL | 10.00 µL | 40.00 µL | 20.00 µL |
|  |  |  |  |  |  |  |  |  |
| Länge Produkt |  |  | 2200 bp |  |  |  | 2200 | 394 |
|  |  |  |  |  |  |  |  |  |
| ρ final | 67 ng/µL | 100 ng/µL | 10 ng/µL | 3 ng/µL | 6 ng/µL | 10 ng/µL | 23 ng/µL | 100 ng/µL |

Probes were incubated for 1h at 37°C and following 20 min at 80°C.

Restriction 1.2,3.9,16 and 17 were applied to a 1% agarose gel and purified by using the “GeneJet Gel Extraction Kit” from Thermo Fisher.

![](https://static.igem.org/mediawiki/2018/0/0a/T--Hamburg--WunderschoenesGelbildFuerNici.png)

Restriction 2 and 17 didn’t work.

Ligation

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Product | | pSB1C3\_RNAG120\_MlcRE-pSB1C3-E0240 | pSB1C3-BBa\_K206000-34sulA | pSB1C3-I13453-34-AOX1 |
|  | |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-BBa\_K206000 | pSB1C3-I13453 |
| Tube | **1** | **9** | **16** |
| ρ | 67 ng/µL | 6 ng/µL | 23 ng/µL |
| Länge | 2697 bp | 2200 bp | 2200 bp |
| V | 0.35 µL | 4.25 µL | 0.67 µL |
| **Part 1** | Part 1 | pSB1C3-E0240 | 34sulA | 34-AOX1 |
| Tube | **2** | **12** | **6** |
| ρ | 100 ng/µL | 10 ng/µL | 3 ng/µL |
| Länge | 878 bp | 565 bp | 1200 bp |
| V | 0.77 µL | 7.20 µL | 33.80 µL |
| **Part 2** | Part 2 |  |  |  |
| Tube |  |  |  |
| ρ |  |  |  |
| Länge |  |  |  |
| V |  |  |  |
| T4 Buffer | | 1.00 µL | 2.00 µL | 4.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 7.38 µL | 5.63 µL | 1.02 µL |
| ad | | 10.00 µL | 20.00 µL | 40.00 µL |

Probes were incubated at 37°C for 1h.

The ligations were transformed into DH5α.

28. 150 µl Competent cell aliquots were thawed on ice for 15-30 min.
29. 10 µl DNA were added and mixed
30. Cells were incubated for 35 min on ice
31. Heat-shock: 42°C for 45 sec, then put back on ice for 2-5 min
32. Pre-warm 850 µl LB-Medium
33. Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
34. Cetrifuge 4500 rpm 2 min to collect cells.
35. Put cells on appropriate plates or medium
36. Incubation at 37°C overnight.

## Lysis and protein assay of BjaIT

The stored pellet of the 21st of September was lysed.

1. Pellets were resuspended in 50 µl PBS & incubated for 5 min on ice
2. Lysate was sonified in a waterbath for 30 sec
3. Incubation on ice for 15 min
4. Zentrifugation 3 min; 11000rpm; 11°C

Bradford-Assay:

1. 99 µl ddH2O + 1 µl lysate were mixed with 200 µl Bradford-Reagent
2. OD-measurement at 595 nm

A linear regression was made by using BSA

790 µl H2O + 10 µl BSA

780 µl H2O + 20 µl BSA

796 µl H2O + 4 µl BSA

792 µl H2O + 8 µl BSA

784 µl H2O + 16 µl BSA

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| concentration [µg/µL] | Extinction linear regression |  | measured extinction of the probes (induced) | calculated concentration [µg/µL] |
| 1 | 0.079 |  | 0.062 | 0.06963788301 |
| 2 | 0.126 |  | 0.067 | 0.208913649 |
| 4 | 0.24 |  |  |  |
| 8 | 0.332 |  | measured extinction of the probes (not induced) |  |
| 16 | 0.363 |  | 0.055 | -0.1253481894 |
|  |  |  | 0.053 | -0.1810584958 |
|  |  |  |  |  |
|  |  |  |  | y = 0.0359x + 0.0595 |

## Preparing of the needed buffers for Coomassie-staining

Fixing-buffer

|  |  |
| --- | --- |
| Methanol | 300 ml |
| Acidic acid | 100 ml |
| ddH2O | 600 ml |

Destaining-buffer

|  |  |
| --- | --- |
| Ethanol (70%) | 50 ml |
| Acidic acid | 100 ml |
| ddH2O | 850 ml |

## ONCs from following cryostocks were set up

180829LD03

180518BK20

180802BK01

180826LP03

180712BK04

## Control digestion of 180918AK13

Mastermix

|  |  |
| --- | --- |
| DNA | 5 µl |
| EcoRI | 0.5 µl |
| FastAP | 0.5 µl |
| NEBBuffer 2.1 | 1 µl |
| ddH2O | 3 µl |

The probe was incubated for 1h at 37°C and following 20 min at 80°C. The probe was applied to a 1% agarose gel.

## <https://static.igem.org/mediawiki/2018/0/0a/T--Hamburg--WunderschoenesGelbildFuerNici.png>

## Interlab study Marburg

Preparation of electrocompetent cells & electroporation.

The ONC from the 23rd of September was used to inoculate 500 ml LB-media + V2 salt (204mM NaCl, 4.2 mM KCl, 23.14 mm MgCl2).

1. The culture was incubated at 37°C; 200rpm for 3h.
2. OD was measured (0.502)
3. The culture was incubated for 15 min on ice
4. Cells were centrifuged at 3000g; 4°C; 20min
5. Pellet was resuspended in 10 ml electroporation buffer
6. Step 4 and 5 were repeated 3 times
7. Volume adjusted to OD600=16

Transformation through electroporation

1. 1 µl DNA was added to 80 µl *Vibrio natrigens*
2. Cells were transformed by 900 V; 25 mF; 200 Ω
3. Afterwards cells were added to recovery media and incubated at 37°C for 1.5 h
4. Cells were centrifuged at 3000g; 2 min; room temperature
5. Cells were applied to plates and incubated at 37°C overnight.

## cPCR to check the transformations from 180924 and of pSB1C3-MlcRE

Master mix for 20 samples:

* 40 µL Taq buffer
* 8 µL dNTPs
* 12 µL MgCl2
* 12 µL VF2
* 12 µL VR
* 8 µL Taq polymerase
* 316 µL H2Odest

|  |  |
| --- | --- |
| Clones | Part |
| 1-8 | pSB1C3-BBa\_K20600-BBa\_0034-sulA |
| 9-16 | pSB1C3-MlcRE (from 180518BK01) |
| 17-19 | pSB1C3-I13453-BBa\_0034-AOX1 |

PCR program:

|  |  |
| --- | --- |
| 95°C | 3 min |
| 95°C | 30 s |
| 55°C | 30 s |
| 72°C | 2 min |
| Go to step 2 30x | |
| 72°C | 5 min |

Only clone 14 was inoculated for the overnight cultures. All the other clones had the wrong band.

## SDS-PAGE of ldhA

The running buffer (10x) was diluted 1:10 and filled up in the electrophoresis chamber. The gel ran for 120 min by 80 V.

Loading plan:

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| DH5α-control | | | Marker | ldhA with Arabinose | | | ldhA without Arabinose | | |
| 2 µL | 10 µL | 20 µL | 5 µL | 2 µL | 10 µL | 20 µL | 2 µL | 10 µL | 20 µL |
|  |  |  |  |  |  |  |  |  |  |

The stacking gel was cut out and the separating gel was washed with H2Odest. Afterwards the gel fixed with 15 mL fix solution for 20 min. Then washed two times with H2Odest and after that colored with 15 mL staining solution.

## Preparation of polyacrylamid gel (15%)

|  |  |  |
| --- | --- | --- |
|  | separating gel (15%) | stacking gel |
| Acrylamid | 6.75 mL | 750 µL |
| Separating gel buffer | 4.5 mL | - |
| Stacking gel buffer | - | 1.7 mL |
| H2Odest | 6.75 mL | 4.25 mL |
| 10% APS | 60 µL | 20 µL |
| TEMED | 13.5 µL | 12 µL |

## Minipreps of the overnight cultures from 180924

The miniprep was made with the GeneJET Plasmid Miniprep Kit by Thermo Fischer.

|  |  |  |
| --- | --- | --- |
| Lot number | Part | DNA-concentration [ng/µL] |
| 180925NF01 | pSB1C3-RNAG120-Operator-MlcRE | 139.946 |
| 180925NF02 | pSB1C3-E0240 | 144.673 |
| 180925NF03 | pSB1C3-K206000 | 130.21 |
| 180925NF04 | pSB1C3-BBa\_I13453 | 126.15 |
| 180925NF05 | pSB1C3-MlcRE-BBa\_E0840 | 120.535 |

## PCR of the missing parts and the contaminated GGA PCR products

|  |  |  |  |
| --- | --- | --- | --- |
| Template | Part | DNA-concentration [ng/µL] | Lot number |
| 180921BK05 | accC (Bsa1) | 13.8 | 180925BK01 |
| 180921BK06 | accD (Bsa1) | 7.4 | 180925BK02 |
| 180921BK10 | ADH2 (Bsa1) | 14.9 | 180925BK03 |
| 180921BK11 | kivD (Bsa1) | 13 | 180925BK04 |
| 180921BK16 | pBAD (Bsa1) | 118.73 | 180925BK05 |
| 180921BK17 | HlybB (Bsa1) | 12.84 | 180925BK06 |
| *E.coli* | LeuD (Bsa1) | - | - |

PCR program:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | |  | For leuD |  |
| 98°C | 20 s |  | 98°C | 3 min |
| 98°C | 10 s |  | 98°C | 10 s |
| 60°C | 20 s |  | 52°C | 20 s |
| 72°C | 2 min |  | 72°C | 30 s |
| Go to step 2 30x | | | | |
| 72°C | 2 min |  | 72°C | 3 min |

## Gelelectrophoresis

We made a 1% agarose gel.

## <https://static.igem.org/mediawiki/2018/7/7d/T--Hamburg--180925cPCR.JPG>

## PCR for linear pSB1C3

Mastermix:

* 10 µL 5x Phusion HF buffer
* 1 µL dNTPs
* 35 µL SB-prep-3P-1
* 35 µL SB-prep-2Ea
* 5 µL Template pSB1C3-I20270 (180509NF01)
* 5 µL Phusion Polymerase
* 3 µL H2Odest

PCR program:

|  |  |
| --- | --- |
| 98°C | 2 min |
| 98°C | 30 s |
| 55°C | 30 s |
| 72°C | 3 min |
| Go to step 2 30x | |
| 72°C | 10 min |

## Set up overnight cultures

Overnight cultures set up of:

|  |  |
| --- | --- |
| Lot number | Part |
| 180803NB01 | pGGA-pBAO-BjalT-OmpT-Site-FLAG-HlyA |
| 180531NF03 | pSB1C3-BBa\_B0032-cbtA |
| 180703LP02 | pSB1C3-BBa\_K206000-BBa\_B0034-ldhA |

## Restriction

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** | **15** | **16** | **17** |
|  | Part | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-E0240 | BBa\_J45503 | pSB1C3-oHybB | 34-AOX1 | pSB1C3 | mraz | pSB1C3-BBa\_K206000 | 31sulA | 32sulA | 34sulA | cspD | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-BBa\_B0032\_cbtA |
|  | Lot# | 180921OM02 | 180921OM01 | 180817LD10 | 180625NiF12 | 180921OM03 | 180920LP01 | 180906LP02 | 180622NB08 | neue Miniprep | neue Miniprep | neue Miniprep | 180905AW03 | neue Miniprep | neue Miniprep | 180924AW1 | 180925NaF04 | 180731NF04 |
|  | **ρ** | 113 ng/µL | 123 | 200 ng/µL | 45 ng/µL | 186 ng/µL | 575 ng/µL | 138 ng/µL | 44.175 | 50 ng/µL | 28 ng/µL | 40 ng/µL | 43 ng/µL | 48 ng/µL | 81 ng/µL | 95 ng/µL | 126.15 | 173.5 |
|  | Use | vector | vector | vector | single insert | vector | single insert | vector | single insert | vector | single insert | single insert | single insert | single insert | vector | vector | vector | vector |
|  | target m | 2000 ng | 2000 ng | 2000 ng | 100 ng | 2000 ng | 100 ng | 2000 ng | 100 ng | 2000 ng | 100 ng | 100 ng | 100 ng | 100 ng | 2000 ng | 2000 ng | 2000 ng | 2000 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 17.70 µL | 16.26 µL | 10.00 µL | 2.24 µL | 10.75 µL | 0.17 µL | 14.49 µL | 2.26 µL | 40.00 µL | 3.57 µL | 2.50 µL | 2.33 µL | 2.08 µL | 24.84 µL | 21.15 µL | 15.85 µL | 11.53 µL |
| NEBuffer 2.1 | 3.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 2.00 µL | 4.00 µL | 2.00 µL | 1.00 µL | 5.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 3.00 µL | 3.00 µL | 2.00 µL | 2.00 µL |
| EcoRI-HF |  |  | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |
| XbaI |  | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL |  | 0.50 µL |
| SpeI | 0.50 µL |  |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL |  |  |  |  | 0.50 µL |  | 0.50 µL |  |
| PstI | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL |  | 0.50 µL |  | 0.50 µL |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | 7.80 µL | 0.24 µL | 6.50 µL | 5.76 µL | 5.75 µL | 34.83 µL | 2.01 µL | 5.74 µL | 3.50 µL | 4.43 µL | 5.50 µL | 5.67 µL | 5.92 µL | 0.66 µL | 4.35 µL | 0.65 µL | 4.97 µL |
|  | ad | 30.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 20.00 µL | 40.00 µL | 20.00 µL | 10.00 µL | 50.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 30.00 µL | 30.00 µL | 20.00 µL | 20.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt |  |  |  |  | 2200 bp |  |  |  |  |  |  |  |  |  |  | 2200 | 394 |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 67 ng/µL | 100 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 3 ng/µL | 100 ng/µL | 10 ng/µL | 6 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 8 ng/µL | 67 ng/µL | 7 ng/µL | 100 ng/µL |

## Ligation

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** |
| Product | | pSB1C3\_RNAG120\_MlcRE-pSB1C3-E0240 | pSB1C3-E0240-BBa\_J45503 | pSB1C3-oHybB-34-AOX1 | pSB1C3-mraz | pSB1C3-BBa\_K206000-32sulA | pSB1C3-BBa\_K206000-34sulA | pSB1C3-BBa\_B0032-cspD | pSB1C3-34-AOX1 | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-pSB1C3-BBa\_B0032\_cbtA | pSB1C3-oHybB-pSB1C3-E0240 |
|  | |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-oHybB | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-oHybB |
| Tube | **1** | **3** | **5** | **7** | **9** | **9** | **14** | **15** | **16** | **16** | **5** |
| ρ | 67 ng/µL | 10 ng/µL | 10 ng/µL | 100 ng/µL | 6 ng/µL | 6 ng/µL | 8 ng/µL | 67 ng/µL | 7 ng/µL | 7 ng/µL | 10 ng/µL |
| Länge | 2697 bp | 2948 bp | 2200 bp | 2070 bp | 2200 bp | 2200 bp | 2083 bp | 2070 bp | 2200 bp | 2200 bp | 2200 bp |
| V | 0.35 µL | 4.29 µL | 1.55 µL | 0.61 µL | 4.25 µL | 4.25 µL | 5.74 µL | 0.22 µL | 2.21 µL | 5.12 µL | 2.00 µL |
| **Part 1** | Part 1 | pSB1C3-E0240 | BBa\_J45503 | 34-AOX1 | mraz | 32sulA | 34sulA | cspD | 34-AOX1 | 34-AOX1 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-E0240 |
| Tube | **2** | **4** | **6** | **8** | **11** | **12** | **13** | **6** | **6** | **17** | **2** |
| ρ | 100 ng/µL | 10 ng/µL | 3 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 3 ng/µL | 3 ng/µL | 100 ng/µL | 100 ng/µL |
| Länge | 878 bp | 393 bp | 1200 bp | 458 bp | 565 bp | 565 bp | 245 bp | 1200 bp | 1200 bp | 394 bp | 878 bp |
| V | 0.77 µL | 5.71 µL | 33.80 µL | 10.00 µL | 7.20 µL | 7.20 µL | 5.40 µL | 34.12 µL | 33.80 µL | 0.64 µL | 0.80 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |  |  |  |
| T4 Buffer | | 1.00 µL | 2.00 µL | 4.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 4.00 µL | 5.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 7.38 µL | 7.50 µL | 0.15 µL | 6.89 µL | 5.63 µL | 5.63 µL | 6.35 µL | 1.16 µL | 8.48 µL | 2.74 µL | 5.70 µL |
| ad | | 10.00 µL | 20.00 µL | 40.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 40.00 µL | 50.00 µL | 10.00 µL | 10.00 µL |

## Measurement of the DNA-concentration

The samples were measured by Nano Drop λ = 600 nm.

|  |  |
| --- | --- |
| Tube | DNA-concentration [ng/µL] |
| 1 | 17.756 |
| 5 | 26.167 |
| 16 | 28.349 |

## Title: cPCR of transformation (24.09.18) and pSB1C3-MIcRE (plated out again)

Mix for 20 approaches of PCR:

|  |  |
| --- | --- |
| Taq-Buffer | 40 μL |
| dNTPs | 8 μL |
| MgCl2 | 12 μL |
| VP2 | 12 μL |
| VR | 12 μL |
| Taq-Polymerase | 8 μL |
| H2O | 316 μL |

PCR-program:

|  |  |
| --- | --- |
| temperature | time |
| 95 °C | 3:00 Min |
| 95 °C | 0:30 Min |
| 55 °C | 0:30 Min |
| 72 °C | 2:00 Min |
| 72 °C | 5:00 Min |
| 4 °C | ∞ |

clones 1-8: pSB1C3-BBa\_K206000-BBa\_0034-SulA (L6)

clones 9-16: pSB1C3-MIcRE (derived from 180618BK01)

clones 17-19: pSB1C3-I13453-BBa\_0034-AOX1 (L9)

* Overnight culture of clone 14 (pSB1C3-MIcRE) (all other clones showed not estimated bands on gel)

## SDS-PAGE of ldhA

Dilute 10x running buffer until 1x concentration is reached then pouring into run chamber.

Be aware of installing glas plates opposite of the gel to prevent run buffer from escaping the chamber!

Loadingscheme of gel:

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| DH5α | | | Page Ruler | ldhA + arabinose (+) | | | ldhA + arabinose (-) | | |
| 2 μL | 10 μL | 20 μL | 5 μL | 2 μL | 10 μL | 20 μL | 2 μL | 10 μL | 20 μL |
|  |  |  |  |  |  |  |  |  |  |

Gel was runned für 120 minutes at 80 V. Gathering gel was removed. Seperation gel was washed for 2 minutes with ddH2O and afterwards incubated for 20 minutes in 15 mL fixing solution. The gel was washed twice with ddH2O. Incubation of the gel for 20 minutes in 15 mL coloring solution.

(gel lost)

Pouring of PAA-gels (15 %)

|  |  |  |
| --- | --- | --- |
|  | seperation gel | gathering gel |
| acryl amide | 6.75 mL | 750 μL |
| seperation gel buffer | 4.5 mL | - |
| gathering gel buffer | - | 1.7 mL |
| H2O | 6.75 mL | 4.25 mL |
| 10 % APS | 60 μL | 20 μL |
| TEMED | 13.5 μL | 12 μL |

## Repetition of the PCR for generating LeuD

Two different protocols were used.

Mastermix 1

|  |  |
| --- | --- |
| Substance | 1x volume (µl) |
| 2x Q5 Mastermix | 12.5 |
| F-Primer | 0.2 |
| R-Primer | 0.2 |
| DNA-Template (DH5α) | 1 |

Mastermix 2

|  |  |
| --- | --- |
| Substance | 1x volume (µl) |
| 5x Phusion buffer | 4 |
| F-Primer | 0.2 |
| R-Primer | 0.2 |
| 10 mM dNTPs | 0.4 |
| Phusion polymerase | 0.2 |
| DNA-Template (DH5α) | 1 |

PCR-program (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 98 | 120 |
| 98 | 10 |
| 55 | 20 |
| 72 | 30 |
| 72 | 300 |

Both PCR-products were applied to an 1% agarose gel. The PCR using Mastermix 1 didn’t work therefore just one product was cut up and purified using the “GenJet Gel Extraction Kit” from Thermo Fisher.

## <https://static.igem.org/mediawiki/2018/c/ce/T--Hamburg--180926.jpg>

## Restriction of pGGA

Mastermix

|  |  |
| --- | --- |
| Substance | 1x volume (µl) |
| DNA-Template (pGGA) | 10 |
| BsaI-HF | 0.5 |
| 10x CutSmart buffer | 2 |
| ddH2O | 7.5 |

The product was applied to a 1% agarose gel and purified using the “GenJet Gel Extraction Kit” from Thermo Fisher.

## <https://static.igem.org/mediawiki/2018/c/ce/T--Hamburg--180926.jpg>

## GGA-Assembly

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Calculation** | Länge (bp) | Dichte (ng/µL) | Volumen (µL) | 3MB |
| Promotor | 161 | 66 | 0.2 | pBAD |
| Part 1 | 1050 | 14.9 | 3.6 | ADH2 |
| Part 2 | 1650 | 13 | 5.5 | kivD |
| Part 3 | 1590 | 40 | 2.4 | LeuA |
| Part 4 | 1100 | 44 | 1.7 | LeuB |
| Part 5 | 1410 | 47 | 2 | LeuC |
| Part 6 | 630 | 29.3 | 1.4 | LeuD |
| Buffer |  |  | 2 |  |
| Mix |  |  | 1 |  |
| pGGA |  |  | 3 |  |
| H2O |  |  | 0 |  |
|  |  |  |  |  |
|  |  |  |  |  |
|  |  |  |  |  |
| **Calculation** | Länge (bp) | Dichte (ng/µL) | Volumen (µL) | Excretion |
| Promotor | 161 | 66 | 0.2 | pBAD |
| Part 1 | 3872 | 12.84 | 13 | HlyB+HlyD |
| Part 2 | 1296 | 186 | 0.4807549633 | TolC |
| Part 3 | 975 | 105 | 0.6406886582 | ompT |
| Buffer |  |  | 2 |  |
| Mix |  |  | 1 |  |
| pGGA |  |  | 3 |  |
| H2O |  |  | 0 |  |
|  |  |  |  |  |
| **Calculation** | Länge (bp) | Dichte (ng/µL) | Volumen (µL) | Myristic Acid |
| Promotor | 161 | 66 | 0.2 | pBAD |
| Part 1 | 993 | 163 | 0.4203328799 | accA |
| Part 2 | 489 | 204 | 0.1653904432 | accB |
| Part 3 | 1358 | 170 | 0.5511661886 | accC |
| Part 4 | 925 | 209 | 0.3053705603 | accD |
| Part 5 | 679 | 74 | 0.6330962978 | tesA |
| Buffer |  |  | 2 |  |
| Mix |  |  | 1 |  |
| pGGA |  |  | 3 |  |
| H2O |  |  | 11.72464363 |  |
|  |  |  |  |  |
| **Calculation** | Länge (bp) | Dichte (ng/µL) | Volumen (µL) | Growth Inhibition |
| Promotor | 161 | 66 | 0.2 | pBAD |
| Part 1 | 410 | 58.154 | 0.4864475091 | 34cbtA |
| Part 2 | 260 | 65.224 | 0.2750411264 | 34cspD |
| Part 3 | 500 | 66.294 | 0.5203882713 | 34mraZ |
| Part 4 | 565 | 87.334 | 0.4463718673 | 34sulA |
| Buffer |  |  | 2 |  |
| Mix |  |  | 1 |  |
| pGGA |  |  | 1 |  |
| H2O |  |  | 14.07175123 |  |

## Preparation of cell lysate including ldhA and BjaIT

The ONCs prepared on the 25th of September were used to inoculate 200 ml cultures.

|  |  |  |  |
| --- | --- | --- | --- |
| Culture | Induction | Start-OD | OD after 6 h |
| DH5α (control) | - | 0.076 | 5.22 |
| ldhA (not induced) | - | 0.036 | 8.02 |
| ldhA (induced) | 0.248ml arabinose | 0.026 | 5.06 |
| BjaIT (not induced) | - | 0.182 | 2.4 |
| BjaIT (induced) | 0.224 ml arabinose | 0.110 | 3.52 |

The ldhA and DH5α cultures got centrifuged at 5000rpm for 20 min. The pellet was stored at -20°C. The BjaIT cultures were incubated further at 16°C overnight.

## Restriction, ligation and transformation

Restriction

|  |  |  |
| --- | --- | --- |
| **Tube** | **2** | **17** |
| **Part** | pSB1C3-E0240 | pSB1C3-BBa\_B0032\_cbtA |
| **Lot#** | 180925NaF02 | 180926NaF03 |
| **ρ** | 144.673 | 93.011 |
| Use | vector | vector |
| target m | 1000 ng | 1000 ng |
|  |  |  |
| DNA | 6.91 µL | 10.75 µL |
| NEBuffer 2.1 | 1.00 µL | 2.00 µL |
| EcoRI-HF |  |  |
| XbaI | 0.50 µL | 0.50 µL |
| SpeI |  |  |
| PstI | 0.50 µL | 0.50 µL |
| FastAP |  |  |
| H2O | 1.09 µL | 6.25 µL |
| ad | 10.00 µL | 20.00 µL |
|  |  |  |
| Länge Produkt | 878 bp | 394 |
|  |  |  |
| ρ final | 11 ng/µL | 7 ng/µL |

The probes were incubated at 37°C for 1h following another 20 min at 80°C. The products were then applied to a 1% agarose gel and purified using the “GenJet Gel Extraction Kit” from Thermo Fisher.

![](https://static.igem.org/mediawiki/2018/c/ce/T--Hamburg--180926.jpg)

pSB1C3\_RNAG120\_MlcRE, pSB1C3-oHybB, 34-AOX1, pSB1C3-I13453 were already digested on September 25.

Ligation

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L9** | **L10** | **L11** |
| Product | | pSB1C3\_RNAG120\_MlcRE-pSB1C3-E0240 | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-pSB1C3-BBa\_B0032\_cbtA | pSB1C3-oHybB-pSB1C3-E0240 |
|  | |  |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-oHybB |
| Tube | **1** | **16** | **16** | **5** |
| ρ | 18 ng/µL | 28 ng/µL | 28 ng/µL | 26 ng/µL |
| Länge | 2697 bp | 2200 bp | 2200 bp | 2200 bp |
| V | 1.32 µL | 0.55 µL | 1.27 µL | 0.76 µL |
| **Part 1** | Part 1 | pSB1C3-E0240 | 34-AOX1 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-E0240 |
| Tube | **2** | **6** | **17** | **2** |
| ρ | 11 ng/µL | 3 ng/µL | 7 ng/µL | 11 ng/µL |
| Länge | 878 bp | 1200 bp | 394 bp | 878 bp |
| V | 6.71 µL | 33.80 µL | 8.79 µL | 7.01 µL |
| **Part 2** | Part 2 |  |  |  |  |
| Tube |  |  |  |  |
| ρ |  |  |  |  |
| Länge |  |  |  |  |
| V |  |  |  |  |
| T4 Buffer | | 1.00 µL | 4.00 µL | 2.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 0.47 µL | 1.15 µL | 7.44 µL | 0.72 µL |
| ad | | 10.00 µL | 40.00 µL | 20.00 µL | 10.00 µL |

The ligations were incubated at 37°C for 1 h.

The ligations were transformed into DH5α.

37. 150 µl Competent cell aliquots were thawed on ice for 15-30 min.
38. 10 µl DNA were added and mixed
39. Cells were incubated for 35 min on ice
40. Heat-shock: 42°C for 45 sec, then put back on ice for 2-5 min
41. Pre-warm 850 µl LB-Medium
42. Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
43. Cetrifuge 4500 rpm 2 min to collect cells.
44. Put cells on appropriate plates or medium
45. Incubation at 37°C overnight.

## RIPA-Buffer was prepared

Recipe for 50 ml

|  |  |
| --- | --- |
| Substance | Volume |
| 25 mM Tris | 0.151g |
| 150 mM NaCl | 0.435g |
| 0.5% Sodium desoxycholat | 0.25g |
| 1% NP-40 Alternative | 500µl |
| ddH2O | ad 50ml |

1 tablet complete was added and the pH was adjusted to 7.5.

## Two 12.5% SDS-PAGE gels were prepared

|  |  |  |
| --- | --- | --- |
|  | Stacking gel | Separate gel |
| Stacking gel buffer | 0.675ml | - |
| Separate gel buffer | - | 2.6ml |
| ddH2O | 3.425ml | 3.2ml |
| Acrylamid | 0.875ml | 4.1ml |
| APS | 30µl | 60µl |
| TEMED | 15µl | 30µl |

Gels were stored at 4°C.

## Cryostock and miniprep of pSB1C3-MlcRE and pSB1C3-BBa\_B0032-cbta

The prepared ONCs from September 25 was purified by using the “GeneJet Plasmid Miniprep Kit” from Thermo Fisher.

## Control digestion of pSB1C3-MlcRE

Mastermix

|  |  |
| --- | --- |
| Substance | 1x volume (µl) |
| DNA | 5 |
| NEBBuffer 2.1 | 1 |
| EcoRI | 0.5 |
| PstI | 0.5 |
| FastAP | 0.5 |
| ddH2O | 3 |

The product war applied to an 1% agarose gel.

## <https://static.igem.org/mediawiki/2018/c/ce/T--Hamburg--180926.jpg>

## Transfer buffer was prepared for Western-Blot

|  |  |
| --- | --- |
| Substance | Volume |
| Methanol | 200ml |
| Tris | 3.03g |
| Glycin | 14.4g |
| ddH2O | Ad 1l |

## Preparing of cell lysates

The day before BjalT grew so slow, so this part was incubated overnight at 16°C. The cultures was centrifugated at 5000 rpm and 4°C. The supernatant was discarded. Then added xxx µL RIPA-Buffer to the pellets and resuspend on ice at 4°C. The samples incubated 30 min on ice at 4°C and vortexed every 10 min. After that sonificated the samples for 30 s in Sonifier-Waterbath. Then the samples incubated on ice for 15 min at 4°C. After that centrifugated for 3 min at 1100 rpm and 4°C. The supernatant were transferred into new tubes.

## Proteinconcentration by Bradford

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| µg/µL | Extinktion 1 | Extinktion 2 | Mean |  |  |  |  |  |  |  |  |
| 1 | 0.074 | 0.055 | 0.0645 |  |  |  | DH5a | ldha - | ldha + | bjaIT - | bjaIT + |
| 2 | 0.113 | 0.115 | 0.114 |  |  | Extinktion 1 | 0.098 | 0.08 | 0.11 | 0.098 | 0.171 |
| 4 | 0.203 | 0.212 | 0.2075 |  |  | Extinktion 2 | 0.103 | 0.076 | 0.102 | 0.122 | 0.151 |
| 8 | 0.285 | 0.277 | 0.281 |  |  | Mean | 0.1005 | 0.078 | 0.106 | 0.11 | 0.161 |
| 16 | 0.318 | 0.305 | 0.3115 |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  | Mean | Konzentration [µg/µL] | Input 1400 µg Protein | ad 1100 µL H2O | Konzentration Stamm[µg/µL] |
|  |  |  |  |  |  | DH5a | 0.1005 | 1.879 | 745.274 | 354.726 | 1.273 |
|  |  |  |  |  |  | ldha+ | 0.106 | 1.981 | 706.604 | 393.396 | 1.273 |
|  |  |  |  |  |  | ldha- | 0.078 | 1.458 | 960.256 | 139.744 | 1.273 |
|  |  |  |  |  |  | bjaIT+ | 0.161 | 3.009 | 465.217 | 634.783 | 1.273 |
|  |  |  |  |  |  | bjaIT- | 0.11 | 2.056 | 680.909 | 419.091 | 1.273 |
|  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  | Input 20µg | Input 30 | Input 40 µg |  |  |
|  |  |  |  |  |  | Ansatz | 15.71 | 23.57 | 31.43 |  |  |
|  |  |  |  |  |  | Wasser | 15.71 | 7.86 | 0.00 |  |  |

The standard curve set up analog to 180924. After the measurement of the concentration, the samples were adjusted on 1400 µg protein. We added 5x SDS-Loading Dye and incubated for 10 min at 95°C. The samples used for a Western Blot and stored at -20°C.

## SDS-Page of ldhA and BjalT

The gels were clamped and added 1x running buffer to the chamber.

Loading plan:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Gel 1 | Marker | 30 µg DH5α | 30 µg ldhA | 30 µg ldhA |
|  |  | Control | Without Arabinose | With Arabinose |

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Gel 2 | DH5α | BjalT- | BjalT+ | DH5α | BjalT- | BjalT+ | DH5α | BjalT- | BjalT+ |
|  | 20 µg protein | | | 30 µg protein | | | 40 µg protein | | |

The current was 8 mA in each stacking gel and 12 mA in each separate gel. After that the gel was washed 2 min with H2Odest. Afterwards the gel fixed with 15 mL fix solution for 20 min. Then washed two times with H2Odest and after that colored with 15 mL staining solution. Then destained overnight in destain buffer.

## Western Blot of BjalT

The transfer buffer was stored at 4°C. The first membrane (PVPF) and four Whatmanfilter cut out for each gel (6x9 cm). After that the membrane was activated by putting it in methanol.

The device for the blot was built up:

* Anode
* Sponge
* 2 filter papers
* Membrane
* 2 filter papers
* Sponge
* Cathode

We added an ice pack and a stirring bar to the transfer buffer and blotted for 1 h at 100 V. We put the membrane in 5% milk powder and TBS overnight at 4°C.

## TBS buffer

For 1 L buffer we used:

* 50 mM Tris = 6.07 g
* 150 mM NaCl = 8.77 g
* The pH was set on 8

## cPCR of 3A Assembly

|  |  |  |  |
| --- | --- | --- | --- |
| Mastermix | 1x | 40x | 10x for GGA |
| 10x Dream Taq buffer | 2 µL | 80 µL | 20 µL |
| dNTPs (10 mM) | 0.4 µL | 16 µL | 8 µL |
| MgCl2 (50 mM) | 1.2 µL | 48 µL | 6 µL |
| Fw Primer VF2 | 0.6 µL | 24 µL | 6 µL (GGA\_cPCR\_f) |
| Rv Primer vR | 0.6 µL | 24 µL | 6 µL (GGA\_cPCR\_r) |
| Taq Polymerase | 0.1 µL | 4 µL | 1 µL |
| H2Odest | 15.1 µL | 604 µL | 151 µL |

|  |  |  |
| --- | --- | --- |
| Ligation | Part | Tube |
| L1 | pSB1C3-RNAG120\_MlcRE-pSB1C3-E0240 | 1-4 |
| L9 | pSB1C3-I13453-34-AOX1 | 5-12 |
| L10 | pSB1C3-I13453-BBa\_B0032\_cbtA | 13-25 |
| L11 | pSB1C3-oHybB-E0240 | 26-38 |

PCR-Program:

|  |  |
| --- | --- |
| 95°C | 3 min |
| 95°C | 30 s |
| 55°C | 30 s |
| 72°C | 1:45 min |
| Go to step 2 35x | |
| 72°C | 5 min |

GGA Program:

|  |  |
| --- | --- |
| 95°C | 3 min |
| 94°C | 30 s |
| 55°C | 30 s |
| 72°C | 7 min |
| Go to step 2 30x | |
| 72°C | 10 min |

## Repetition of the transformation of the GGA parts + I732018 of the Kit

The transformation was made with protocol by Zhang Ghong. The samples were stored overnight at 37°C.

## Continuation of the Western Blot

Washed the membrane with 20 mL TBST for 10 min.

The primary antibody Sigma F3165 Monoclonal Anti-Flag M2 Antibody contained 5 µL AK (4 µg/µL) on 2 mL TBST, 1% milk powder (20 mg) for a AK-concentration of 10 µg/µL.

First the membrane was incubated in a 2 mL AK-solution for 30 min at room temperature. To make sure, that all antibodies got contact with the membrane, we welded the membrane with the AK-solution and incubated it for another 90 min at room temperature. After that the membrane washed with 20 mL TBST two times for 10 min at room temperature.

The secondary antibody solution contained 20 mL TBST 1% milk powder (200 mg) with 2 µL goat-anti-mouse. The antibody was incubated for 1 h at room temperature.

After that washed with 20 mL TBST two time for 10 min at room temperature.

## Set up of chemiluminescent substrate

Solution 1:

* 100 µL Luminol stock
* 44 µL p-Cumanic acid stock
* 1 mL Tris (1 M)/ pH with HCl to 8.5
* 85 mL H2Odest

Solution 2:

* 6 µL 30% H2O2
* 1 mL Tris (1 M)/ pH with HCl to 8.5
* 9 mL H2Odest

Both solutions were added to the blot and incubated for 2 min. We made photos with a Imager. The program was ImageLab.

## <https://static.igem.org/mediawiki/2018/2/22/T--Hamburg--Imager_2018-09-28_16hr_06min%2BImager_2018-09-28_16hr_04min.tiff>

## Transformation of the GGA-constructs and I732018 from 180927

We made the transformation with the iGEM Transformation protocol.

## Restriktion

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Tube** | **5** | **6** | **7** | **13** | **19** | **20** | **21** | **22** |
| **Part** | pSB1C3-oHybB | 34-AOX1 | pSB1C3 | cspD | accD | ldhA | ompT | RNAG120 |
| **Lot#** | 180921OM03 | 180920LP01 | 180914LP01 | 180622NB04 | 180921BK09 | 180622NB01 | 180827LP01 | 180909BK09 |
| **ρ** | 186 ng/µL | 575 ng/µL | 89 ng/µL | 50 ng/µL | 213 ng/µL | 57 ng/µL | 68 ng/µL | 77 ng/µL |
| Use | vector | single insert | vector | single insert | single insert | single insert | single insert | single insert |
| target m | 1000 ng | 100 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng | 100 ng |
|  |  |  |  |  |  |  |  |  |
| DNA | 5.38 µL | 0.17 µL | 11.24 µL | 2.00 µL | 0.47 µL | 1.77 µL | 1.47 µL | 1.30 µL |
| NEBuffer 2.1 | 1.00 µL | 4.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| XbaI |  | 0.50 µL |  |  |  |  |  |  |
| SpeI | 0.50 µL |  |  |  |  |  |  |  |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL |  | 0.50 µL |  |  |  |  |  |
| H2O | 2.12 µL | 34.83 µL | 5.26 µL | 6.00 µL | 7.53 µL | 6.23 µL | 6.53 µL | 6.70 µL |
| ad | 10.00 µL | 40.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |
| Länge Produkt | 2200 bp | 1200 bp | 2070 | 222 | 915 | 987 | 954 | 120 |
|  |  |  |  |  |  |  |  |  |
| ρ final | 10 ng/µL | 3 ng/µL | 50 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |

The probes were incubated at 37°C for 1h following another 20 min at 80°C. Probe 1 and 2 were applied to a 1% agarose gel and purified using the “GeneJet Gel Extraction Kit” from Thermo Fisher.

![](https://2018.igem.org/File:T--Hamburg--180929Gelex.png)

Since the concentrations were very low no ligations were possible.

Probes were stored at 4°C overnight.

## PCR of pSB1C3

3 samples were set up.

Mastermix

|  |  |
| --- | --- |
| Substances | 3x volume (µl) |
| DNA-Template | 1.5 |
| F-Primer | 1.75 |
| R-Primer | 1.75 |
| 5x Phusion HF-buffer | 30 |
| dNTPs | 3 |
| Phusion polymerase | 1.5 |
| ddH2O | 111.9 |

PCR-Program (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 98 | 120 |
| 98 | 30 |
| 55 | 30 |
| 72 | 180 |
| 72 | 600 |

## Transformation

The transformation of pSB1C3-BBa-I732018 was repeated since the plate performed on September 28 didn’t show any colonies.

46. 150 µl Competent cell aliquots were thawed on ice for 15-30 min.
47. 10 µl DNA were added and mixed
48. Cells were incubated for 35 min on ice
49. Heat-shock: 42°C for 45 sec, then put back on ice for 2-5 min
50. Pre-warm 850 µl LB-Medium
51. Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
52. Cetrifuge 4500 rpm 2 min to collect cells.
53. Put cells on appropriate plates or medium
54. Incubation at 37°C overnight.

## Restriction, ligation and transformation

Restriction

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Tube** | **1** | **2** | **6** | **7** | **13** | **16** | **17** | **19** | **20** | **21** | **22** |
| **Part** | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | 34-AOX1 | pSB1C3 | cspD | pSB1C3-I13453 | pSB1C3-BBa\_B0032\_cbtA | accD | ldhA | ompT | RNAG120 |
| **Lot#** | 180925NaF01 | 180925NaF02 | 180920LP01 | 180914LP01 | 180622NB04 | 180925NaF04 | 180926NaF03 | 180921BK09 | 180622NB01 | 180827LP01 | 180909BK09 |
| **ρ** | 140 ng/µL | 144.673 | 575 ng/µL | 89 ng/µL | 50 ng/µL | 126 ng/µL | 93.011 | 213 ng/µL | 57 ng/µL | 68 ng/µL | 77 ng/µL |
| Use | Vector | vector | single insert | vector | single insert | vector | vector | single insert | single insert | single insert | single insert |
| target m | 1000 ng | 1000 ng | 100 ng | 1000 ng | 100 ng | 1000 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |
| DNA | 7.15 µL | 6.91 µL | 0.17 µL | 11.24 µL | 2.00 µL | 7.93 µL | 10.75 µL | 0.47 µL | 1.77 µL | 1.47 µL | 1.30 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 4.00 µL | 2.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF |  |  |  | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| XbaI |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL |  |  |  |  |
| SpeI | 0.50 µL |  |  |  |  | 0.50 µL |  |  |  |  |  |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL |  |  | 0.50 µL |  | 0.50 µL |  |  |  |  |  |
| H2O | 0.35 µL | 1.09 µL | 34.83 µL | 5.26 µL | 6.00 µL | 8.57 µL | 6.25 µL | 7.53 µL | 6.23 µL | 6.53 µL | 6.70 µL |
| ad | 10.00 µL | 10.00 µL | 40.00 µL | 20.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |
| Länge Produkt | 2697 bp | 878 bp | 1200 bp | 2070 | 222 | 2200 | 394 | 915 | 987 | 954 | 120 |
|  |  |  |  |  |  |  |  |  |  |  |  |
| ρ final | 100 ng/µL | 8 ng/µL | 3 ng/µL | 19 ng/µL | 10 ng/µL | 11 ng/µL | 5 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |

The probes were incubated for 1h at 37°C following another 20 min at 80°C.

Restriction 1.2,7.16 and 17 were applied to a 1% agarose gel and purified using the “GeneJet Gel Extraction Kit” of Thermo Fisher.

![](https://static.igem.org/mediawiki/2018/0/06/T--Hamburg--180930Gelex_restr.png)

PSB1C3-oHybB had been digested on September 14 and was kept and therefor used for the following ligations. The digested parts of September 29 were used as well.

Ligation

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L9** | **L10** | **L11** | **L13** | **L14** | **L15** | **L16** | **L17** |
| Product | | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-pSB1C3-BBa\_B0032\_cbtA | pSB1C3-oHybB-pSB1C3-E0240 | pSB1C3-cspD | pSB1C3-accD | pSB1C3-ldhA | pSB1C3-ompT | pSB1C3-RNAG120 |
|  | |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-oHybB | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 |
| Tube | **16** | **16** | **5** | **7** | **7** | **7** | **7** | **7** |
| ρ | 11 ng/µL | 11 ng/µL | 28 ng/µL | 19 ng/µL | 19 ng/µL | 19 ng/µL | 19 ng/µL | 19 ng/µL |
| Länge | 2200 bp | 2200 bp | 2200 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp |
| V | 1.41 µL | 3.26 µL | 0.72 µL | 2.54 µL | 0.97 µL | 0.91 µL | 0.94 µL | 3.33 µL |
| **Part 1** | Part 1 | 34-AOX1 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-E0240 | cspD | accD | ldhA | ompT | RNAG120 |
| Tube | **6** | **17** | **2** | **13** | **19** | **20** | **21** | **22** |
| ρ | 3 ng/µL | 5 ng/µL | 8 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |
| Länge | 1200 bp | 394 bp | 878 bp | 222 bp | 915 bp | 987 bp | 954 bp | 120 bp |
| V | 33.80 µL | 12.83 µL | 10.00 µL | 5.17 µL | 8.16 µL | 8.27 µL | 8.22 µL | 3.67 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |
| T4 Buffer | | 4.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 0.29 µL | 1.41 µL | 6.79 µL | 0.79 µL | 8.37 µL | 8.32 µL | 8.34 µL | 1.50 µL |
| ad | | 40.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL |

The probes were incubated for 1.5h at 37°C.

The products were transformed into DH5α.

55. 150 µl Competent cell aliquots were thawed on ice for 15-30 min.
56. 10 µl DNA were added and mixed
57. Cells were incubated for 35 min on ice
58. Heat-shock: 42°C for 45 sec, then put back on ice for 2-5 min
59. Pre-warm 850 µl LB-Medium
60. Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
61. Cetrifuge 4500 rpm 2 min to collect cells.
62. Put cells on appropriate plates or medium
63. Incubation at 37°C overnight.

## Gelextraction of pSB1C3

The 3 PCR samples made on September 29 were applied to a gel.

![](https://static.igem.org/mediawiki/2018/9/9f/T--Hamburg--180930_PCR%2BGelEx2.png)

Since all results seemed wrong the probes were discarded.

## PCR to amplify pSB1C3 and pSB1K3

2 samples of each part were set up.

Mastermix

|  |  |  |
| --- | --- | --- |
| Substances | 1x volume (µl) | 5x volume (µl) |
| DNA-Template | 0.1 | 0.5 |
| F-Primer | 0.35 | 1.75 |
| R-Primer | 0.35 | 1.75 |
| 5x Phusion HF-buffer | 10 | 50 |
| dNTPs | 1 | 5 |
| Phusion polymerase | 0.5 | 2.5 |
| ddH2O | 37.7 | 188.5 |

PCR-Program (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 98 | 120 |
| 98 | 30 |
| 55 | 30 |
| 72 | 180 |
| 72 | 600 |

5 µl of each product was applied to a 1% agarose gel. The rest was purified using the “GeneJET Gel Extraction Kit” of Thermo Fisher.

## <https://static.igem.org/mediawiki/2018/c/cd/T--Hamburg--180930PCR_test.png>

## PCR of cspD

Mastermix

|  |  |  |
| --- | --- | --- |
| Substances | 1x volume (µl) | 5x volume (µl) |
| DNA-Template | 0.1 | 0.5 |
| F-Primer | 0.35 | 1.75 |
| R-Primer | 0.35 | 1.75 |
| 5x Phusion HF-buffer | 10 | 50 |
| dNTPs | 1 | 5 |
| Phusion polymerase | 0.5 | 2.5 |
| ddH2O | 37.7 | 188.5 |

PCR-Program (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 98 | 120 |
| 98 | 30 |
| 55 | 30 |
| 72 | 180 |
| 72 | 600 |

## <https://static.igem.org/mediawiki/2018/1/1c/T--Hamburg--180930_cPCR_Pcr_test.png>

## Colony PCR of pSB1C3-BBa\_I732018

The transformed part set up on September 29 was used.

Mastermix

|  |  |  |
| --- | --- | --- |
| Substance | 1x (volume in µl) | 5x (volume in µl) |
| 10x DreamTaq Buffer | 2 | 10 |
| dNTPs 10mM | 0.4 | 2 |
| MgCl2 50mM | 1.2 | 6 |
| Fw-Primer VF2 | 0.6 | 3 |
| Rv-Primer VR | 0.6 | 3 |
| Taq-Polymerase | 0.1 | 0.5 |
| ddH2O ad 20µl | 15.1 | 75.5 |

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Program (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 95 | 180 |
| 95 | 30 |
| 55 | 30 |
| 72 | 150 |
| 72 | 180 |

The products were applied to a 1% agarose gel.

## <https://static.igem.org/mediawiki/2018/1/1c/T--Hamburg--180930_cPCR_Pcr_test.png>

## Set up of ONCs

ONCs were set up using cryostocks for new minipreps of pSB1C3-BBa\_B0032-cspD, pSB1C3-oHybB, pSB1C3‑BBa\_K206000-BBa\_B0034-ldhA. The transformation of pSB1C3-BBa\_I732018 was also set up as an ONC.

## PCR for the production of pSB1C3 and pSB1K3

The PCR reactions were prepared as follows:

|  |  |
| --- | --- |
|  | **1x** |
| 5x Phusion HF-Buffer | 10 µL |
| dNTPs | 1 µL |
| SB-Prep-3P1 | 0.35 µL |
| SB-Prep-2Ea | 0.35 µL |
| Phusion Polymerase | 0.5 µL |
| H2O (ad 50) | 37.7 µL |
| Template (je 2x pSB1K3 und psB1C3) | 0.1 µL |

The PCR programme was as follows:

|  |  |
| --- | --- |
| **Temperatur** | **Zeit** |
| 98 °C | 2:00 |
| 98 °C | 0:30 |
| 55 °C | 0:30 |
| 72 °C | 3:00 |
| go to step to (30x) |  |
| 72 °C | 10:00 |
| 4 °C | ∞ |

## Purification of cspD from the PCR from the 30.09.18

The Nanodrop measured a concentration of 36.662 ng/µL for 181001NB01.

## Restriction and ligation 1

 The restriction reactions were prepared as follows:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** | **15** | **16** | **17** | **18** | **19** | **20** | **21** | **22** |
|  | Part | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-E0240 | BBa\_J45503 | pSB1C3-oHybB | 34-AOX1 | pSB1C3 | mraz | pSB1C3-BBa\_K206000 | 31sulA | 32sulA | 34sulA | cspD | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-BBa\_I732018 | accD | ldhA | ompT | RNAG120 |
|  | Lot# | 180925NaF01 | 180925NaF02 | 180817LD10 | 180625NiF12 | 180921OM03 | 180920LP01 | 180914LP01 | 180622NB08 | neue Miniprep | neue Miniprep | neue Miniprep | 180905AW03 | 180622NB04 | neue Miniprep | 180924AW1 | 180925NaF04 | 180926NaF03 |  | 180921BK09 | 180622NB01 | 180827LP01 | 180909BK09 |
|  | **ρ** | 140 ng/µL | 144.673 | 200 ng/µL | 45 ng/µL | 186 ng/µL | 575 ng/µL | 89 ng/µL | 44.175 | 50 ng/µL | 28 ng/µL | 40 ng/µL | 43 ng/µL | 50 ng/µL | 81 ng/µL | 95 ng/µL | 126 ng/µL | 93.011 |  | 213 ng/µL | 57 ng/µL | 68 ng/µL | 77 ng/µL |
|  | Use | Vector | vector | vector | single insert | vector | single insert | vector | single insert | vector | single insert | single insert | single insert | single insert | vector | vector | vector | vector | vector | single insert | single insert | single insert | single insert |
|  | target m | 1000 ng | 1000 ng | 1000 ng | 100 ng | 1000 ng | 100 ng | 1000 ng | 100 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 7.15 µL | 6.91 µL | 5.00 µL | 2.24 µL | 5.38 µL | 0.17 µL | 11.24 µL | 2.26 µL | 20.20 µL | 3.57 µL | 2.50 µL | 2.33 µL | 2.00 µL | 12.42 µL | 10.57 µL | 7.93 µL | 10.75 µL |  | 0.47 µL | 1.77 µL | 1.47 µL | 1.30 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 4.00 µL | 2.00 µL | 1.00 µL | 3.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF |  |  | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL |  |  |  |  | 0.50 µL |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| XbaI |  | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  | 0.50 µL | 0.50 µL |  |  |  |  |
| SpeI | 0.50 µL |  |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL |  |  |  |  | 0.50 µL |  | 0.50 µL |  |  |  |  |  |  |
| PstI | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL |  | 0.50 µL |  | 0.50 µL |  | 0.50 µL |  | 0.50 µL |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  |
| H2O | 0.35 µL | 1.09 µL | 2.50 µL | 5.76 µL | 2.12 µL | 34.83 µL | 5.26 µL | 5.74 µL | 5.30 µL | 4.43 µL | 5.50 µL | 5.67 µL | 6.00 µL | 4.08 µL | 5.93 µL | 8.57 µL | 6.25 µL | 8.00 µL | 7.53 µL | 6.23 µL | 6.53 µL | 6.70 µL |
|  | ad | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 40.00 µL | 20.00 µL | 10.00 µL | 30.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt | 2697 bp | 878 bp |  |  | 2200 bp | 1200 bp | 2070 |  |  |  |  |  | 222 |  | 2070 | 2200 | 394 | 252 | 915 | 987 | 954 | 120 |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 8 ng/µL | 6 ng/µL | 10 ng/µL | 10 ng/µL | 28 ng/µL | 3 ng/µL | 19 ng/µL | 10 ng/µL | 6 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 8 ng/µL | 50 ng/µL | 11 ng/µL | 5 ng/µL | 0 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |

The ligations were prepared as follows:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** | **L12** | **L13** | **L14** | **L15** | **L16** | **L17** |
| Product | | pSB1C3\_RNAG120\_MlcRE-pSB1C3-E0240 | pSB1C3-E0240-BBa\_J45503 | pSB1C3-oHybB-34-AOX1 | pSB1C3-mraz | pSB1C3-BBa\_K206000-32sulA | pSB1C3-BBa\_K206000-34sulA | pSB1C3-BBa\_B0032-cspD | pSB1C3-34-AOX1 | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-pSB1C3-BBa\_B0032\_cbtA | pSB1C3-oHybB-pSB1C3-E0240 | pSB1C3\_RNAG120\_MlcRE-pSB1C3-BBa\_I732018 | pSB1C3-cspD | pSB1C3-accD | pSB1C3-ldhA | pSB1C3-ompT | pSB1C3-RNAG120 |
|  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-oHybB | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-oHybB | pSB1C3\_RNAG120\_MlcRE | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 |
| Tube | **1** | **3** | **5** | **7** | **9** | **9** | **14** | **15** | **16** | **16** | **5** | **1** | **7** | **7** | **7** | **7** | **7** |
| ρ | 8 ng/µL | 10 ng/µL | 28 ng/µL | 19 ng/µL | 6 ng/µL | 6 ng/µL | 8 ng/µL | 50 ng/µL | 11 ng/µL | 11 ng/µL | 28 ng/µL | 8 ng/µL | 19 ng/µL | 19 ng/µL | 19 ng/µL | 19 ng/µL | 19 ng/µL |
| Länge | 2697 bp |  | 2200 bp | 2070 bp |  |  |  | 2070 bp | 2200 bp | 2200 bp | 2200 bp | 2697 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp |
| V | 2.97 µL |  | 0.55 µL | 0.61 µL | 4.25 µL | 4.25 µL | 0.00 µL | 0.29 µL | 1.41 µL | 3.26 µL | 0.72 µL | 6.54 µL | 2.54 µL | 0.97 µL | 0.91 µL | 0.94 µL | 3.33 µL |
| **Part 1** | Part 1 | pSB1C3-E0240 | BBa\_J45503 | 34-AOX1 | mraz | 32sulA | 34sulA | cspD | 34-AOX1 | 34-AOX1 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-E0240 | pSB1C3-BBa\_I732018 | cspD | accD | ldhA | ompT | RNAG120 |
| Tube | **2** | **4** | **6** | **8** | **11** | **12** | **13** | **6** | **6** | **17** | **2** | **18** | **13** | **19** | **20** | **21** | **22** |
| ρ | 6 ng/µL | 10 ng/µL | 3 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 3 ng/µL | 3 ng/µL | 5 ng/µL | 6 ng/µL |  | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |
| Länge | 878 bp |  | 1200 bp |  |  |  | 222 bp | 1200 bp | 1200 bp | 394 bp | 878 bp | 252 bp | 222 bp | 915 bp | 987 bp | 954 bp | 120 bp |
| V | 12.75 µL |  | 33.80 µL | 10.00 µL |  |  | 10.00 µL | 34.12 µL | 33.80 µL | 12.83 µL | 12.97 µL |  | 5.17 µL | 8.16 µL | 8.27 µL | 8.22 µL | 3.67 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| T4 Buffer | | 2.00 µL | 1.00 µL | 4.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 4.00 µL | 4.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 1.78 µL | 8.50 µL | 1.14 µL | 6.89 µL | 5.63 µL | 5.63 µL | 7.50 µL | 1.09 µL | 0.29 µL | 1.41 µL | 3.82 µL | 1.96 µL | 0.79 µL | 8.37 µL | 8.32 µL | 8.34 µL | 1.50 µL |
| ad | | 20.00 µL | 10.00 µL | 40.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 40.00 µL | 40.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL |

![](https://static.igem.org/mediawiki/2018/c/c6/T--Hamburg--181001.jpg)

The Gel extration of pSB1C3-RNAG120-MlcRE (~2100 bp) & E0240 (Bande bei ~900 bp) was performed according to the kit’s protocol. Nanodrop measurement showed results as follows:

181001DDW01    pSB1C3-RNAG120-MlcRE : 7.903 ng/µL

181001DDW02    E0240 : 6.166 ng/µL

The concentrations measured after the PCR purification were as follows:

pSB1C3: 25.1 ng/µL

pSB1K3: 41.411 ng/µL

L1 was transformed and incubated at 37°C overnight.

## ldhA characterisation

The characterisation was not carried out, because ldhA was already verified on a SDS-gel. Andreas said, that activity tests would be too time consuming and susceptible to errors. Instead, we made an easy prove of the presence of the enzyme with the gel.

## cPCR for golden gate assemblies

The reactions and the results of the GGA are summarised in the following table:

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Part | fw-Primer | rv-Primer | Template | Tube | concentration (ng/µL) |
| accC | BsaI-accC-f | BsaI-accC-r | 180925BK01 | 1 | multiple bands |
| accD | BsaI-accD-f | BsaI-accD-r | 180925BK02 | 2 | did not work |
| ADH2 | BsaI-ADH2-f | BsaI-ADH2-r | 180925BK03 | 3 | multiple bands |
| kivD | BsaI-kivD-f | BsaI-kivD-r | 180925BK04 | 4 | did not work |
| pBAD | dapAP-BsaI-f | BsaI-pBAD-r | 180925BK05 | 5 |  |
| HlyB | BsaI-HlyB-f | BsaI-HlyB-r | 180925BK06 | 6 | did not work |
| RnaG120 + Terminator | dapAP-BsaI-f | RnaG120-r | 180925NaF01 | 7 |  |

![](https://static.igem.org/mediawiki/2018/8/89/T--Hamburg--181001_PCR_GGA1.JPG)

The PCR programme was performed as follows:

|  |  |  |
| --- | --- | --- |
| Temperature | Time | Steps |
| 98 | 0:20 | 1 |
| 98 | 0:10 | 30 |
| 60 | 0:20 | 30 |
| 72 | 2:00 | 30 |
| 72 | 5:00 | 1 |

PCR Purification of Tube 5 (181001BK01) and Tube 7 (181001BK02).

## cPCR of the transformation from the 30.09.2018

The cPCR reactions were prepared as follows:

|  |  |  |
| --- | --- | --- |
|  | **1x** | **65x** |
| 10x DreamTaq Buffer | 2 µL | 130 µL |
| dNTPs | 0.4 µL | 26 µL |
| VF2 | 0.6 µL | 39 µL |
| VR | 0.6 µL | 39 µL |
| MgCl2 50 mM | 1.2 µL | 78 µL |
| Dream Taq | 0.5 µL | 32.5 µL |
| H2O (ad 20) | 15.1 µL | 981.5 µL |
| Template (von Platten) | 0.1 µL |  |

The cPCR programme was performed as follows:

|  |  |
| --- | --- |
| **Temperatur** | **Zeit** |
| 98 °C | 3:00 |
| 98 °C | 0:30 |
| 55 °C | 0:30 |
| 72 °C | 1:30 |
| go to step to (30x) |  |
| 72 °C | 5:00 |
| 4 °C | ∞ |

![](https://static.igem.org/mediawiki/2018/f/fb/T--Hamburg--181001-1_beschr..png)
![](https://static.igem.org/mediawiki/2018/8/83/T--Hamburg--181001-2_beschr..png)

The marked clones were used to prepare an overnight culture.

## 181001 – 7

## Overnight culture of pBAD-AOX1 (180908NF01) for AOX1 characterisation

The cryostock 180908NF01 was used to set up an ONC.

## 181002 – 1

## Characterisation of AOX1

We have a room temperature of 21.5 °C. The incubator shows a temperature of 25.6 °C. Cultures of 100ml were set up at an OD of 0.1. Induced at 120 min (and respective OD at 120 minutes) with 1 mL of 0.1 mol/L Arabinose. After measurement at 150 min, temperature and OD were measured in 1 h intervalls. After measurement of 570 min, 2 mL of culture was transferred into New medium (100 mL) and Arabinose (1 mL, 0.1 mM) was added to the induced cultures. Cultures were incubated overnight at 16\*C to control for late induction of the promoter.

3 x 100 mL pBAD-AOX1 (induced)

1.

|  |  |  |
| --- | --- | --- |
| Time | OD | Temperature (°C) |
| 0 | 0.1 | 15.5 |
| 30 | 0.13 | 24 |
| 60 | 0.138 | 25 |
| 90 | 0.184 | 26 |
| 120 | 0.234 | 26 |
| 150 | 0.274 | 25.5 |
| 210 | 0.568 | 27.5 |
| 270 | 0.968 | 28 |
| 330 | 1.960 | 28 |
| 390 | 2.260 | 28 |
| 450 | 2.76 | 28.5 |
| 510 | 3.26 (contaminated) | 28.5 |
| 570 | 2.66 | 28.5 |

2.

|  |  |  |
| --- | --- | --- |
| Time | OD | Temperature (°C) |
| 0 | 0.1 | 15.5 |
| 30 | 0.13 | 24 |
| 60 | 0.14 | 25 |
| 90 | 0.164 | 26 |
| 120 | 0.202 | 26 |
| 150 | 0.244 | 25.5 |
| 210 | 0.396 | 27 |
| 270 | 0.786 | 28 |
| 330 | 1.96 | 28 |
| 390 | 2.26 | 28 |
| 450 | 2.80 | 28.5 |
| 510 | 3.52 (contaminated) | 28.5 |
| 570 | 3.30 | 28 |

3.

|  |  |  |
| --- | --- | --- |
| Time | OD | Temperature (°C) |
| 0 | 0.1 | 15.5 |
| 30 | 0.14 | 23.5 |
| 60 | 0.156 | 26 |
| 90 | 0.196 | 26 |
| 120 | 0.276 | 26 |
| 150 | 0.314 | 25 |
| 210 | 0.660 | 27.5 |
| 270 | 0.920 | 28 |
| 330 | 2.06 | 28 |
| 390 | 2.36 | 28 |
| 450 | 3.40 | 28 |
| 510 | 3.28 (contaminated) | 28 |
| 570 | 3.30 | 28 |

1 x 100 mL pBAD-AOX1 (not induced)

|  |  |  |
| --- | --- | --- |
| Time | OD | Temperature (°C) |
| 0 | 0.1 | 15 |
| 30 | 0.11 | 24 |
| 60 | 0.122 | 25 |
| 90 | 0.178 | 26 |
| 120 | 0.190 | 26 |
| 150 | 0.266 | 25 |
| 210 | 0.442 | 27 |
| 270 | 0.956 | 27 |
| 330 | 2.18 | 27 |
| 390 | 2.98 | 27.5 |
| 450 | 3.38 | 28 |
| 510 | 4.36 (contaminated) | 28 |
| 570 | 3.24 | 27.5 |

1 x 100 mL DH5a

|  |  |  |
| --- | --- | --- |
| Time | OD | Temperature (°C) |
| 0 | 0.1 |  |
| 30 | 0.11 | 23.5 |
| 60 | 0.116 | 26 |
| 90 | 0.152 | 25.5 |
| 120 | 0.176 | 26 |
| 150 | 0.198 | 24.5 |
| 210 | 0.385 | 26.5 |
| 270 | 0.764 | 27 |
| 330 | 2.0 | 27 |
| 390 | 2.60 | 27 |
| 450 | 3.38 | 27.5 |
| 510 | 3.97 (contaminated) | 27.5 |
| 570 | 3.90 | 27.5 |

## 181002 – 2

## PCR of pSB1C3 and missing GGA parts

The plan for the PCR was as follows:

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Part | fw-Primer | rv-Primer | Template | Tube | result |
| BsaI-ADH2 | BsaI-ADH2-f | BsaI-ADH2-r | 180726LP03 | 1 | wrong length |
| BsaI-kivD | BsaI-kivD-f | BsaI-kivD-r | 180726LP01 | 2 | no bands |
| BsaI-HlyB | BsaI-HlyB-f | BsaI-HlyB-r | K554013 | 3 | multiple bands |
| pSB1C3 | SB-Prep-3p1 | SB-Prep-2ea | pSB1C3 aus Kit | 4 | no bands |

For HlyB gel extraction was performed.

The PCR programme was as follows:

|  |  |  |
| --- | --- | --- |
| Temperature | Time | Steps |
| 98 | 0:20 | 1 |
| 98 | 0:10 | 30 |
| 60 | 0:20 | 30 |
| 72 | 2:30 (pSB1C3 2:00) | 30 |
| 72 | 5:00 | 1 |

The PCR was prepared as follows:

|  |  |
| --- | --- |
| Volume (µL) | Chemical |
| 20 | H2O |
| 2.5 | rv-Primer |
| 2.5 | fw-Primer |
| 25 | Q5 2X Master Mix |
| 0.25 (pSB1C3 0.5) | Template |

## 181002 – 3

## Minipreps

Minipreps were performed according to the kit’s protocol. The nanodrop measured the concentration as follows:

|  |  |  |
| --- | --- | --- |
| LOT number | part | concentration [ng/µL] |
| 181002NB06 | pSB1C3-cspD | 76.138 |
| 181002NB07 | pSB1C3-oHybB-BBa\_E0240 | 150.571 |
| 181002NB08 | pSB1C3-BBa\_I13453-BBa\_0032-cbtA | 157.52 |
| 181002NB09 | pSB1C3-ldhA | 65.203 |
| 181002NB10 | pSB1C3-ompt | 106.305 |

## 181002 – 4

## Preparation for Sequencing

overall low visibility in the control restriction and light bands

180726LP04 → psB1C3\_accC #10 → too short

180920OM01 → pSB1C3\_OmpA-Aox1 #17 → smear

180914AW02 → pSB1C3-MlcRE → no band

181002NB06 → pSB1C3-cspD → no band

181002NB07 → pSB1C3-oHybB-E0240 → no band

181002NB08 → pSB1C3-BBa\_I13453-BBa\_0032-cbtA → light band (little high but could be right (vf2 vr)

181002NB09 → pSB1C3-ldhA → light right band (vf2 vr)

181002NB010 → pSB1C3-ompT → no band

180912LP02 → pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT → no band

180920OM02 → pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT (#16) → multiple bands, one right band →  retrafo

|  |  |  |
| --- | --- | --- |
| **compound** | **a-h** | **i and j** |
| DNA | 5 µL | 5 µL |
| EcoRI | 0.5 µL |  |
| BsaI |  | 0.5 µL |
| FastAP | 0.5 µL | 0.5 µL |
| Buffer 2.1 | 1 µL |  |
| Buffer CutSmart |  | 1 µL |
| H2O | 3 µL | 3 µL |

## 181002 – 5

## Restriction, ligation and transformation of multiple parts

The Restriction was prepared as follows:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** | **15** | **16** | **17** | **18** | **19** | **20** | **21** | **22** | **23** | **24** | **25** | **26** | **27** |
|  | Part | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-E0240 | BBa\_J45503 | pSB1C3-oHybB | 34-AOX1 | pSB1C3 | mraz | pSB1C3-BBa\_K206000 | 31sulA | 32sulA | 34sulA | cspD | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-BBa\_I732018 | accD | ldhA | ompT | RNAG120 | pSB1K3 | pSB1A3-BBa\_B0034 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA |
|  | Lot# | 180925NaF01 | 180925NaF02 | 180817LD10 | 180625NiF12 | 181001NB05 | 180920LP01 | 180914LP01 | 180622NB08 | neue Miniprep | neue Miniprep | neue Miniprep | 180905AW03 | 180622NB04 | neue Miniprep | 180924AW1 | 180925NaF04 | 180926NaF03 | 181001NB03 | 180921BK09 | 180622NB01 | 180827LP01 | 180909BK09 | 181001NB07 | 180514NB02 | 180912LP02 | 180920OM02 | 180803NB02 |
|  | **ρ** | 140 ng/µL | 144.673 | 200 ng/µL | 45 ng/µL | 107 ng/µL | 575 ng/µL | 89 ng/µL | 44.175 | 50 ng/µL | 28 ng/µL | 40 ng/µL | 43 ng/µL | 50 ng/µL | 81 ng/µL | 95 ng/µL | 126 ng/µL | 93.011 | 162 ng/µL | 213 ng/µL | 57 ng/µL | 68 ng/µL | 77 ng/µL | 41.411 | 65 | 129.25 | 800 ng/µL | 235.7 |
|  | Use | Vector | vector | vector | single insert | vector | single insert | vector | single insert | vector | single insert | single insert | single insert | single insert | vector | vector | vector | vector | vector | single insert | single insert | single insert | single insert | Vector | Vector | Vector | Vector | Vector |
|  | target m | 1000 ng | 1000 ng | 1000 ng | 100 ng | 1000 ng | 100 ng | 1000 ng | 100 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 7.15 µL | 6.91 µL | 5.00 µL | 2.24 µL | 9.34 µL | 0.17 µL | 11.24 µL | 2.26 µL | 20.20 µL | 3.57 µL | 2.50 µL | 2.33 µL | 2.00 µL | 12.42 µL | 10.57 µL | 7.93 µL | 10.75 µL | 6.17 µL | 0.47 µL | 1.77 µL | 1.47 µL | 1.30 µL | 24.15 µL | 15.38 µL | 7.74 µL | 2.50 µL | 8.49 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 4.00 µL | 2.00 µL | 1.00 µL | 3.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 3.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 2.00 µL |
| EcoRI-HF |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL |  |  |  |  | 0.50 µL |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.60 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| XbaI | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |
| SpeI |  |  |  | 0.50 µL |  |  |  |  | 0.50 µL |  |  |  |  | 0.50 µL |  | 0.50 µL |  |  |  |  |  |  |  |  |  |  |  |
| PstI | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.60 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL |  | 0.50 µL |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  | 0.50 µL | 0.50 µL |  |  |  |
| H2O | 0.85 µL | 0.59 µL | 2.50 µL | 5.76 µL | 7.66 µL | 34.83 µL | 5.26 µL | 5.74 µL | 5.30 µL | 4.43 µL | 5.50 µL | 5.67 µL | 6.00 µL | 4.08 µL | 5.93 µL | 8.57 µL | 6.25 µL | 1.83 µL | 7.53 µL | 6.23 µL | 6.53 µL | 6.70 µL | 1.15 µL | 1.12 µL | 9.26 µL | 5.50 µL | 8.51 µL |
|  | ad | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 40.00 µL | 20.00 µL | 10.00 µL | 30.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 30.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 20.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt | 2697 bp | 878 bp |  |  | 2200 bp | 1200 bp | 2070 |  |  |  |  |  | 222 |  | 2070 | 2200 | 394 | 252 | 915 | 987 | 954 | 120 | 2070 | 2070 | 6500 | 6500 | 602 |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | 7 ng/µL | 6 ng/µL | Gelex | Gelex | Gelex | 3 ng/µL | 50 ng/µL | 10 ng/µL | 6 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 8 ng/µL | 50 ng/µL | 7 ng/µL | 50 ng/µL | 100 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 33 ng/µL | Gelex | Gelex | Gelex | Gelex |

The ligation was prepared as follows:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** | **L12** | **L13** | **L14** | **L15** | **L16** | **L17** | **L18** | **L19** | **L20** | **L21** | **L22** | **L23** | **L24** | **L25** | **L26** |
| Product | | pSB1C3\_RNAG120\_MlcRE-E0240 | pSB1C3-E0240-BBa\_J45503 | pSB1C3-oHybB-34-AOX1 | pSB1C3-mraz | pSB1C3-BBa\_K206000-32sulA | pSB1C3-BBa\_K206000-34sulA | pSB1C3-BBa\_B0032-cspD | pSB1C3-34-AOX1 | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-pSB1C3-BBa\_B0032\_cbtA | pSB1C3-oHybB-E0240 | pSB1C3\_RNAG120\_MlcRE-pSB1C3-BBa\_I732018 | pSB1C3-cspD | pSB1C3-accD | pSB1C3-ldhA | pSB1C3-ompT | pSB1C3-RNAG120 | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA |
|  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-oHybB | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-oHybB | pSB1C3\_RNAG120\_MlcRE | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1K3 | pSB1K3 | pSB1K3 | pSB1A3-BBa\_B0034 | pSB1A3-BBa\_B0034 | pSB1A3-BBa\_B0034 |
| Tube | **1** | **3** | **5** | **7** | **9** | **9** | **14** | **15** | **16** | **16** | **5** | **1** | **7** | **7** | **7** | **7** | **7** | **7** | **7** | **7** | **23** | **23** | **23** | **24** | **24** | **24** |
| ρ | 7 ng/µL | Gelex | Gelex | 50 ng/µL | 6 ng/µL | 6 ng/µL | 8 ng/µL | 50 ng/µL | 7 ng/µL | 7 ng/µL | Gelex | 7 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 33 ng/µL | 33 ng/µL | 33 ng/µL | Gelex | Gelex | Gelex |
| Länge | 2697 bp |  | 2200 bp | 2070 bp |  |  |  | 2070 bp | 2200 bp | 2200 bp | 2200 bp | 2697 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp |
| V | 3.15 µL |  |  | 0.61 µL | 4.25 µL | 4.25 µL | 0.00 µL | 0.29 µL | 2.21 µL | 5.12 µL |  | 6.93 µL | 0.97 µL | 0.37 µL | 0.35 µL | 0.36 µL | 1.27 µL | 0.06 µL | 0.12 µL | 1.02 µL | 0.19 µL | 0.19 µL | 1.54 µL |  |  |  |
| **Part 1** | Part 1 | pSB1C3-E0240 | BBa\_J45503 | 34-AOX1 | mraz | 32sulA | 34sulA | cspD | 34-AOX1 | 34-AOX1 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-E0240 | pSB1C3-BBa\_I732018 | cspD | accD | ldhA | ompT | RNAG120 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA |
| Tube | **2** | **4** | **6** | **8** | **11** | **12** | **13** | **6** | **6** | **17** | **2** | **18** | **13** | **19** | **20** | **21** | **22** | **25** | **26** | **27** | **25** | **26** | **27** | **25** | **26** | **27** |
| ρ | 6 ng/µL | Gelex | 3 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 3 ng/µL | 3 ng/µL | 50 ng/µL | 6 ng/µL | 100 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | Gelex | Gelex | Gelex | Gelex | Gelex | Gelex | Gelex | Gelex | Gelex |
| Länge | 878 bp |  | 1200 bp |  |  |  | 222 bp | 1200 bp | 1200 bp | 394 bp | 878 bp | 252 bp | 222 bp | 915 bp | 987 bp | 954 bp | 120 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp |
| V | 6.00 µL |  | 33.80 µL | 10.00 µL |  |  | 10.00 µL | 34.12 µL | 33.80 µL | 1.28 µL | 12.97 µL | 0.48 µL | 5.17 µL | 8.16 µL | 8.27 µL | 8.22 µL | 3.67 µL |  |  |  |  |  |  |  |  |  |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| T4 Buffer | | 2.00 µL | 1.00 µL | 4.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 4.00 µL | 5.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 8.35 µL | 8.50 µL | 1.70 µL | 6.89 µL | 5.63 µL | 5.63 µL | 7.50 µL | 1.09 µL | 8.48 µL | 2.10 µL | 4.53 µL | 1.09 µL | 2.36 µL | -0.02 µL | 8.89 µL | -0.07 µL | 3.56 µL | 8.44 µL | 8.38 µL | 7.48 µL | 8.31 µL | 8.31 µL | 6.96 µL | 8.50 µL | 8.50 µL | 8.50 µL |
| ad | | 20.00 µL | 10.00 µL | 40.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 40.00 µL | 50.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |

Only L1 was performed.

![](https://static.igem.org/mediawiki/2018/b/bf/T--Hamburg--181002.png)

## Preparation of several stuff

1. Of new agar plates with Kan and Amp
2. New stocks of cAMP

## Overnight cultures

1. a) Overnight cultures were prepared from the minipreps of this morning, because gDNA was visible in the test digest

|  |  |  |
| --- | --- | --- |
| **LOT Nummer Plasmid** | **Insert** | **LOT Nummer Cryostock** |
| 180726LP04 | psB1C3\_accC #10 | 180727DK04 |
| 180920OM01 | pSB1C3\_OmpA-Aox1 #17 | No cryostock available |
| 180914AW02 | pSB1C3-MlcRE | 180908NF03 |
| 181002NB06 | pSB1C3-cspD | 181002NB01 |
| 181002NB07 | pSB1C3-oHybB-E0240 | 181002NB02 |
| 181002NB08 | pSB1C3-BBa\_I13453-BBa\_0032-cbtA | 181002NB03 |
| 181002NB09 | pSB1C3-ldhA | 181002NB04 |
| 181002NB010 | pSB1C3-ompT | 181002NB05 |
| 180912LP02 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | 180912LP01 |
| 180920OM02 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT (#16) | 180917AK02 |

1. c) Overnight culture of pSB1C3-BBa\_K206000-BBa\_0034-ldhA for production of cell lysates for the characterisation on Wednesday → 180518BK11
2. d) Check, if there are still enough sample for restriction for tomorrow, otherwise new overnight cultures need to be prepared. Result: everything is empty, so new overnight cultures were prepared from the following lot numbers:

180912LP01

180917AK02

180803NB01 → pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

## 181003 – 1

## Characterisation of AOX1

Reactions from yesterday were cultured overnight at 16 °C and shook.

3 x 100 mL pBAD-AOX1 (induced)

1.

|  |  |  |
| --- | --- | --- |
| Time [am] | OD | Temperature (°C) |
| 9:30 |  | 17.5 |
| 10:30 |  | 18 |
| 11:30 | 1.88 | 18 |

2.

|  |  |  |
| --- | --- | --- |
| Time [am] | OD | Temperature (°C) |
| 9:30 |  | 18.5 |
| 10:30 |  | 18 |
| 11:30 | 1.60 | 18 |

3.

|  |  |  |
| --- | --- | --- |
| Time [am] | OD | Temperature (°C) |
| 9:30 |  | 18 |
| 10:30 |  | 18 |
| 11:30 | 2.10 | 18.5 |

1x 100ml pBAD-AOX1 not induced

|  |  |  |
| --- | --- | --- |
| Time [am] | OD | Temperature (°C) |
| 9:30 |  | 18 |
| 10:30 |  | 18 |
| 11:30 | 2.66 | 18 |

1x 100ml DH5a

|  |  |  |
| --- | --- | --- |
| Time [am] | OD | Temperature (°C) |
| 9:30 |  | 18 |
| 10:30 |  | 18.2 |
| 11:30 | 2.74 | 19 |

The experiment was cancelled after 11:30 h, because there was no observable change anymore.

## 181003 – 2

## Miniprep purification of the overnight cultures of yesterday

The products of the minipreps got new lot numbers afterwards:

|  |  |  |
| --- | --- | --- |
| 181003NB01 | 03.10.2018 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT (#16) |
| 181003NB02 | 03.10.2018 | pSB1C3-BBa\_I13453-BBa\_0032-cbtA |
| 181003NB03 | 03.10.2018 | pSB1C3-MlcRE |
| 181003NB04 | 03.10.2018 | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA |
| 181003NB05 | 03.10.2018 | psB1C3\_accC #10 |
| 181003NB06 | 03.10.2018 | pSB1C3-oHybB-BBa\_E0240 |
| 181003NB07 | 03.10.2018 | pSB1C3-cspD |
| 181003NB08 | 03.10.2018 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT |
| 181003NB09 | 03.10.2018 | pSB1C3-ldhA |
| 181003NB10 | 03.10.2018 | pSB1C3-ompt |

## 181003 – 3

## Restriction, ligation, transformation

The restriction was prepared as follows:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** | **15** | **16** | **17** | **18** | **19** | **20** | **21** | **22** | **23** | **24** | **25** | **26** | **27** | **28** | **29** | **30** |
|  | Part | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-E0240 | BBa\_J45503 | pSB1C3-oHybB | 34-AOX1 | pSB1C3 | mraz | pSB1C3-BBa\_K206000 | 31sulA | 32sulA | 34sulA | cspD | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-BBa\_I732018 | accD | ldhA | ompT | RNAG120 | pSB1K3 | pSB1A3-BBa\_B0034 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3 | pSB1C3 | pSB1A3-BBa\_B0034 |
|  | Lot# | 180925NaF01 | 180925NaF02 | 180817LD10 | 180625NiF12 | 181001NB05 | 180920LP01 | backbone | 180622NB08 | neue Miniprep | neue Miniprep | neue Miniprep | 180905AW03 | 180622NB04 | neue Miniprep | 180924AW1 | 180925NaF04 | 180926NaF03 | 181001NB03 | 180921BK09 | 180622NB01 | 180827LP01 | 180909BK09 | backbone | 180514NB02 | 180912LP02 | 180920OM02 | 181003NB04 | backbone | backbone | 180514NB02 |
|  | **ρ** | 140 ng/µL | 144.673 | 200 ng/µL | 45 ng/µL | 107 ng/µL | 575 ng/µL | 25 ng/µL | 44.175 | 50 ng/µL | 28 ng/µL | 40 ng/µL | 43 ng/µL | 50 ng/µL | 81 ng/µL | 95 ng/µL | 126 ng/µL | 93.011 | 162 ng/µL | 213 ng/µL | 57 ng/µL | 68 ng/µL | 77 ng/µL | 25 ng/µL | 65 | 129 ng/µL | 800 ng/µL | 267 ng/µL | 25 ng/µL | 25 ng/µL | 65 |
|  | Use | Vector | vector | vector | single insert | vector | single insert | vector | single insert | vector | single insert | single insert | single insert | single insert | vector | vector | vector | vector | vector | single insert | single insert | single insert | single insert | vector | vector | vector | vector | vector | vector | vector | vector |
|  | target m | 1000 ng | 1000 ng | 1000 ng | 100 ng | 1000 ng | 100 ng | 500 ng | 100 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng | 200 ng | 1000 ng | 1000 ng | 1000 ng | 2000 ng | 100 ng | 200 ng | 200 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 7.15 µL | 6.91 µL | 5.00 µL | 2.24 µL | 9.34 µL | 0.17 µL | 20.00 µL | 2.26 µL | 20.20 µL | 3.57 µL | 2.50 µL | 2.33 µL | 2.00 µL | 12.42 µL | 10.57 µL | 7.93 µL | 10.75 µL | 12.34 µL | 0.47 µL | 1.77 µL | 1.47 µL | 1.30 µL | 8.00 µL | 30.77 µL | 15.47 µL | 2.50 µL | 20.00 µL | 8.00 µL | 8.00 µL | 8.00 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 4.00 µL | 3.00 µL | 1.00 µL | 3.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 4.00 µL | 2.00 µL | 1.00 µL | 3.00 µL | 2.00 µL | 2.00 µL | 2.00 µL |
| EcoRI-HF |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL |  |  |  |  | 0.50 µL |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| XbaI | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |  |  |  |
| SpeI |  |  |  | 0.50 µL |  |  |  |  | 0.50 µL |  |  |  |  | 0.50 µL |  | 0.50 µL |  |  |  |  |  |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| PstI | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |
| FastAP |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL |  | 0.50 µL |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | 0.85 µL | 0.59 µL | 2.50 µL | 5.76 µL | 7.66 µL | 34.83 µL | 5.50 µL | 5.74 µL | 5.30 µL | 4.43 µL | 5.50 µL | 5.67 µL | 6.00 µL | 4.08 µL | 5.93 µL | 8.57 µL | 6.25 µL | 4.66 µL | 7.53 µL | 6.23 µL | 6.53 µL | 6.70 µL | 8.50 µL | 3.73 µL | 1.53 µL | 5.50 µL | 6.00 µL | 8.50 µL | 8.50 µL | 8.50 µL |
|  | ad | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 40.00 µL | 30.00 µL | 10.00 µL | 30.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 40.00 µL | 20.00 µL | 10.00 µL | 30.00 µL | 20.00 µL | 20.00 µL | 20.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt | 2697 bp | 878 bp |  |  | 2200 bp | 1200 bp | 2070 |  |  |  |  |  | 222 |  | 2070 | 2200 | 394 | 252 | 915 | 987 | 954 | 120 | 2070 | 2070 | 6500 | 6500 | 602 | 2070 | 2070 | 2070 |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | Gelex | 17 ng/µL | Gelex | Gelex | 5 ng/µL | Gelex | 30 ng/µL | 10 ng/µL | 33 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 8 ng/µL | 50 ng/µL | 8 ng/µL | 8 ng/µL | 7 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 19 ng/µL | 16 ng/µL | Gelex | Gelex | 11 ng/µL | 16 ng/µL | 14 ng/µL | 26 ng/µL |

The restriction was performed at 37 °C for 1h followed by 80 °C for 20 min.

Afterwards, the samples were put on a 1 % agarose gel for gel electrophoresis.

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA was cut three times, because the double digest results in two fragments of the same size, of which one is the desired one and the second part of the vector (third enzyme NcoI cuts only in the vector fragment)

a double restriction reaction is necessary, because NcoI is available only from Thermo Fisher

![](https://static.igem.org/mediawiki/2018/f/f6/T--Hamburg--181003-beschriftet.jpg)

Framed bands were cut out and used for ligation.

Samples 180518BK15, 181001NB02 were empty and were re-produced with a new overnight culture.

![](https://static.igem.org/mediawiki/2018/2/2c/T--Hamburg--181003-beschriftet_%28ua_PCR%29.jpg)

Second digest of pGGA-pBAD-BjalT-OmpT-FLAG-HlyA, each upper band was cut out for ligation

The ligation was prepared as follows:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** | **L12** | **L13** | **L14** | **L15** | **L16** | **L17** | **L18** | **L19** | **L20** | **L21** | **L22** | **L23** | **L24** | **L25** | **L26** |
| Product | | pSB1C3\_RNAG120\_MlcRE-pSB1C3-E0240 | pSB1C3-E0240-BBa\_J45503 | pSB1C3-oHybB-34-AOX1 | pSB1C3-mraz | pSB1C3-BBa\_K206000-32sulA | pSB1C3-BBa\_K206000-34sulA | pSB1C3-BBa\_B0032-cspD | pSB1C3-34-AOX1 | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-pSB1C3-BBa\_B0032\_cbtA | pSB1C3-oHybB-pSB1C3-E0240 | pSB1C3\_RNAG120\_MlcRE-pSB1C3-BBa\_I732018 | pSB1C3-cspD | pSB1C3-accD | pSB1C3-ldhA | pSB1C3-ompT | pSB1C3-RNAG120 | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA |
|  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-oHybB | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-oHybB | pSB1C3\_RNAG120\_MlcRE | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1K3 | pSB1K3 | pSB1K3 | pSB1A3-BBa\_B0034 | pSB1A3-BBa\_B0034 | pSB1A3-BBa\_B0034 |
| Tube | **1** | **3** | **5** | **7** | **9** | **9** | **14** | **15** | **16** | **16** | **5** | **1** | **7** | **7** | **7** | **7** | **7** | **7** | **7** | **29** | **23** | **23** | **28** | **24** | **24** | **30** |
| ρ | Gelex | Gelex | 5 ng/µL | 30 ng/µL | 33 ng/µL | 33 ng/µL | 8 ng/µL | 50 ng/µL | 8 ng/µL | 8 ng/µL | 5 ng/µL | Gelex | 30 ng/µL | 30 ng/µL | 30 ng/µL | 30 ng/µL | 30 ng/µL | 30 ng/µL | 30 ng/µL | 14 ng/µL | 19 ng/µL | 19 ng/µL | 16 ng/µL | 16 ng/µL | 16 ng/µL | 26 ng/µL |
| Länge | 2697 bp |  | 2200 bp | 2070 bp |  |  |  | 2070 bp | 2200 bp | 2200 bp | 2200 bp | 2697 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp |
| V |  |  | 2.90 µL | 0.61 µL | 4.25 µL | 4.25 µL | 0.00 µL | 0.29 µL | 1.92 µL | 4.45 µL | 3.75 µL |  | 1.62 µL | 0.62 µL | 0.58 µL | 0.60 µL | 2.13 µL | 0.10 µL | 0.10 µL | 1.83 µL | 0.16 µL | 0.16 µL | 1.60 µL | 0.19 µL | 0.19 µL | 0.98 µL |
| **Part 1** | Part 1 | pSB1C3-E0240 | BBa\_J45503 | 34-AOX1 | mraz | 32sulA | 34sulA | cspD | 34-AOX1 | 34-AOX1 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-E0240 | pSB1C3-BBa\_I732018 | cspD | accD | ldhA | ompT | RNAG120 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA |
| Tube | **2** | **4** | **6** | **8** | **11** | **12** | **13** | **6** | **6** | **17** | **2** | **18** | **13** | **19** | **20** | **21** | **22** | **25** | **26** | **27** | **25** | **26** | **27** | **25** | **26** | **27** |
| ρ | 17 ng/µL | Gelex | Gelex | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | Gelex | Gelex | 8 ng/µL | 17 ng/µL | 7 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | Gelex | Gelex | 11 ng/µL | Gelex | Gelex | 11 ng/µL | Gelex | Gelex | 11 ng/µL |
| Länge | 878 bp |  | 1200 bp |  |  |  | 222 bp | 1200 bp | 1200 bp | 394 bp | 878 bp | 252 bp | 222 bp | 915 bp | 987 bp | 954 bp | 120 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp |
| V | 4.54 µL |  |  | 10.00 µL |  |  | 10.00 µL |  |  | 8.54 µL | 4.75 µL | 7.20 µL | 5.17 µL | 8.16 µL | 8.27 µL | 8.22 µL | 3.67 µL |  |  | 6.88 µL |  |  | 6.88 µL |  |  | 6.88 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| T4 Buffer | | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 3.96 µL | 8.50 µL | 5.60 µL | 6.89 µL | 5.63 µL | 5.63 µL | 7.50 µL | 8.21 µL | 6.58 µL | 4.51 µL | 0.00 µL | 1.30 µL | 1.70 µL | 8.72 µL | 8.65 µL | 8.68 µL | 2.70 µL | 8.40 µL | 8.40 µL | 8.79 µL | 8.34 µL | 8.34 µL | 0.02 µL | 8.31 µL | 8.31 µL | 0.64 µL |
| ad | | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |

## 181003 – 4

## cPCR of the two plates pSB1C3-NOT-MlcRE-E0240

One plate was from yesterday, one from the 01.10.

The master mix was prepared as follows:

|  |  |  |
| --- | --- | --- |
|  | **1x** | **22x** |
| 10x Dream Taq Buffer | 2 µL | 44 µL x |
| dNTPs 10 mM | 0.4 µL | 8.8 µLx |
| MgCl2 | 1.2 µL | 26.4 µLx |
| Dream Taq Polymerase | 0.1 µL | 2.2 µL |
| fw Primer VF2 | 0.6 µL | 13.2 µL |
| rv Primer VR | 0.6 µL | 13.2 µL |
| H2O ad 20 µL | 15.1 | 332.2 µL x |

The cPCR programme was as follows:

|  |  |
| --- | --- |
| Temperature | Time [min] |
| 98 °C | 3:00 |
| 98 °C | 0:30 |
| 55 °C | 0:30 |
| 72 °C | 2:00 |
| go to step to (30x) |  |
| 72 °C | 5:00 |
| 4 °C | ∞ |

The plate from the 1.10.2018 had just one grown colony which was picked, the plate from yesterday hat a lot more colonies, of which 18 were picked.

![](https://static.igem.org/mediawiki/2018/b/be/T--Hamburg--181003_cPCR_NOT-MlcRE-E0240.JPG)

No clone was positive, a new restriction is necessary.

## 181003 – 5

## Preparation for the activation assay of ldha

The part used for the activation assay was pSB1C3-BBa\_K206000-BBa\_0034-ldha.

Preparation of 3 x 200 mL cultures.

|  |  |  |  |
| --- | --- | --- | --- |
| Medium | Inoculated with | Arabinose-Induction | OD at point of induction |
| 200ml LB  plus cAMP | 180703LP02 | 2ml (0.1mol/L) | 1 |
| 200ml LB plus cAMP | 180703LP02 | - | - |
| 200ml LB | DH5a | - | - |

Samples were centrifuged (20 min, 5000 rpm, 4 °C) to get a pellet. This pellet was stored at – 20 °C.

## 181003 – 6

## PCR of synthetic DNA RNAG120-MlcRE

This was done in prospect to further experiments of the restriction for the next day.

PCR preparation according to the following table:

|  |  |
| --- | --- |
| 10x Dream Taq Buffer | 2 µL |
| dNTPs 10 mM | 0.4 µL |
| MgCl2 | 1.2 µL |
| Dream Taq Polymerase | 0.1 µL |
| fw Primer VF2 | 0.6 µL |
| rv Primer VR | 0.6 µL |
| H2O ad 20 µL | 15.1 µL |

PCR Programm as follows:

|  |  |
| --- | --- |
| Temperature | Zeit [min] |
| 95 °C | 3:00 |
| 95 °C | 0:30 |
| 55 °C | 0:30 |
| 72 °C | 0:45 |
| go to step to (30x) |  |
| 72 °C | 5:00 |
| 4 °C | ∞ |

Purification was done using the PCR purification kit following the protocol. The final concentration was measured with Nanodrop:

Lot number: 181003NiF01 with the concentration of 82.666 ng/µL.

## Restriction, Ligation and transformation of pSB1C3-NOT-MlcRE-E0240

Restriction:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** |
|  | Part | RNAG120-MlcRE | pSB1K3 | pSB1C3-E0240 |
|  | Lot# | 181003NiF01 | backbone | 180925NaF02 |
|  | **ρ** | 83 ng/µL | 25 ng/µL | 145 ng/µL |
|  | Use | single insert | vector | vector |
|  | target m | 100 ng | 200 ng | 1000 ng |
|  |  |  |  |  |
| Volumes | DNA | 1.21 µL | 8.00 µL | 6.91 µL |
| NEBuffer 2.1 | 1.00 µL | 2.00 µL | 1.00 µL |
| EcoRI-HF | 0.50 µL | 0.50 µL |  |
| XbaI |  |  | 0.50 µL |
| SpeI | 0.50 µL |  |  |
| PstI |  | 0.50 µL | 0.50 µL |
| FastAP |  | 0.50 µL | 0.50 µL |
| H2O | 6.79 µL | 8.50 µL | 0.59 µL |
|  | ad | 10.00 µL | 20.00 µL | 10.00 µL |
|  |  |  |  |  |
|  | Länge Produkt | 546 | 2070 | 878 |
|  |  |  |  |  |
|  | ρ final | 10 ng/µL | 13 ng/µL | 2 ng/µL |

## Ligation:

|  |  |  |
| --- | --- | --- |
| Tube | | **L1** |
| Product | | pSB1K3-RNAG120-MlcRE-pSB1C3-E0240 |
|  | |  |
| **Vector** | Vector | pSB1K3 |
| Tube | **2** |
| ρ | 13 ng/µL |
| Länge | 2070 bp |
| V | 1.00 µL |
| **Part 1** | Part 1 | RNAG120-MlcRE |
| Tube | **1** |
| ρ | 10 ng/µL |
| Länge | 546 bp |
| V | 3.35 µL |
| **Part 2** | Part 2 | pSB1C3-E0240 |
| Tube | **3** |
| ρ | 2 ng/µL |
| Länge | 878 bp |
| V | 24.75 µL |
| T4 Buffer | | 4.00 µL |
| T4 Ligase | | 0.50 µL |
| H2O | | 6.41 µL |
| ad | | 40.00 µL |

## Transformation:

The transformation was made with protocol by Zhang Ghong. Transformation on a kanamycin plate.

## Restriction, Ligation and transformation of pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

Restriction:

|  |  |  |
| --- | --- | --- |
| **Tube** | **27** | **28** |
| **Part** | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3 |
| **Lot#** | 181003NB04 | backbone |
| **ρ** | 267 ng/µL | 25 ng/µL |
| Use | vector | vector |
| target m | 1000 ng | 100 ng |
|  |  |  |
| DNA | 7.49 µL | 4.00 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL |
| EcoRI-HF | 0.50 µL | 0.50 µL |
| XbaI |  |  |
| SpeI | 0.50 µL | 0.50 µL |
| PstI |  |  |
| FastAP |  | 0.50 µL |
| H2O | 0.51 µL | 3.50 µL |
| ad | 10.00 µL | 10.00 µL |
|  |  |  |
| Länge Produkt | 602 | 2070 |
|  |  |  |
| ρ final | Gelex | 10 ng/µL |

## Ligation

|  |  |  |
| --- | --- | --- |
| Tube | | **L23** |
| Product | | pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA |
|  | |  |
| **Vector** | Vector | pSB1K3 |
| Tube | **28** |
| ρ | 10 ng/µL |
| Länge | 2070 bp |
| V | 2.56 µL |
| **Part 1** | Part 1 | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA |
| Tube | **27** |
| ρ | Gelex |
| Länge | 602 bp |
| V |  |
| **Part 2** | Part 2 |  |
| Tube |  |
| ρ |  |
| Länge |  |
| V |  |
| T4 Buffer | | 1.00 µL |
| T4 Ligase | | 0.50 µL |
| H2O | | 5.94 µL |
| ad | | 10.00 µL |

## Transformation

The transformation was made with protocol by Zhang Ghong. Transformation on a kanamycin plate.

## Digestion test

|  |  |  |  |
| --- | --- | --- | --- |
| LOT Number | Part | Test restriction number | Einschicken |
| 181003NB03 | pSB1C3-MlcRE | 1 | ja (mit vr) |
| 181003NB05 | psB1C3\_accC #10 | 2 | ja (vf2) |
| 181003NB06 | pSB1C3-oHybB-BBa\_E0240 | 3 | keine Bande |
| 181003NB07 | pSB1C3-cspD | 4 | ja(aber wahrscheinlich was anderes) |
| 181003NB08 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | 5 |  |

|  |  |
| --- | --- |
| compound | volume |
| DNA | 5 µL |
| EcoRI | 0.5 µL |
| FastAP | 0.5 µL |
| Buffer 2.1 | 1 µL |
| H2O | 3 µL |

## Gelelectrophoresis

Gel of the digestion test.

![](https://static.igem.org/mediawiki/2018/4/43/T--Hamburg--181004_Testverdau_und_Restriktion.png)

## Characterization of ldhA

According to protocol “ldhA Charakterisierung”.

Preparation of the buffer containing 0.5. mM EDTA and 1 mg/ml lysozyme for 40 mL:

* 5.8 mg EDTA
* 40 mg Lysozym
* 40 ml ddH20

Preparation of the cell lysates

* Resuspension of the cell pellets in 2 mL buffer
* Incubation for 1 hour on ice

Activity assay

Mastermix:

* 1300 µL Na-Pyrophosphate buffer
* 100 µL NAD+
* 200 µL INT
* 200 µL PMS

* 100 µL 40% Lactic acid solution (0.0013 M)
* 100 µL cell lysate (1000 µg protein)

Measurement

3 min in 20sec Abständen messen

Results:

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| DH5 alpha 1000 µg |  |  | ldhA + 1000 µg |  |  | ldhA- 1000 µg |  |
| Time | Durchschnittliche Extinktion |  | time | Durchschnitt Extinktion |  | time | Extinktion |
| 0 | 0 |  | 0 | 0 |  | 0 | 0 |
| 20 | 1.14525 |  | 20 | 0.379 |  | 20 | 0.616 |
| 40 | 1.264 |  | 40 | 0.426 |  | 40 | 0.681 |
| 60 | 1.283 |  | 60 | 0.459 |  | 60 | 0.721 |
| 80 | 1.31925 |  | 80 | 0.484 |  | 80 | 0.7513333333 |
| 100 | 1.3325 |  | 100 | 0.504 |  | 100 | 0.7803333333 |
| 120 | 1.35375 |  | 120 | 0.524 |  | 120 | 0.585 |
| 140 | 1.36475 |  | 140 | 0.538 |  | 140 | 0.8676666667 |
| 160 | 1.3845 |  | 160 | 0.552 |  | 160 | 0.8496666667 |
| 180 | 1.28225 |  | 180 | 0.566 |  | 180 | 0.853 |

## Minipreps of 180518BK15, 181001NB02

PCR of constructs from 03.10.2018 and L1 from 02.10.2018:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** | **L12** | **L13** | **L14** | **L15** | **L16** | **L17** | **L18** | **L19** | **L20** | **L21** | **L22** | **L23** | **L24** | **L25** | **L26** |
| Product | | pSB1C3\_RNAG120\_MlcRE-pSB1C3-E0240 | pSB1C3-E0240-BBa\_J45503 | pSB1C3-oHybB-34-AOX1 | pSB1C3-mraz | pSB1C3-BBa\_K206000-32sulA | pSB1C3-BBa\_K206000-34sulA | pSB1C3-BBa\_B0032-cspD | pSB1C3-34-AOX1 | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-pSB1C3-BBa\_B0032\_cbtA | pSB1C3-oHybB-pSB1C3-E0240 | pSB1C3\_RNAG120\_MlcRE-pSB1C3-BBa\_I732018 | pSB1C3-cspD | pSB1C3-accD | pSB1C3-ldhA | pSB1C3-ompT | pSB1C3-RNAG120 | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA |
|  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-oHybB | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-oHybB | pSB1C3\_RNAG120\_MlcRE | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1K3 | pSB1K3 | pSB1K3 | pSB1A3-BBa\_B0034 | pSB1A3-BBa\_B0034 | pSB1A3-BBa\_B0034 |
| Tube | **1** | **3** | **5** | **7** | **9** | **9** | **14** | **15** | **16** | **16** | **5** | **1** | **7** | **7** | **7** | **7** | **7** | **7** | **7** | **29** | **23** | **23** | **28** | **24** | **24** | **30** |
| ρ | Gelex | Gelex | 5 ng/µL | 30 ng/µL | 33 ng/µL | 33 ng/µL | 8 ng/µL | 50 ng/µL | 8 ng/µL | 8 ng/µL | 5 ng/µL | Gelex | 30 ng/µL | 30 ng/µL | 30 ng/µL | 30 ng/µL | 30 ng/µL | 30 ng/µL | 30 ng/µL | 14 ng/µL | 19 ng/µL | 19 ng/µL | 16 ng/µL | 16 ng/µL | 16 ng/µL | 26 ng/µL |
| Länge | 2697 bp |  | 2200 bp | 2070 bp |  |  |  | 2070 bp | 2200 bp | 2200 bp | 2200 bp | 2697 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp |
| V |  |  | 2.90 µL | 0.61 µL | 4.25 µL | 4.25 µL | 0.00 µL | 0.29 µL | 1.92 µL | 4.45 µL | 3.75 µL |  | 1.62 µL | 0.62 µL | 0.58 µL | 0.60 µL | 2.13 µL | 0.10 µL | 0.10 µL | 1.83 µL | 0.16 µL | 0.16 µL | 1.60 µL | 0.19 µL | 0.19 µL | 0.98 µL |
| **Part 1** | Part 1 | pSB1C3-E0240 | BBa\_J45503 | 34-AOX1 | mraz | 32sulA | 34sulA | cspD | 34-AOX1 | 34-AOX1 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-E0240 | pSB1C3-BBa\_I732018 | cspD | accD | ldhA | ompT | RNAG120 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA |
| Tube | **2** | **4** | **6** | **8** | **11** | **12** | **13** | **6** | **6** | **17** | **2** | **18** | **13** | **19** | **20** | **21** | **22** | **25** | **26** | **27** | **25** | **26** | **27** | **25** | **26** | **27** |
| ρ | 17 ng/µL | Gelex | Gelex | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | Gelex | Gelex | 8 ng/µL | 17 ng/µL | 7 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | Gelex | Gelex | 11 ng/µL | Gelex | Gelex | 11 ng/µL | Gelex | Gelex | 11 ng/µL |
| Länge | 878 bp |  | 1200 bp |  |  |  | 222 bp | 1200 bp | 1200 bp | 394 bp | 878 bp | 252 bp | 222 bp | 915 bp | 987 bp | 954 bp | 120 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp |
| V | 4.54 µL |  |  | 10.00 µL |  |  | 10.00 µL |  |  | 8.54 µL | 4.75 µL | 7.20 µL | 5.17 µL | 8.16 µL | 8.27 µL | 8.22 µL | 3.67 µL |  |  | 6.88 µL |  |  | 6.88 µL |  |  | 6.88 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| T4 Buffer | | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 3.96 µL | 8.50 µL | 5.60 µL | 6.89 µL | 5.63 µL | 5.63 µL | 7.50 µL | 8.21 µL | 6.58 µL | 4.51 µL | 0.00 µL | 1.30 µL | 1.70 µL | 8.72 µL | 8.65 µL | 8.68 µL | 2.70 µL | 8.40 µL | 8.40 µL | 8.79 µL | 8.34 µL | 8.34 µL | 0.02 µL | 8.31 µL | 8.31 µL | 0.64 µL |
| ad | | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** | **L12** | **L13** | **L14** | **L15** | **L16** | **L17** | **L18** | **L19** | **L20** | **L21** | **L22** | **L23** | **L24** | **L25** | **L26** |
| Product | | pSB1C3\_RNAG120\_MlcRE-E0240 | pSB1C3-E0240-BBa\_J45503 | pSB1C3-oHybB-34-AOX1 | pSB1C3-mraz | pSB1C3-BBa\_K206000-32sulA | pSB1C3-BBa\_K206000-34sulA | pSB1C3-BBa\_B0032-cspD | pSB1C3-34-AOX1 | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-pSB1C3-BBa\_B0032\_cbtA | pSB1C3-oHybB-E0240 | pSB1C3\_RNAG120\_MlcRE-pSB1C3-BBa\_I732018 | pSB1C3-cspD | pSB1C3-accD | pSB1C3-ldhA | pSB1C3-ompT | pSB1C3-RNAG120 | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA |
|  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-oHybB | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-oHybB | pSB1C3\_RNAG120\_MlcRE | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1K3 | pSB1K3 | pSB1K3 | pSB1A3-BBa\_B0034 | pSB1A3-BBa\_B0034 | pSB1A3-BBa\_B0034 |
| Tube | **1** | **3** | **5** | **7** | **9** | **9** | **14** | **15** | **16** | **16** | **5** | **1** | **7** | **7** | **7** | **7** | **7** | **7** | **7** | **7** | **23** | **23** | **23** | **24** | **24** | **24** |
| ρ | 7 ng/µL | Gelex | Gelex | 50 ng/µL | 6 ng/µL | 6 ng/µL | 8 ng/µL | 50 ng/µL | 7 ng/µL | 7 ng/µL | Gelex | 7 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 50 ng/µL | 33 ng/µL | 33 ng/µL | 33 ng/µL | Gelex | Gelex | Gelex |
| Länge | 2697 bp |  | 2200 bp | 2070 bp |  |  |  | 2070 bp | 2200 bp | 2200 bp | 2200 bp | 2697 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp |
| V | 3.15 µL |  |  | 0.61 µL | 4.25 µL | 4.25 µL | 0.00 µL | 0.29 µL | 2.21 µL | 5.12 µL |  | 6.93 µL | 0.97 µL | 0.37 µL | 0.35 µL | 0.36 µL | 1.27 µL | 0.06 µL | 0.12 µL | 1.02 µL | 0.19 µL | 0.19 µL | 1.54 µL |  |  |  |
| **Part 1** | Part 1 | pSB1C3-E0240 | BBa\_J45503 | 34-AOX1 | mraz | 32sulA | 34sulA | cspD | 34-AOX1 | 34-AOX1 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-E0240 | pSB1C3-BBa\_I732018 | cspD | accD | ldhA | ompT | RNAG120 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA |
| Tube | **2** | **4** | **6** | **8** | **11** | **12** | **13** | **6** | **6** | **17** | **2** | **18** | **13** | **19** | **20** | **21** | **22** | **25** | **26** | **27** | **25** | **26** | **27** | **25** | **26** | **27** |
| ρ | 6 ng/µL | Gelex | 3 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 3 ng/µL | 3 ng/µL | 50 ng/µL | 6 ng/µL | 100 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | Gelex | Gelex | Gelex | Gelex | Gelex | Gelex | Gelex | Gelex | Gelex |
| Länge | 878 bp |  | 1200 bp |  |  |  | 222 bp | 1200 bp | 1200 bp | 394 bp | 878 bp | 252 bp | 222 bp | 915 bp | 987 bp | 954 bp | 120 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp |
| V | 6.00 µL |  | 33.80 µL | 10.00 µL |  |  | 10.00 µL | 34.12 µL | 33.80 µL | 1.28 µL | 12.97 µL | 0.48 µL | 5.17 µL | 8.16 µL | 8.27 µL | 8.22 µL | 3.67 µL |  |  |  |  |  |  |  |  |  |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| ρ |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Länge |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| V |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| T4 Buffer | | 2.00 µL | 1.00 µL | 4.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 4.00 µL | 5.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 8.35 µL | 8.50 µL | 1.70 µL | 6.89 µL | 5.63 µL | 5.63 µL | 7.50 µL | 1.09 µL | 8.48 µL | 2.10 µL | 4.53 µL | 1.09 µL | 2.36 µL | -0.02 µL | 8.89 µL | -0.07 µL | 3.56 µL | 8.44 µL | 8.38 µL | 7.48 µL | 8.31 µL | 8.31 µL | 6.96 µL | 8.50 µL | 8.50 µL | 8.50 µL |
| ad | | 20.00 µL | 10.00 µL | 40.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 40.00 µL | 50.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |

Mastermix cPCR:

|  |  |  |
| --- | --- | --- |
|  | **1x** | **60 x** |
| 10x Dream Taq Buffer | 2 µL | 120 µL |
| dNTPs 10 mM | 0.4 µL | 24 µL |
| MgCl2 | 1.2 µL | 72 µL |
| Dream Taq Polymerase | 0.1 µL | 6 µL |
| fw Primer VF2 | 0.6 | 36 µL |
| rv Primer VR | 0.6 | 36 µL |
| H2O ad 20 µL | 15.1 | 906 µL |

## Gelelectrophoresis

![](https://static.igem.org/mediawiki/2018/3/31/T--Hamburg--181004_cPCR.png)

## Excretion Module Assembly

Following things were incubated overnight for 10 hours and inactivated at 50 °C for 10 mins and 80°C for 10 mins: 4µL BsaI-HlyB, 0.8 µL BsaI-TolC, 0.8 µL BsaI-OmpT, 0.2 µL BsaI-pBAD, 2.2 µL H2O, 0.5 µL BsaI-HF, 0.5 µL T4 Ligase, 1µL Ligase buffer.

0.5 µL of this reaction was used as a PCR-Template with 1.25 µL of each BBSyn-f- and BB-Syn-r-Primer, 10 µL H2O and 12 µL Q5 2X MasterMix. The band at roughly 6000 bp was extracted and restriction and ligation was performed according to the tables. Transformation was performed using the Zhang protocol.

![](https://static.igem.org/mediawiki/2018/a/af/T--Hamburg--181004_GGA_PCR.JPG)

## Restriction

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** |
|  | Part | pSB1C3 | pBAD-HlyB-HlyD-TolC-OmpT | pBAD-HlyB-HlyD-TolC-OmpT |
|  | Lot# | aus Kit | R | P |
|  | **ρ** | 25 ng/µL | 15 ng/µL | 10 ng/µL |
|  | Use |  |  |  |
|  | target m |  |  |  |
|  |  |  |  |  |
| Volumes | DNA | 4.00 µL | 7.00 µL | 7.00 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF | 0.50 µL | 0.50 µL | 0.50 µL |
| XbaI |  |  |  |
| SpeI |  |  |  |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL |  |  |
| H2O | 3.50 µL | 1.00 µL | 1.00 µL |
|  | ad | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |
|  | Länge Produkt | 2000 | 6300 | 6300 |
|  |  |  |  |  |
|  | ρ final | 10 ng/µL | 11 ng/µL | 7 ng/µL |

## Ligation

|  |  |  |  |
| --- | --- | --- | --- |
| Tube | | **L1** | **L2** |
| Product | | pSB1C3-pBAD-HlyB-HlyD-TolC-OmpT | pSB1C3-pBAD-HlyB-HlyD-TolC-OmpT |
|  | |  |  |
| **Vector** | Vector | pSB1C3 | pSB1C3 |
| Tube | **1** | **1** |
| ρ | 10 ng/µL | 10 ng/µL |
| Länge | 2000 bp | 2000 bp |
| V | 0.50 µL | 0.50 µL |
| **Part 1** | Part 1 | pBAD-HlyB-HlyD-TolC-OmpT | pBAD-HlyB-HlyD-TolC-OmpT |
| Tube | **2** | **3** |
| ρ | 11 ng/µL | 7 ng/µL |
| Länge | 6300 bp | 6300 bp |
| V | 8.00 µL | 8.00 µL |
| **Part 2** | Part 2 |  |  |
| Tube |  |  |
| ρ |  |  |
| Länge |  |  |
| V |  |  |
| T4 Buffer | | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL |
| H2O | | 0.00 µL | 0.00 µL |
| ad | | 10.00 µL | 10.00 µL |

## Restriction/Ligation/Trafo

Restriction:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** | **15** | **16** | **17** | **18** | **19** | **20** | **21** | **22** | **23** | **24** | **25** | **26** | **27** | **28** | **29** | **30** | **31** | **32** | **33** |
|  | Part | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-E0240 | BBa\_J45503 | pSB1C3-oHybB | 34-AOX1 | pSB1C3 | mraz | pSB1C3-BBa\_K206000 | 31sulA | 32sulA | 34sulA | cspD | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-BBa\_I732018 | accD | ldhA | ompT | RNAG120 | pSB1K3 | pSB1A3 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3 | pSB1C3 | pSB1A3 | RNAG120-MlcRE | pSB1K3 | pSB1C3-E0240 |
|  | Lot# | 180925NF01 | 180925NF02 | 180817LD10 | 180625NiF12 | 181001NB05 | 180920LP01 | backbone | 180622NB08 | neue Miniprep | neue Miniprep | neue Miniprep | 180905AW03 | 180622NB04 | neue Miniprep | 180924AW1 | 180925NaF04 | 180926NaF03 | 181004NF01 | 180921BK09 | 180622NB01 | 180827LP01 | 180909BK09 | backbone | backbone | 181003NB08 | 180920OM02 | 181003NB04 | backbone | backbone | backbone | 181003NiF01 | backbone | 180925NaF02 |
|  | **ρ** | 140 ng/µL | 144.673 | 200 ng/µL | 45 ng/µL | 107 ng/µL | 575 ng/µL | 25 ng/µL | 44.175 | 50 ng/µL | 28 ng/µL | 40 ng/µL | 43 ng/µL | 50 ng/µL | 81 ng/µL | 95 ng/µL | 126 ng/µL | 93.011 | 93 ng/µL | 213 ng/µL | 57 ng/µL | 68 ng/µL | 77 ng/µL | 25 ng/µL | 25 | 184 ng/µL | 800 ng/µL | 6 ng/µL | 25 ng/µL | 25 ng/µL | 25 | 83 ng/µL | 25 ng/µL | 145 ng/µL |
|  | Use | Vector | vector | vector | single insert | vector | single insert | vector | single insert | vector | single insert | single insert | single insert | single insert | vector | vector | vector | vector | vector | single insert | single insert | single insert | single insert | vector | vector | vector | vector | vector | vector | vector | vector | single insert | vector | vector |
|  | target m | 1000 ng | 1000 ng | 1000 ng | 100 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng | 200 ng | 200 ng | 1000 ng | 1000 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng | 100 ng | 1000 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 7.15 µL | 6.91 µL | 5.00 µL | 2.24 µL | 9.34 µL | 0.17 µL | 4.00 µL | 2.26 µL | 20.20 µL | 3.57 µL | 2.50 µL | 2.33 µL | 2.00 µL | 12.42 µL | 10.57 µL | 7.93 µL | 10.75 µL | 21.43 µL | 0.47 µL | 1.77 µL | 1.47 µL | 1.30 µL | 8.00 µL | 8.00 µL | 10.90 µL | 2.50 µL | 15.00 µL | 4.00 µL | 4.00 µL | 4.00 µL | 1.21 µL | 4.00 µL | 6.91 µL |
| NEBuffer 2.1 | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 4.00 µL | 1.00 µL | 1.00 µL | 3.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 3.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL |  |  |  |  | 0.50 µL |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |
| XbaI | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 0.50 µL |
| SpeI |  |  |  | 0.50 µL |  |  |  |  | 0.50 µL |  |  |  |  | 0.50 µL |  | 0.50 µL |  |  |  |  |  |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |
| PstI | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  | 0.50 µL | 0.50 µL |
| FastAP |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  | 0.50 µL |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL |  |
| H2O | 0.85 µL | 0.59 µL | 2.50 µL | 5.26 µL | 7.66 µL | 34.83 µL | 3.50 µL | 5.74 µL | 5.30 µL | 4.43 µL | 5.50 µL | 5.67 µL | 6.00 µL | 4.08 µL | 5.93 µL | 8.57 µL | 6.25 µL | 4.57 µL | 7.53 µL | 6.23 µL | 6.53 µL | 6.70 µL | 8.50 µL | 8.50 µL | 6.10 µL | 5.50 µL | 2.00 µL | 3.50 µL | 3.50 µL | 3.50 µL | 6.79 µL | 3.50 µL | 1.09 µL |
|  | ad | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 40.00 µL | 10.00 µL | 10.00 µL | 30.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 30.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt | 2697 bp | 878 bp |  |  | 2200 bp | 1200 bp | 2070 |  |  |  |  |  | 222 |  | 2070 | 2200 | 394 | 252 | 915 | 987 | 954 | 120 | 2070 | 2070 | 6500 | 6500 | 602 | 2070 | 2070 | 2070 | 546 | 2070 | 878 |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | Gelex | Gelex | Gelex | Gelex | 8 ng/µL | Gelex | 20 ng/µL | 10 ng/µL | 33 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 8 ng/µL | 50 ng/µL | Gelex | Gelex | 11 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | PCR Purification | Gelex | Gelex | Gelex | 6 ng/µL | 5 ng/µL | 5 ng/µL | 5 ng/µL | 10 ng/µL | 2 ng/µL | Gelex |

## Ligation

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** | **L12** | **L13** | **L14** | **L15** | **L16** | **L17** | **L18** | **L19** | **L20** | **L21** | **L22** | **L23** | **L24** | **L25** | **L26** | **L27** |
| Product | | pSB1C3\_RNAG120\_MlcRE-pSB1C3-E0240 | pSB1C3-E0240-BBa\_J45503 | pSB1C3-oHybB-34-AOX1 | pSB1C3-mraz | pSB1C3-BBa\_K206000-32sulA | pSB1C3-BBa\_K206000-34sulA | pSB1C3-BBa\_B0032-cspD | pSB1C3-34-AOX1 | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-pSB1C3-BBa\_B0032\_cbtA | pSB1C3-oHybB-pSB1C3-E0240 | pSB1C3\_RNAG120\_MlcRE-pSB1C3-BBa\_I732018 | pSB1C3-cspD | pSB1C3-accD | pSB1C3-ldhA | pSB1C3-ompT | pSB1C3-RNAG120 | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-RNAG120-MlcRE-pSB1C3-E0240 |
|  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-oHybB | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-oHybB | pSB1C3\_RNAG120\_MlcRE | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1K3 | pSB1K3 | pSB1K3 | pSB1A3 | pSB1A3 | pSB1A3 | pSB1K3 |
| Tube | **1** | **3** | **5** | **7** | **9** | **9** | **14** | **15** | **16** | **16** | **5** | **1** | **7** | **7** | **7** | **7** | **7** | **7** | **7** | **29** | **23** | **23** | **28** | **24** | **24** | **30** | **32** |
| ρ | Gelex | Gelex | 8 ng/µL | 20 ng/µL | 33 ng/µL | 33 ng/µL | 8 ng/µL | 50 ng/µL | Gelex | Gelex | 8 ng/µL | Gelex | 20 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 20 ng/µL | 5 ng/µL | PCR Purification | PCR Purification | 5 ng/µL | Gelex | Gelex | 5 ng/µL | 2 ng/µL |
| Länge | 2697 bp |  | 2200 bp | 2070 bp |  |  |  | 2070 bp | 2200 bp | 2200 bp | 2200 bp | 2697 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp |
| V |  |  | 1.93 µL | 0.61 µL | 4.25 µL | 4.25 µL | 0.00 µL | 0.29 µL |  |  | 2.50 µL |  | 2.46 µL | 0.94 µL | 0.88 µL | 0.91 µL | 3.22 µL | 0.16 µL | 0.16 µL | 4.90 µL |  |  | 5.51 µL |  |  | 5.32 µL | 5.50 µL |
| **Part 1** | Part 1 | pSB1C3-E0240 | BBa\_J45503 | 34-AOX1 | mraz | 32sulA | 34sulA | cspD | 34-AOX1 | 34-AOX1 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-E0240 | pSB1C3-BBa\_I732018 | cspD | accD | ldhA | ompT | RNAG120 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | RNAG120-MlcRE |
| Tube | **2** | **4** | **6** | **8** | **11** | **12** | **13** | **6** | **6** | **17** | **2** | **18** | **13** | **19** | **20** | **21** | **22** | **25** | **26** | **27** | **25** | **26** | **27** | **25** | **26** | **27** | **31** |
| ρ | Gelex | Gelex | Gelex | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | Gelex | Gelex | Gelex | Gelex | 11 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | Gelex | Gelex | 6 ng/µL | Gelex | Gelex | 6 ng/µL | Gelex | Gelex | 6 ng/µL | 10 ng/µL |
| Länge | 878 bp |  | 1200 bp |  |  |  | 222 bp | 1200 bp | 1200 bp | 394 bp | 878 bp | 252 bp | 222 bp | 915 bp | 987 bp | 954 bp | 120 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp | 546 bp |
| V |  |  |  | 10.00 µL |  |  | 10.00 µL |  |  |  |  | 4.30 µL | 5.17 µL | 8.16 µL | 8.27 µL | 8.22 µL | 3.67 µL |  |  | 6.00 µL |  |  | 6.00 µL |  |  | 6.00 µL | 3.35 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | pSB1C3-E0240 |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | **33** |
| ρ |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | Gelex |
| Länge |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 878 bp |
| V |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| T4 Buffer | | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 8.50 µL | 8.50 µL | 6.57 µL | 6.89 µL | 5.63 µL | 5.63 µL | 7.50 µL | 8.21 µL | 8.50 µL | 8.50 µL | 6.00 µL | 4.20 µL | 0.87 µL | 8.41 µL | 8.35 µL | 8.37 µL | 1.61 µL | 8.34 µL | 8.34 µL | 6.60 µL | 8.50 µL | 8.50 µL | 5.99 µL | 8.50 µL | 8.50 µL | 6.18 µL | 8.65 µL |
| ad | | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL |

## Transformation

Transformation on plates, protocol by Zhang Ghong.

## Digestion test

|  |  |
| --- | --- |
| compound | volume |
| DNA | 5 µL |
| EcoRI | 0.5 µL |
| FastAP | 0.5 µL |
| Buffer 2.1 | 1 µL |
| H2O | 3 µL |

## Gelelectrophoresis

We made a 1% agarose gel. It ran for 45 min at 80 V.

![](https://static.igem.org/mediawiki/2018/4/43/T--Hamburg--181004_Testverdau_und_Restriktion.png)

## Digestion test

|  |  |
| --- | --- |
| compound | volume |
| DNA | 5 µL |
| EcoRI | 0.5 µL |
| FastAP | 0.5 µL |
| Buffer 2.1 | 1 µL |
| H2O | 3 µL |

## Gelelectrophoresis

We made a 1% agarose gel. It ran for 45 min at 80 V.

![](https://static.igem.org/mediawiki/2018/d/d3/T--Hamburg--181004_Restr2.png)

## Gel extraction of 27 (pSB1K3-RNAG120-MlcRE-pSB1C3-E0240)

Gelextraction was made with GeneJET Gelextraction Kit by Thermo Fischer. Gel: 430 mg -> 430µl BindingBuffer added -> Concentration Nanodrop: 5.728 ng/µl.

## Ligation

|  |  |  |  |
| --- | --- | --- | --- |
| **L17** | **L20** | **L23** | **L26** |
| pSB1C3-RNAG120 | pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA |

So just 6 µl of DNA were used for ligation 20, 23, 26 in a 20 µl approach instead of 30 µl and incubated for 1 h at 37°C.

The transformation was made with protocol by Zhang Ghong.

## ONCs of (start: 3:00 a.m)

L11 #3: pSB1C3\_oHybB\_E0240 -> Camp

L11 #1: pSB1C3\_oHybB\_E0240 -> Camp

L13 #1: pSB1C3\_cspD -> Camp

L13 #2: pSB1C3\_cspD -> Camp

L13 #5: pSB1C3\_cspD -> Camp

L14 #3: pSB1C3\_accD -> Camp

L16 #4: pSB1C3\_ompT -> Camp

L16 #5: pSB1C3\_ompT -> Camp

L1 #1: pSB1C3\_NOT\_MlcRE\_E0240 -> Camp

L1 #3: pSB1C3\_NOT\_MlcRE\_E0240 -> Camp

L1 #4: pSB1C3\_NOT\_MlcRE\_E0240 -> Camp

L1 #5: pSB1C3\_NOT\_MlcRE\_E0240 -> Camp

L1 #6: pSB1C3\_NOT\_MlcRE\_E0240 -> Camp

L1 #7: pSB1C3\_NOT\_MlcRE\_E0240 -> Camp

L1 #8: pSB1C3\_NOT\_MlcRE\_E0240 -> Camp

180803NB01 -> Camp

180826LP03 -> Camp = 180925NaF02 !!! for restriction 2 & 33 (pSB1C3\_E0240)

180908NF01 -> Camp

DH5a -> no antibiotic.

## Preparing LB-Medium

LB medium (Luria/Miller) 25 g for 1 L, pH 7.0, autoclaved.

## Characterization of AOX1

Two Cultures of 100 mL were prepared. For the culture containing pBAD-AOX1, chloramphenicol (1:1000) and arabinose (1 mL of 0.1 mol/L) was added. A starter culture of each 3 mL overnight culture was transferred into the according media at an OD of 0.01 and grown to an OD of 0.2. After 120 min wt DH5alpha at OD 0.3 and pBAD-AOX1 cultures were transferred into an incubator at 15 °C and measured every half hour.

1 x 100 mL pBAD-AOX1 (induziert):

|  |  |  |
| --- | --- | --- |
| Time [min] | OD | Temperature [°C] |
| 0 | 0.01 | 37 |
| 60 | 0.02 | 37 |
| 120 | 0.26 | 37 |
| 150 | 0.27 | 18.4 |
| 180 | 0.27 | 16.1 |
| 210 | 0.28 | 18.0 |
| 240 | 0.31 | 17.4 |
| 270 | 0.33 | 17.6 |
| 300 | 0.37 | 16.8 |
| 330 |  |  |
| 360 |  |  |
| 390 |  |  |
| 420 |  |  |

1 x 100 mL DH5alpha:

|  |  |  |
| --- | --- | --- |
| Time [min] | OD | Temperature [°C] |
| 0 | 0.01 | 37 |
| 60 | 0.02 | 37 |
| 120 | 0.34 | 37 |
| 150 | 0.54 | 18.1 |
| 180 | 0.64 | 15.7 |
| 210 | 0.69 | 17.7 |
| 240 | 0.71 | 17.2 |
| 270 | 0.73 | 17.5 |
| 300 | 0.88 | 16.8 |
| 330 |  |  |
| 360 |  |  |
| 390 |  |  |
| 420 |  |  |

## Minipreps and cryostocks of Ligations from 1801004

Cryostocks were prepared for the following samples:

|  |  |
| --- | --- |
| Lot No. | Part |
| 181005AK01 | pSB1C3-accD #3 |
| 181005AK02 | pSB1C3-ompT #4 |
| 181005AK03 | pSB1C3-ompT #5 |
| 181005AK04 | pSB1C3- NOT-MlCRE-E0240 #1 |
| 181005AK05 | pSB1C3- NOT-MlCRE-E0240 #3 |
| 181005AK06 | pSB1C3- NOT-MlCRE-E0240 #4 |
| 181005AK07 | pSB1C3- NOT-MlCRE-E0240 #5 |
| 181005AK08 | pSB1C3- NOT-MlCRE-E0240 #6 |
| 181005AK09 | pSB1C3- NOT-MlCRE-E0240 #7 |
| 181005AK10 | pSB1C3- NOT-MlCRE-E0240 #8 |
| 181005AK11 | pSB1C3-oHybB-E0240 #1 |
| 181005AK12 | pSB1C3-oHybB-E0240 #3 |
| 181005AK13 | pSB1C3-cspD #1 |
| 181005AK14 | pSB1C3-cspD #2 |
| 181005AK15 | pSB1C3-cspD #5 |

After revision of the agarose gel from 181004, the samples marked in grey were discarded.

## Minipreps

Minipreps were performed using a GeneAID Presto Mini Plasmid Kit. The following Lot number were given:

|  |  |  |
| --- | --- | --- |
| Lot number | Part | Concentration [ng/µL] |
| 181005LD01 | pSB1C3-E0240 | 40.7 |
| 181005LD02 | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | 37.8 |
| 181005LD03 | pSB1C3-ompT #4 | 80.4 |
| 181005LD04 | pSB1C3-ompT #5 | 96.6 |
| 181005LD05 | pSB1C3-oHybB-E0240 #1 | 110.3 |
| 181005LD06 | pSB1C3-oHybB-E0240 #3 | 81.9 |
| 181005LD07 | pSB1C3-cspD #1 | 71 |
| 181005LD08 | pSB1C3-cspD #2 | 96.8 |

## Sequencing

|  |  |
| --- | --- |
| Lot number | Seq. ID |
| 181003NB06 | ID 37BC69 |
| 181003NB07 | ID 37BC70 |
| 181003NB10 | ID 37BC72 |
| 181005LD03 | ID 37BC73 |
| 181005LD04 | ID 37BC74 |
| 181005LD05 | ID 37BC75 |
| 181005LD06 | ID 37BC76 |
| 181005LD07 | ID 37BC77 |
| 181005LD08 | ID 37BC78 |

## Digestion test

|  |  |
| --- | --- |
| compound | volume |
| DNA | 5 µL |
| EcoRI | 0.5 µL |
| FastAP | 0.5 µL |
| Buffer 2.1 | 1 µL |
| H2Odest | 3 µL |

## Gelelectrophoresis

We made a 1% agarose gel. It ran for 45 min by 80 V.

![](https://static.igem.org/mediawiki/2018/d/de/T--Hamburg--181005_Testverdau.JPG)

## Set up the cPCRs from 181004

The transformed cultures of the ligations from 181004:

L1 (pSB1C3-pBAD-HlyB-HlyD-TolC-OmpT)

L2 (pSB1C3-pBAD-HlyB-HlyD-TolC-OmpT)

L23 (pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA)

L17 (pSB1C3-RNAG120)

Plate from 181003 where 2 colonies grew (pSB1K3-pBAD-BjalT-ompT-FLAg-HlyA)

Mastermix:

|  |  |  |
| --- | --- | --- |
|  | **1x** | **40 x** |
| 10x Dream Taq Buffer | 2 µL | 80 µL |
| dNTPs 10 mM | 0.4 µL | 16 µL |
| MgCl2 | 1.2 µL | 48 µL |
| Dream Taq Polymerase | 0.1 µL | 4 µL |
| fw Primer VF2 | 0.6 | 24 µL |
| rv Primer VR | 0.6 | 24 µL |
| H2O ad 20 µL | 15.1 | 604 µL |

PCR program:

|  |  |
| --- | --- |
| Temperature | Time |
| 98 °C | 3:00 |
| 98 °C | 0:30 |
| 55 °C | 0:30 |
| 72 °C | 3:00 |
| go to step 2 30x |  |
| 72 °C | 5:00 |
| 4 °C | ∞ |

## Gelelectrophoresis

We made a 1% agarose gel. It ran for 1 h by 100 V.

![](https://static.igem.org/mediawiki/2018/3/36/T--Hamburg--181005_beschriftet.jpg)

## PCRs of pSB1C3

To control the PCR preparation two PCR samples were prepared, with one of them using the Phusion HF and the other using Q5 Polymerase.

Mastermix:

|  |  |
| --- | --- |
| Sample 1 |  |
| 5x Phusion HF | 10 µL |
| dNTPs 10 mM | 1 µL |
| SB-prep-3P1 | 0.35 µL |
| SB-prep-2Ea | 0.35 µL |
| Template (181001NB04) | 0.5 |
| Phusion Polymerase | 0.5 |
| H2O ad 20 µL | 37.3 |

|  |  |
| --- | --- |
| Sample 2 |  |
| Q5 2x Mastermix | 10 µL |
| SB-prep-3P1 | 0.2 µL |
| SB-prep-2Ea | 0.2 µL |
| Template (181001NB04) | 0.5 µL |
| H2O ad 20 µL | 37.3 |

PCR program:

|  |  |
| --- | --- |
| Temperature | Time |
| 98 °C | 3:00 |
| 98 °C | 0:30 |
| 55 °C | 0:30 |
| 72 °C | 2:00 |
| go to step 2 30x |  |
| 72 °C | 10:00 |
| 4 °C | ∞ |

## Overnight cultures

Starting time: 1:30 am.

180712BK04

180826LP03 (2x)

180803NB01 (2x)

180712BK02

DH5a (no camp)

L1 #3  - pSB1C3\_RNAG120\_MlcRE-E0240

L20 #5 - pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA

L23#1 - pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

L23 #5 - pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

## Restriction

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **7** | **13** | **18** | **19** | **21** |
|  | Part | pSB1C3 | cspD | pSB1C3-BBa\_I732018 | accD | ompT |
|  | Lot# | 181001NB06 | 181001NB01 | 181004NF01 | 180921BK09 | 180827LP01 |
|  | **ρ** | 25 ng/µL | 37 ng/µL | 93 ng/µL | 213 ng/µL | 68 ng/µL |
|  | Use | vector | single insert | vector | single insert | single insert |
|  | target m | 200 ng | 100 ng | 1000 ng | 100 ng | 100 ng |
|  |  |  |  |  |  |  |
| Volumes | DNA | 8.00 µL | 2.73 µL | 10.72 µL | 0.47 µL | 1.47 µL |
| NEBuffer 2.1 | 2.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL |
| EcoRI-HF | 0.50 µL | 0.50 µL |  | 0.50 µL | 0.50 µL |
| XbaI |  |  | 0.50 µL |  |  |
| SpeI |  |  |  |  |  |
| PstI | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP | 0.50 µL |  |  |  |  |
| H2O | 8.50 µL | 5.27 µL | 6.28 µL | 7.53 µL | 6.53 µL |
|  | ad | 20.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL |
|  |  |  |  |  |  |  |
|  | Länge Produkt | 2070 | 222 | 252 | 915 | 954 |
|  |  |  |  |  |  |  |
|  | ρ final | 10 ng/µL | 10 ng/µL | 2 ng/µL | 10 ng/µL | 10 ng/µL |

## Ligation

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** | **L12** | **L13** | **L14** | **L15** | **L16** | **L17** | **L18** | **L19** | **L20** | **L21** | **L22** | **L23** | **L24** | **L25** | **L26** | **L27** | **L28** | **L29** | **L30** | **L31** |
| Product | | pSB1C3\_RNAG120\_MlcRE-pSB1C3-E0240 | pSB1C3-E0240-BBa\_J45503 | pSB1C3-oHybB-34-AOX1 | pSB1C3-mraz | pSB1C3-BBa\_K206000-32sulA | pSB1C3-BBa\_K206000-34sulA | pSB1C3-BBa\_B0032-cspD | pSB1C3-34-AOX1 | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-pSB1C3-BBa\_B0032\_cbtA | pSB1C3-oHybB-pSB1C3-E0240 | pSB1C3\_RNAG120\_MlcRE-pSB1C3-BBa\_I732018 | pSB1C3-cspD | pSB1C3-accD | pSB1C3-ldhA | pSB1C3-ompT | pSB1C3-RNAG120 | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-RNAG120-MlcRE-pSB1C3-E0240 | pSB1C3-RnaG120-Operator | pSB1K3-RNAG120-MlcRE-pSB1C3-BBa\_B0034-ldhA | pSB1C3-RNAG120-MlcRE-pSB1C3-BBa\_B0034-ldhA | pSB1C3-RNAG120-MlcRE-pSB1C3-E0240 |
|  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-oHybB | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-oHybB | pSB1C3\_RNAG120\_MlcRE | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1K3 | pSB1K3 | pSB1K3 | pSB1A3 | pSB1A3 | pSB1A3 | pSB1K3 | pSB1C3 | pSB1K3 | pSB1C3 | pSB1C3 |
| Tube | **1** | **3** | **5** | **7** | **9** | **9** | **14** | **15** | **16** | **16** | **5** | **1** | **7** | **7** | **7** | **7** | **7** | **7** | **7** | **29** | **23** | **23** | **28** | **24** | **24** | **30** | **32** | **29** | **32** | **7** | **7** |
| ρ | Gelex | Gelex | Gelex | 10 ng/µL | 33 ng/µL | 33 ng/µL | 8 ng/µL | 50 ng/µL | Gelex | Gelex | Gelex | Gelex | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | PCR Purification | PCR Purification | 3 ng/µL | PCR Purification | PCR Purification | 6 ng/µL | 14 ng/µL | 10 ng/µL | 14 ng/µL | 10 ng/µL | 10 ng/µL |
| Länge | 2697 bp |  | 2200 bp | 2070 bp |  |  |  | 2070 bp | 2200 bp | 2200 bp | 2200 bp | 2697 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp |
| V |  |  |  | 0.61 µL | 4.25 µL | 4.25 µL | 0.00 µL | 0.29 µL |  |  |  |  | 4.83 µL | 1.84 µL | 1.73 µL | 1.78 µL | 6.33 µL | 0.31 µL | 0.31 µL | 2.56 µL |  |  | 9.18 µL |  |  | 3.95 µL | 0.92 µL | 4.08 µL | 0.86 µL | 1.18 µL | 1.27 µL |
| **Part 1** | Part 1 | pSB1C3-E0240 | BBa\_J45503 | 34-AOX1 | mraz | 32sulA | 34sulA | cspD | 34-AOX1 | 34-AOX1 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-E0240 | pSB1C3-BBa\_I732018 | cspD | accD | ldhA | ompT | RNAG120 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | RNAG120-MlcRE | RnaG120-Operator | RNAG120-MlcRE | RNAG120-MlcRE | RNAG120-MlcRE |
| Tube | **2** | **4** | **6** | **8** | **11** | **12** | **13** | **6** | **6** | **17** | **2** | **18** | **13** | **19** | **20** | **21** | **22** | **25** | **26** | **27** | **25** | **26** | **27** | **25** | **26** | **27** | **31** | **35** | **31** | **31** | **31** |
| ρ | Gelex | Gelex | Gelex | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | Gelex | Gelex | Gelex | Gelex | 2 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | Gelex | Gelex | 8 ng/µL | Gelex | Gelex | 8 ng/µL | Gelex | Gelex | 8 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL |
| Länge | 878 bp |  | 1200 bp |  |  |  | 222 bp | 1200 bp | 1200 bp | 394 bp | 878 bp | 252 bp | 222 bp | 915 bp | 987 bp | 954 bp | 120 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp | 546 bp | 300 bp | 546 bp | 546 bp | 546 bp |
| V |  |  |  | 10.00 µL |  |  | 10.00 µL |  |  |  |  | 21.34 µL | 5.17 µL | 8.16 µL | 8.27 µL | 8.22 µL | 3.67 µL |  |  | 9.79 µL |  |  | 9.79 µL |  |  | 9.79 µL | 3.35 µL | 5.92 µL | 3.11 µL | 3.11 µL | 3.35 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | pSB1C3-E0240 |  | pSB1C3-BBa\_B0034-ldhA | pSB1C3-BBa\_B0034-ldhA | pSB1C3-E0240 |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | **33** |  | **34** | **34** | **33** |
| ρ |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 3 ng/µL |  | Gelex | Gelex | 3 ng/µL |
| Länge |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 878 bp |  | 1000 bp | 1000 bp | 878 bp |
| V |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 17.42 µL |  |  |  | 17.42 µL |
| T4 Buffer | | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 3.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 3.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 3.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 3.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 8.50 µL | 8.50 µL | 8.50 µL | 6.89 µL | 5.63 µL | 5.63 µL | 7.50 µL | 8.21 µL | 8.50 µL | 8.50 µL | 8.50 µL | 5.16 µL | 7.50 µL | 7.50 µL | 7.50 µL | 7.50 µL | 7.50 µL | 8.19 µL | 8.19 µL | 5.15 µL | 8.50 µL | 8.50 µL | 7.52 µL | 8.50 µL | 8.50 µL | 3.76 µL | 4.82 µL | 7.50 µL | 4.53 µL | 4.20 µL | 4.47 µL |
| ad | | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 30.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 30.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 30.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 30.00 µL |

## PCR purification of 28, 30, 32

The purification was made with the GeneJET PCR Purification Kit by Thermo Fischer. Eluted in 20 µL H2Odest.

## Vector Gelextraction of 18, 27, 33, (34 were negative)

The gelextraction was made by GeneJET Gelextraction Kit by Thermo Fischer.

![](https://static.igem.org/mediawiki/2018/1/18/T--Hamburg--181005_beschriftet_Restriktion.jpg)

## Ligation

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| L12 | L13 | L14 | L16 | L20 | L23 | L26 | L27 | L28 | L31 |
| pSB1C3\_RNAG120\_MlcRE-pSB1C3-BBa\_I732018 | pSB1C3-cspD | pSB1C3-accD | pSB1C3-ompT | pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-RNAG120-MlcRE-pSB1C3-E0240 | pSB1C3-RnaG120-Operator | pSB1C3-RNAG120-MlcRE-pSB1C3-E0240 |

Incubated for 1 h at room temperature.

## Competent cells

The competent cells was made with protocol of Zhang Ghong. Stored at -4°C.

## Transformation

Transformation of L13, L14, L16, L20 L28, L31 on camp plates. Transformation of L23, L27 on kanamycin plates and of Amp plates.

## Repetition of the digestion test from 181005

|  |  |  |
| --- | --- | --- |
| LOT Number | Part | Test restriction number |
| 181005LD03 | pSB1C3-ompT | 3 |
| 181005LD04 | pSB1C3-ompT #5 | 4 |
| 181005LD05 | pSB1C3-oHybB-E0240 #1 | 5 |
| 181005LD06 | pSB1C3-oHybB-E0240 #3 | 6 |
| 181005LD07 | pSB1C3-cspD #1 | 7 |
| 181005LD08 | pSB1C3-cspD #2 | 8 |

|  |  |
| --- | --- |
| compound | volume |
| DNA | 5 µL |
| EcoRI | 0.5 µL |
| FastAP | 0.5 µL |
| Buffer 2.1 | 1 µL |
| H2O | 3 µL |

## Gelelectrophoresis

We made a 1% agarose gel. It ran for 45 min by 80 V.

![](https://static.igem.org/mediawiki/2018/4/4f/T--Hamburg--181006_Testverdau.JPG)

## Minipreps

The minipreps were performed on the ONC, the following Lot No. were given:

|  |  |  |
| --- | --- | --- |
| Lot No. | Part | Concentration [ng/µL] |
| 181006LD01 | pSB1C3-RnaG120-Operator + MlcRE | 53.364 |
| 181006LD02 | pSB1C3-E0240 | 64.174 |
| 181006LD03 | pSB1C3-E0240 | 71.938 |
| 181006LD04 | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | 61.228 |
| 181006LD05 | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | 53.201 |
| 181006LD06 | L1 #3  - pSB1C3\_RNAG120\_MlcRE-E0240 | 73.904 |
| 181006LD07 | L20 #5 - pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA | 72.132 |
| 181006LD08 | L23#1 - pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA | 38.318 |
| 181006LD09 | L23 #5 - pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA | 59.710 |

## Cryostocks

|  |  |
| --- | --- |
| Lot No | Part |
| 181006LD10 | L1 #3  - pSB1C3\_RNAG120\_MlcRE-E0240 |
| 181006LD11 | L20 #5 - pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA |
| 181006LD12 | L23#1 - pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA |
| 181006LD13 | L23 #5 - pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA |

## Characterization of AOX1

Preparation of Arabinose solution (0.1 M): To 10 mL H2O 0.15 g Arabinose (L+, Roth) were added and mixed.

Characterization of AOX1: Two Cultures of 100 mL were prepared. For the culture containing pBAD-AOX1, chloramphenicol (1:1000) and arabinose (1 mL of 0.1 mol/L) was added. A starter culture of each 5 mL overnight culture was transferred into the according media at an OD of 0.01 and grown to an OD of 0.2. After 120 min wt DH5alpha at OD 0.3 and pBAD-AOX1 cultures were transferred into an incubator at 15 °C and measured every half hour. 300 min after induction arabinose was given a second time.

1 x 100 mL pBAD-AOX1 (induced):

|  |  |  |
| --- | --- | --- |
| Time | OD | Temperature (°C) |
| 0 | 0.01 | 37 |
| 60 | 0.138 | 37 |
| 120 | 0.264 | 37 |
| 150 |  |  |
| 180 | 0.389 | 21.2 |
| 210 | 0.399 | 18.2 |
| 240 | 0.366 | 17.5 |
| 270 | 0.409 | 17.5 |
| 300 | 0.430 | 17.5 |
| 330 | 0.451 | 17.5 |
| 360 | 0.482 | 17.5 |
| 390 | 0.701 | 17.5 |

1 x 100 mL DH5alpha:

|  |  |  |
| --- | --- | --- |
| Time | OD | Temperature (°C) |
| 0 | 0.01 | 37 |
| 60 | 0.234 | 37 |
| 120 | 0.296 (nach Angleich) | 37 |
| 150 |  |  |
| 180 | 0.768 | 19.9 |
| 210 | 0.831 | 17.1 |
| 240 | 0.996 | 17.0 |
| 270 | 1.069 | 17.2 |
| 300 | 1.416 | 17.0 |
| 330 | 1.611 | 17.2 |
| 360 | 1.648 | 17.3 |
| 390 | 4.409 | 17.2 |

## ONC ( starting time: 5:30 pm)

180712BK02 = pGGA-dapAP-30cbtA-30cspD-30mraZ-34sulA

181005AK02 = pSB1C3-ompT #4

181005AK03 = pSB1C3-ompT #5

181005AK11 = pSB1C3-oHybB-E0240 #1

181005AK12 = pSB1C3-oHybB-E0240 #2

181005AK14 = pSB1C3-cspD #2

## ONC for the Characterization of AOX1  ( starting time: 7:15 pm)

Prepared the cultures DH5α and 2x pSB1C3-K206000-BBa\_B0034-AOX1.

## Miniprep

The miniprep was made with GeneJET Miniprep Kit by Thermo Fischer. Following ONC were miniprepped:

180712BK02 = pGGA-dapAP-30cbtA-30cspD-30mraZ-34sulA

181005AK02 = pSB1C3-ompT #4

181005AK03 = pSB1C3-ompT #5

181005AK11 = pSB1C3-oHybB-E0240 #1

181005AK12 = pSB1C3-oHybB-E0240 #2

181005AK14 = pSB1C3-cspD #2

## Cryostocks

|  |  |  |
| --- | --- | --- |
| Cryostock | new LOT number for plasmid | concentration [ng/µL] |
| 181005AK02 | 181007NB01 | 111.553 |
| 181005AK03 | 181007NB02 | 91.844 |
| 181005AK11 | 181007NB03 | 92.949 |
| 181005AK12 | 181007NB04 | 53.723 |
| 181005AK14 | 181007NB05 | 76.105 |
| 180712BK02 | 181007NB06 | 98.611 |

## cPCR performed with the transformation of the 181006 and 181004

|  |  |
| --- | --- |
| Tubes | Part |
| 01-03 | pSB1C3-ompT |
| 04-12 | pSB1C3-RnaG120-MlcRE-E0240 |
| 13-18 | pSB1C3-RnaG120operator |
| 19-21 | pSB1C3-pBAD-BjaIT-OmptSITE-FLAG |
| 22-25 | pSB1C3-cspD |
| 26-30 | pSB1C3-RnaG120-MlcRE-E0840 |
| 31-34 | pSB1K3-pBAD-Bjait-OmpTsite-FLAG |
| 35 | pSB1A3-pBAD-Bjait-OmpTsite-FLAG |
| 36-41 | L1 Excretion GGA |
| 42-48 | L2 Excretion GGA |

Mastermix cPCR:

|  |  |  |  |
| --- | --- | --- | --- |
|  | **1x** | **45 x (for Parts in pSB1C3)** | **5 x (for others)** |
| 10x Dream Taq Buffer | 2 µL | 90 µL | 10 |
| dNTPs 10 mM | 0.4 µL | 18 µL | 2 |
| MgCl2 | 0.6 µL | 27 µL | 3 |
| Dream Taq Polymerase | 0.1 µL | 4.5 µL | 0.5 |
| fw Primer VF2 | 0.6 | 27 µL |  |
| rv Primer VR | 0.6 | 27 µL |  |
| H2O ad 20 µL | 15.1 | 679 µL | 75.5 |
| BBSuf\_Syn\_r |  |  | 3 |
| BBPre\_Syn\_f |  |  | 3 |

PCR Protocol:

|  |  |  |
| --- | --- | --- |
| Temperature | time | time (for Excretion GGA) |
| 95 °C | 3:00 | 3:00 |
| 94 °C | 0:30 | 0:30 |
| 55 °C | 0:30 |  |
| 72 °C | 2:00 | 7:00 |
| go to step to (30x) |  |  |
| 72 °C | 5:00 | 10:00 |
| 4 °C | ∞ | ∞ |

## PCR for RNAG120

Mastermix:

|  |  |
| --- | --- |
|  | **1x** |
| Q5 2x Mastermix | 25 µL |
| Primer syn fw | 2.5 µL |
| Primer syn rev | 2.5 µL |
| Template (synthetic DNA of RNAG120) | 0.5 µL |
| H2O ad 50 µL | 19.5 µL |

PCR protocol:

|  |  |
| --- | --- |
| Temperature | Time |
| 98 °C | 0:30 |
| 98 °C | 0:10 |
| 63 °C | 0:30 |
| 72 °C | 0:30 |
| go to step to (30x) |  |
| 72 °C | 2:00 |
| 4 °C | ∞ |

## Gelelectrophoresis

We made a 1% agarose gel.

![](https://static.igem.org/mediawiki/2018/7/70/T--Hamburg--181007_cPCR1_beschriftet.jpg.png)
![](https://static.igem.org/mediawiki/2018/3/34/T--Hamburg--181007_cPCR2_beschriftet.png)

Results of this PCR according to picture, which is shown above. The expected bad was not present (120 bp), instead a band at 600-700 bp was observed.

## PCR for Growth Inhibition with pGGA-dapAP-30cbtA-30cspD-30mraZ-34sulA

Mastermix:

|  |  |
| --- | --- |
|  | **1x** |
| 5x Phusion Buffer | 10 µL |
| dNTPs 10 mM | 1 µL |
| Phusion Polymerase | 0.5 µL |
| Primer P130Pre\_34\_cbtA\_f | 0.35 µL |
| Primer mraZ\_Suf\_r | 0.35 µL |
| Template (181007NB06: pGGA-dapAP-30cbtA-30cspD-30mraZ-34sulA) | 1 µL |
| H2O ad 20 µL | 36.8 µL |

PCR program:

|  |  |
| --- | --- |
| Temperature | Time |
| 95°C | 3:00 |
| 94°C | 0:30 |
| 55°C | 0:30 |
| 72°C | 1:30 |
| go to step 2 30x |  |
| 72°C | 5:00 |
| 4°C | ∞ |

## Gelelectrophoresis

We made a 1% agarose gel.

![](https://static.igem.org/mediawiki/2018/9/91/T--Hamburg--181007_cPCR_excretion_beschriftet.JPG.png)

The PCR product of 30cbtA-30cspD-30mraz was purified via GeneJET PCR Purification Kit by Thermo Fischer. Afterwards the concentration was measured by Nano Drop.

30cbtA-30cspD-30mraz = 28.442 ng/µL

## cPCR was performed with the transformation of 181003 and 181004

|  |  |
| --- | --- |
| Tubes | Part |
| 1-2 | pSB1K3-pBAD-BjaIT-OmptSITE-FLAG (L23) |
| 3-7 | pSB1K3-pBAD-BjaIT-OmptSITE-FLAG (L23) |
| 8-15 | pSB1C3-RnaG120 (L17) |
| 16-23 | pSB1C3-pBAD-BjaIT-OmptSITE-FLAG (L20) |

Mastermix:

|  |  |  |  |
| --- | --- | --- | --- |
|  | **1x** | **17 x (for Parts in pSB1C3)** | **8 x (for others)** |
| 10x Dream Taq Buffer | 2 µL | 34 µL | 16 µL |
| dNTPs 10 mM | 0.4 µL | 6.8 µL | 3.2 µL |
| MgCl2 | 0.6 µL | 10.2 µL | 4.8 µL |
| Dream Taq Polymerase | 0.1 µL | 1.7 µL | 0.8 µL |
| fw Primer VF2 | 0.6 | 10.2 µL | - |
| rv Primer VR | 0.6 | 10.2 µL | - |
| H2O ad 20 µL | 15.7 | 266.9 µL | 125.6 |
| BBSuf\_Syn\_r | 0.6 | - | 4.8 µL |
| BBPre\_Syn\_f | 0.6 | - | 4.8 µL |

PCR program:

|  |  |
| --- | --- |
| Temperature | time |
| 95 °C | 3:00 |
| 94 °C | 0:30 |
| 55 °C | 0:30 |
| 72 °C | 2:00 |
| go to step to (30x) |  |
| 72 °C | 5:00 |
| 4 °C | ∞ |

## Gelelectrophoresis

We prepared a 1% agarose gel.

![](https://static.igem.org/mediawiki/2018/9/98/T--Hamburg--181007_cPCR_vom_03.10._und_04.10..png)

## Preparing 50x TAE buffer for agarose gel electrophoresis

|  |  |
| --- | --- |
| Compound | Mass/Volume |
| Tris | 242.3 g |
| EDTA | 18.6 g |
| Acetic Acid | 57.1 mL |
| H2Odest | ad 1 L |

After addition of 700 mL water, pH was brought to pH = 8.3, then the buffer was filled up to 1L.

## AOX1 Characterization

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| t [h] | T [°C] | OD 600 DH5a | T [°C] | OD 600 AOX1+Arabinose |
| 0 | - | 0.012 | - | 0.004 |
| 2 | 34.5 | 0.021 | 34.5 | 0.007 |
| 3 | 34.5 | 0.089 | 36 | 0.044 |
| 4 (Nach Messung Schüttler auf 15°C gestellt) | 37.1 | 0.416 | 36.6 | 0.242 |
| 5 | 18.5 | 0.367 | 18.2 | 0.349 |
| 6 | 16.5 | 0.47 | 16.2 | 0.384 |
| 7 | 16.5 | 0.577 | 16.7 | 0.443 |
| 8 | 16.5 | 0.711 | 16.5 | 0.445 |
| 9 | 16.5 | 0.576 | 16.7 | 0.474 |
| 10 | 16.8 | 0.667 | 16.7 | 0.493 |

## Production of competent DH5 alpha cells

Production of the competent cells according to Zhang Ghongs protocol.

## Minipreps

|  |  |  |  |
| --- | --- | --- | --- |
| LOT Number | Insert | concentration | LOT number cryostocks |
| 181008DK04 | pSB1C3-RNAG120-Operator | 32.65 | 181008DK01 |
| 181008DK05 | pSB1C3-pBAD-Bjat-ompT-sideFLAG | 70.87 | 181008DK02 |
| 181008DK06 | pSB1C3-RNAG120-MlcRE-E0240 | 53.4 | 181008DK03 |

## Test digestion

|  |  |
| --- | --- |
| **compound** | **volume** |
| DNA | 5 µL |
| EcoRI | 0.5 µL |
| FastAP | 0.5 µL |
| Buffer 2.1 | 1 µL |
| H2O | 3 µL |

## Gelelectrophoresis

We made a 1% agarose gel. It ran for 45 min by 80 V.

|  |  |  |
| --- | --- | --- |
| Probe | LOT Number | Insert |
| 1 | 181008DK04 | pSB1C3-RNAG120-Operator |
| 2 | 181008DK05 | pSB1C3-pBAD-Bjat-ompT-sideFLAG |
| 3 | 181008DK06 | pSB1C3-RNAG120-MlcRE-E0240 |

![](https://static.igem.org/mediawiki/2018/c/c6/T--Hamburg--181008_Testverdau_beschriftet.png)

## Agarose gel electrophoresis of PCR for RNAG120 from 181007

We made a 1% agarose gel. It ran for 45 min by 80 V.

![](https://static.igem.org/mediawiki/2018/3/34/T--Hamburg--181007_cPCR2_beschriftet.png)

## Restriction

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Tube | **1** | **2** | **3** | **4** | **5** | **6** | **7** | **8** | **9** | **10** | **11** | **12** | **13** | **14** | **15** | **16** | **17** | **18** | **19** | **20** | **21** | **22** | **23** | **24** | **25** | **26** | **27** | **28** | **29** | **30** | **31** | **32** | **33** | **34** | **35** | **36** | **37** |
|  | Part | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-E0240 | BBa\_J45503 | pSB1C3-oHybB | 34-AOX1 | pSB1C3 | mraz | pSB1C3-BBa\_K206000 | 31sulA | 32sulA | 34sulA | cspD | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-BBa\_I732018 | accD | ldhA | ompT | RNAG120 | pSB1K3 | pSB1A3 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3 | pSB1C3 | pSB1A3 | RNAG120-MlcRE | pSB1K3 | pSB1C3-E0240 | pSB1C3-BBa\_B0034-ldhA | RnaG120-Operator | cbtA-cspD-mraZ | pSB1C3-BBa\_I13453 |
|  | Lot# | 181006LD01 | 181006LD03 | 180817LD10 | 180625NiF12 | 181001NB05 | 180920LP01 | 181005NiF01 | 180622NB08 | neue Miniprep | neue Miniprep | neue Miniprep | 180905AW03 | 181001NB01 | neue Miniprep | 180924AW1 | 180925NaF04 | 180926NaF03 | 181004NF01 | 180921BK09 | 180622NB01 | 180827LP01 | 181003BK01 | backbone | backbone | neue Miniprep | 180920OM02 | 181006LD04 | backbone | 181005NiF01 | backbone | 181003NiF01 | backbone | 181006LD03 | 180426NK28 | 181003BK01 | 181008BK01 | 180925NaF04 |
|  | **ρ** | 53 ng/µL | 71.938 | 200 ng/µL | 45 ng/µL | 107 ng/µL | 575 ng/µL | 146 ng/µL | 44.175 | 50 ng/µL | 28 ng/µL | 40 ng/µL | 43 ng/µL | 37 ng/µL | 81 ng/µL | 95 ng/µL | 126 ng/µL | 93.011 | 93 ng/µL | 213 ng/µL | 57 ng/µL | 68 ng/µL | 21 ng/µL | 25 ng/µL | 25 |  | 800 ng/µL | 2 ng/µL | 25 ng/µL | 146 ng/µL | 25 ng/µL | 83 ng/µL | 25 ng/µL | 71.938 | 86 ng/µL | 21 ng/µL | 28 ng/µL | 126 ng/µL |
|  | Use | Vector | vector | vector | single insert | vector | single insert | vector | single insert | vector | single insert | single insert | single insert | single insert | vector | vector | vector | vector | vector | single insert | single insert | single insert | single insert | vector | vector | vector | vector | vector | vector | vector | vector | single insert | vector | vector | single insert | single insert |  |  |
|  | target m | 1000 ng | 1000 ng | 1000 ng | 100 ng | 1000 ng | 100 ng | 500 ng | 100 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 100 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng | 1000 ng | 100 ng | 100 ng | 100 ng | 300 ng | 200 ng | 200 ng | 1000 ng | 1000 ng | 500 ng | 100 ng | 500 ng | 100 ng | 300 ng | 200 ng | 1000 ng | 1000 ng | 300 ng | 300 ng | 1000 ng |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Volumes | DNA | 18.74 µL | 13.90 µL | 5.00 µL | 2.24 µL | 9.34 µL | 0.17 µL | 3.42 µL | 2.26 µL | 20.20 µL | 3.57 µL | 2.50 µL | 2.33 µL | 2.70 µL | 12.42 µL | 10.57 µL | 7.93 µL | 10.75 µL | 10.72 µL | 0.47 µL | 1.77 µL | 1.47 µL | 14.29 µL | 8.00 µL | 8.00 µL |  | 1.25 µL | 20.00 µL | 4.00 µL | 3.42 µL | 4.00 µL | 3.61 µL | 8.00 µL | 13.90 µL | 11.69 µL | 14.03 µL | 10.55 µL | 7.93 µL |
| NEBuffer 2.1 | 3.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 4.00 µL | 1.00 µL | 1.00 µL | 3.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 3.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 2.00 µL |
| EcoRI-HF |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL | 0.50 µL |  |  |  |  | 0.50 µL |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  |  |
| XbaI | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  | 0.50 µL |  | 0.50 µL | 0.50 µL |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 0.50 µL | 0.50 µL |  | 0.50 µL |  |
| SpeI |  |  |  | 0.50 µL |  |  |  |  | 0.50 µL |  |  |  |  | 0.50 µL |  | 0.50 µL |  |  |  |  |  |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  | 0.50 µL |
| PstI | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| FastAP |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL |  | 0.50 µL |  |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  |  |  |  |  |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL | 0.50 µL | 0.50 µL |  | 0.50 µL | 0.50 µL |  |  |  | 0.50 µL |
| H2O | 7.26 µL | 2.60 µL | 2.50 µL | 5.76 µL | 7.66 µL | 34.83 µL | 4.08 µL | 5.74 µL | 5.30 µL | 4.43 µL | 5.50 µL | 5.67 µL | 5.30 µL | 4.08 µL | 5.93 µL | 8.57 µL | 6.25 µL | 6.28 µL | 7.53 µL | 6.23 µL | 6.53 µL | 2.71 µL | 8.50 µL | 8.50 µL | 8.00 µL | 6.75 µL | 6.00 µL | 3.50 µL | 4.08 µL | 3.50 µL | 4.39 µL | 8.50 µL | 2.60 µL | 5.31 µL | 2.97 µL | 6.45 µL | 8.57 µL |
|  | ad | 30.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 40.00 µL | 10.00 µL | 10.00 µL | 30.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 30.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 20.00 µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | Länge Produkt | 2697 bp | 878 bp |  |  | 2200 bp | 1200 bp | 2070 |  |  |  |  |  | 222 |  | 2070 | 2200 | 394 | 252 | 915 | 987 | 954 | 120 | 2070 | 2070 | 6500 | 6500 | 602 | 2070 | 2070 | 2070 | 546 | 2070 | 878 | 1000 | 300 |  | 2200 |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  | ρ final | Gelex | Gelex | Gelex | Gelex | Gelex | Gelex | 18 ng/µL | 10 ng/µL | 33 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 8 ng/µL | 50 ng/µL | Gelex | Gelex | Gelex | 10 ng/µL | 10 ng/µL | 10 ng/µL | 15 ng/µL | PCR Purification | PCR Purification | Gelex | Gelex | 2 ng/µL | 19.149 | PCR Purification | PCR Purification | 30 ng/µL | 9 ng/µL | Gelex | 4 ng/µL | 15 ng/µL | 15 ng/µL | 6 ng/µL |
|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  | PCR Purification |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | Gelex | PCR Purification |  |  |  | PCR Purification |  | Gelex |  |  | Gelex |

## Ligation

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Tube | | **L1** | **L2** | **L3** | **L4** | **L5** | **L6** | **L7** | **L8** | **L9** | **L10** | **L11** | **L12** | **L13** | **L14** | **L15** | **L16** | **L17** | **L18** | **L19** | **L20** | **L21** | **L22** | **L23** | **L24** | **L25** | **L26** | **L27** | **L28** | **L29** | **L30** | **L31** | **L32** |
| Product | | pSB1C3\_RNAG120\_MlcRE-pSB1C3-E0240 | pSB1C3-E0240-BBa\_J45503 | pSB1C3-oHybB-34-AOX1 | pSB1C3-mraz | pSB1C3-BBa\_K206000-32sulA | pSB1C3-BBa\_K206000-34sulA | pSB1C3-BBa\_B0032-cspD | pSB1C3-34-AOX1 | pSB1C3-I13453-34-AOX1 | pSB1C3-I13453-pSB1C3-BBa\_B0032\_cbtA | pSB1C3-oHybB-pSB1C3-E0240 | pSB1C3\_RNAG120\_MlcRE-pSB1C3-BBa\_I732018 | pSB1C3-cspD | pSB1C3-accD | pSB1C3-ldhA | pSB1C3-ompT | pSB1C3-RNAG120 | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT | pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA | pSB1K3-RNAG120-MlcRE-pSB1C3-E0240 | pSB1C3-RnaG120-Operator | pSB1K3-RNAG120-MlcRE-pSB1C3-BBa\_B0034-ldhA | pSB1C3-RNAG120-MlcRE-pSB1C3-BBa\_B0034-ldhA | pSB1C3-RNAG120-MlcRE-pSB1C3-E0240 | pSB1C3-BBa\_I13453-cbtA-cspD-mraZ |
|  | |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| **Vector** | Vector | pSB1C3\_RNAG120\_MlcRE | pSB1C3-E0240 | pSB1C3-oHybB | pSB1C3 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_K206000 | pSB1C3-BBa\_B0032 | pSB1C3 | pSB1C3-I13453 | pSB1C3-I13453 | pSB1C3-oHybB | pSB1C3\_RNAG120\_MlcRE | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1C3 | pSB1K3 | pSB1K3 | pSB1K3 | pSB1A3 | pSB1A3 | pSB1A3 | pSB1K3 | pSB1C3 | pSB1K3 | pSB1C3 | pSB1C3 | pSB1C3-BBa\_I13453 |
| Tube | **1** | **3** | **5** | **7** | **9** | **9** | **14** | **15** | **16** | **16** | **5** | **1** | **7** | **7** | **7** | **7** | **7** | **7** | **7** | **29** | **23** | **23** | **28** | **24** | **24** | **30** | **32** | **7** | **32** | **7** | **7** | **37** |
| ρ | Gelex | Gelex | Gelex | 18 ng/µL | 33 ng/µL | 33 ng/µL | 8 ng/µL | 50 ng/µL | Gelex | Gelex | Gelex | Gelex | 18 ng/µL | 18 ng/µL | 18 ng/µL | 18 ng/µL | 18 ng/µL | 18 ng/µL | 18 ng/µL | PCR Purification | PCR Purification | PCR Purification | 19 ng/µL | PCR Purification | PCR Purification | PCR Purification | 9 ng/µL | 18 ng/µL | 9 ng/µL | 18 ng/µL | 18 ng/µL | 6 ng/µL |
| Länge | 2697 bp |  | 2200 bp | 2070 bp |  |  |  | 2070 bp | 2200 bp | 2200 bp | 2200 bp | 2697 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2070 bp | 2200 bp |
| V |  |  |  | 0.61 µL | 4.25 µL | 4.25 µL | 0.00 µL | 0.29 µL |  |  |  |  | 2.65 µL | 1.01 µL | 0.95 µL | 0.98 µL | 3.48 µL | 0.17 µL | 0.17 µL |  |  |  | 1.34 µL |  |  |  | 1.49 µL | 2.24 µL | 1.39 µL | 0.65 µL | 0.70 µL | 2.79 µL |
| **Part 1** | Part 1 | pSB1C3-E0240 | BBa\_J45503 | 34-AOX1 | mraz | 32sulA | 34sulA | cspD | 34-AOX1 | 34-AOX1 | pSB1C3-BBa\_B0032\_cbtA | pSB1C3-E0240 | pSB1C3-BBa\_I732018 | cspD | accD | ldhA | ompT | RNAG120 | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | RNAG120-MlcRE | RnaG120-Operator | RNAG120-MlcRE | RNAG120-MlcRE | RNAG120-MlcRE | cbtA-cspD-mraZ |
| Tube | **2** | **4** | **6** | **8** | **11** | **12** | **13** | **6** | **6** | **17** | **2** | **18** | **13** | **19** | **20** | **21** | **22** | **25** | **26** | **27** | **25** | **26** | **27** | **25** | **26** | **27** | **31** | **35** | **31** | **31** | **31** | **36** |
| ρ | Gelex | Gelex | Gelex | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | Gelex | Gelex | Gelex | Gelex | Gelex | 10 ng/µL | 10 ng/µL | 10 ng/µL | 10 ng/µL | 15 ng/µL | Gelex | Gelex | 2 ng/µL | Gelex | Gelex | 2 ng/µL | Gelex | Gelex | 2 ng/µL | 30 ng/µL | 15 ng/µL | 30 ng/µL | 30 ng/µL | 30 ng/µL | 15 ng/µL |
| Länge | 878 bp |  | 1200 bp |  |  |  | 222 bp | 1200 bp | 1200 bp | 394 bp | 878 bp | 252 bp | 222 bp | 915 bp | 987 bp | 954 bp | 120 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp | 6500 bp | 6500 bp | 602 bp | 546 bp | 300 bp | 546 bp | 546 bp | 546 bp | 1150 bp |
| V |  |  |  | 10.00 µL |  |  | 10.00 µL |  |  |  |  |  | 5.17 µL | 8.16 µL | 8.27 µL | 8.22 µL | 2.45 µL |  |  | 32.71 µL |  |  | 32.71 µL |  |  | 32.71 µL | 1.12 µL | 3.94 µL | 1.04 µL | 1.04 µL | 1.12 µL | 5.60 µL |
| **Part 2** | Part 2 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | pSB1C3-E0240 |  | pSB1C3-BBa\_B0034-ldhA | pSB1C3-BBa\_B0034-ldhA | pSB1C3-E0240 |  |
| Tube |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | **33** |  | **34** | **34** | **33** |  |
| ρ |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | Gelex |  | 4 ng/µL | 4 ng/µL | Gelex |  |
| Länge |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 878 bp |  | 1000 bp | 1000 bp | 878 bp |  |
| V |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | 15.31 µL | 15.31 µL |  |  |
| T4 Buffer | | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 2.00 µL | 2.00 µL | 2.00 µL | 1.00 µL | 1.00 µL | 1.00 µL | 4.00 µL | 1.00 µL | 1.00 µL | 4.00 µL | 1.00 µL | 1.00 µL | 4.00 µL | 1.00 µL | 1.00 µL | 3.00 µL | 2.00 µL | 1.00 µL | 1.00 µL |
| T4 Ligase | | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL | 0.50 µL |
| H2O | | 8.50 µL | 8.50 µL | 8.50 µL | 6.89 µL | 5.63 µL | 5.63 µL | 7.50 µL | 8.21 µL | 8.50 µL | 8.50 µL | 8.50 µL | 8.50 µL | 0.68 µL | 8.33 µL | 8.28 µL | 8.30 µL | 2.58 µL | 8.33 µL | 8.33 µL | 2.79 µL | 8.50 µL | 8.50 µL | 1.45 µL | 8.50 µL | 8.50 µL | 2.79 µL | 5.89 µL | 2.31 µL | 8.76 µL | 0.50 µL | 6.69 µL | 0.11 µL |
| ad | | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 20.00 µL | 20.00 µL | 20.00 µL | 10.00 µL | 10.00 µL | 10.00 µL | 40.00 µL | 10.00 µL | 10.00 µL | 40.00 µL | 10.00 µL | 10.00 µL | 40.00 µL | 10.00 µL | 10.00 µL | 30.00 µL | 20.00 µL | 10.00 µL | 10.00 µL |

## Gelelectroporesis

We made a 1% agarose gel. It ran for 45 min by 80 V.

![](https://static.igem.org/mediawiki/2018/0/06/T--Hamburg--181008_Restriktion_beschriftet.png)

Due to the insufficient amount of SpeI left, the Enzyme was diluted 1:10. 1 µL was used per restriction. The restriction time was raised to 90 min. NcoI digestion of R27:

|  |  |
| --- | --- |
|  | **27** |
|  | pGGA-pBAD-BjalT-OmpT-FLAG-HlyA |
|  | 181006LD04 |
|  | 2 ng/µL |
|  | vector |
|  | 1000 ng |
|  |  |
| Volumen DNA | 20.00 µL |
| FD buffer | 3.00 µL |
| NcoI | 0.50 µL |
| warrer | 6.00 µL |
| sum | 30.00 µL |
|  |  |
| product length | 602 |

## Gelelectrophoresis:

We made a 1% agarose gel. It ran for 45 min by 80 V.

![](https://static.igem.org/mediawiki/2018/c/c6/T--Hamburg--181008_Testverdau_beschriftet.png)

Digestion was not successful, because there were no bands visible.

## Transformation

Ligation 28, 30 and 32 were performed, transformed using the Zhang Gong protocol and plated out.

## PCR of RNAG120

|  |  |
| --- | --- |
| Volume (µL) | Chemical |
| 20 | H2O |
| 2.5 | rv-Primer: rnaG120 rev |
| 2.5 | fw-Primer: BioBrick prefix fw |
| 25 | Q5 2X Master Mix |
| 0.25 (RNAG120 synth. construct) | Template: 181006LD01 |

PCR program:

|  |  |  |
| --- | --- | --- |
| Temperature | Time | Steps |
| 98 | 0:20 | 1 |
| 98 | 0:10 | 30 |
| 55 | 0:20 | 30 |
| 72 | 0:30 | 30 |
| 72 | 5:00 | 1 |

The PCR product was purified with PCR purification kit by Thermo Fischer. New Lot number:  181008NB01.

## Dilution of primers

A new aliquot of Primers VF2 and VR was diluted (10 mM), the old aliquots were discarded.

## Overnight cultures

180518BK06 = oHybB

180712BK04 = NOTMlcRE

180712BK04 = NnAOX1a

180727DK03 = ADH2

## Preparation of samples for DNA submission

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Lotnummer | Well | Part | Konzentration | µl in well | noch genug vorhanden | Cryostock |
| 181003NB03 | 1A | MlcRE | 133.1 | 1.88 | ja | aus 180908NF03 |
| 181009AK03 | 1B | oHybB | 119 | 2.10 | ja | 180518BK06 |
| 181006LD01 | 1C | NOTMlcRE | 53.364 | 4.68 | ja | 180712BK04 |
|  |  | RnaG120 |  | #DIV/0! |  |  |
| 180918AK12 | 1D | icsA | 92 | 2.72 | ja | 180918AK05 |
| 181007NB05 | 1E | cspD | 76.105 | 3.28 | ja |  |
| 180627LP03 | 1F | cbtA | 65.5 | 3.82 | ja | 180627LP06 |
| 180914AW08 | 1G | mraZ | 160 | 1.56 | ja | 180914LD03 |
| 180914AW01 | 1H | sulA | 30 | 8.33 | ja | 180908NF04 |
| 181002NB09 | 2A | ldhA | 65.203 | 3.83 | ja | 181002NB04 |
| 180918AK10 | 2B | accA | 153 | 1.63 | ja | 180918AK03 |
| 180918AK09 | 2C | accB | 65 | 3.85 | ja | 180918AK02 |
| 181003NB05 | 2D | accC | 143.9 | 1.74 | ja | 180727DK04 |
|  |  | accD |  | #DIV/0! |  |  |
|  |  | tesA |  | #DIV/0! |  |  |
|  |  | LeuABCD |  | #DIV/0! |  |  |
| 180726LP01 | 2E | kivD | 107.39 | 2.33 | ja | 180727DK01 |
| 181009AK02 | 2F | ADH2 | 225 | 1.11 | ja | 180727DK03 |
| 181009AK01 | 2G | NnAOX1a | 185 | 1.35 | ja | 180727DK02 |
| 180918AK14 | 2H | BjaIT-Linker-OmpTSite-FLAG | 104 | 2.40 | ja | 180918AK07 |
| 181003NB10 | 3A | OmpT | 137 | 1.82 | ja |  |
|  |  | NOT\_MlcRE-BBa\_B0030-accA-accD-BBa\_B0032-accB-accC-BBa\_B0031-tesA |  | #DIV/0! |  |  |
|  |  | BBa\_K206000-BBa\_B0030-accA-accD-BBa\_B0032-accB-accC-BBa\_B0031-tesA |  | #DIV/0! |  |  |
|  |  | NOT\_MlcRE-ADH2-kivD-LeuABCD |  | #DIV/0! |  |  |
|  |  | BBa\_K206000-ADH2-kivD-LeuABCD |  | #DIV/0! |  |  |
| 180918AK13 | 3B | oHybB-BBa\_B0034-NnAOX1a | 132 | 1.89 | ja | 180918AK06 |
| 180908NF05 | 3C | BBa\_K206000-BBa\_B0034-NnAOX1a | 64.7 | 3.86 | ja | 180908NF01 |
|  |  | NOTMlcRE-BjaIT-OmpTSite-FLAG-HlyA |  | #DIV/0! |  |  |
| 181006LD07 | 3D | BBa\_K206000-BjaIT-OmpTSite-FLAG-HlyA | 72.132 | 3.47 | ja | 181006LD11 |
|  |  | BBa\_K206000-HlyB-HlyD-TolC-OmpT |  | #DIV/0! |  |  |
| 180731NF03 | 3E | BBa\_B0033-cbtA | 174.8 | 1.43 | ja | 180531NF02 |
| 180914AW10 | 3F | BBa\_B0032-cspD | 77 | 3.25 | ja | 180531NF05 |
| 180731NF05 | 3G | BBa\_B0033-cspD | 110.1 | 2.27 | ja | 180531NF04 |
| 180703BK06 | 3H | BBa\_B0032-mraZ | 250 | 1.00 | ja | 180703LP06 |
| 180731NF02 | 4A | BBa\_B0033-mraz | 143.4 | 1.74 | ja | 180531NF01 |
| 180731NF06 | 4B | BBa\_B0032-sulA | 152.5 | 1.64 | ja | 180531NF06 |
| 180426NK28 | 4C | BBa\_B0034-ldhA | 85.514 | 2.92 | ja | 180518BK11 |
| 180703BK05 | 4D | BBa\_B0032-accB | 212 | 1.18 | ja | 180703LP05 |
| 180731NF01 | 4E | MlcRE-BBa\_E0840 | 195.6 | 1.28 | ja | 180518BK02 |
| 180914AW07 | 4F | BBa\_J45503-BBa\_E0840 | 51 | 4.90 | ja | 180914LD02 |
| 180703BK02 | 4F | BBa\_K206000-BBa\_B0034-ldhA | 167 | 1.50 | ja | 180703LP02 |
| 180703BK01 | 4G | BBa\_K206000-BBa\_B0032-ldhA | 153 | 1.63 | ja | 180703LP01 |
| 181002NB08 | 4H | BBa\_I13453-BBa\_B0032-cbtA | 157.52 | 1.59 | ja | 181002NB03 |
| 180914AW09 | 5A | BBa\_K206000-BBa\_B0032-sulA | 104 | 2.40 | ja | 180914LD04 |
| 180817LD02 | 5B | pSB1C3-K206000-33cspD | 132 | 1.89 | ja | 180826LP01 |
| 180426NK20 | 5C | pSB1C3-BBa\_B0032-ldhA | 63.0404 | 3.97 | ja | 180518BK10 |
| 180926NaF03 | 5D | pSB1C3-BBa\_B0032-cbtA | 93.011 | 2.69 | ja | 180531NF03 |
| 180731NF07 | 5E | psB1C3\_BBa\_B0034\_sulA | 167.9 | 1.49 | ja | 180518BK05 |
| 181003NB06 | 5G CAVE | pSB1C3-oHybB-BBa\_E0240 | 167.6 | 1.49 | ja |  |
|  |  | pSB1C3-BBa\_B0031-sulA |  | #DIV/0! |  | 180518BK04 |
| 181006LD06 | 5F CAVE | pSB1C3\_RNAG120\_MlcRE-BBa\_E0240 L1 #3 | 74 | 3.38 | ja |  |
| 181008DK04 | 5H | RNAG120-Operator | 32.6 | 7.67 |  |  |
| 181010BK01 | 6A | BBa\_I13453-BBa\_B0034-cbtA-BBa\_B0032-cspD-BBa\_B0032-mraZ |  | #DIV/0! |  |  |

## Preparation of cAMP agar plates

Mixed 1 L agar and 1 mL cAmp.

## PSB1C3-NOT-MlcRE-BBa\_E0240 characterization

Precultures were incubated until OD600 0.6 at 37°C. Present GFP-background was eliminated by incubation under highlight for 10 min. Precultures were then split and added into 10 ml cultures with a star-OD of 0.1. Follow approaches were made each 3 times:

1. DH5alpha
2. DH5alpha + pSB1C3-NOT\_MlcRE-BBa\_E0240 no glucose
3. DH5alpha + pSB1C3-NOT\_MlcRE-BBa\_E0240 + glucose (2 g/L)
4. DH5alpha + pSB1C3-NOT\_MlcRE-BBa\_E0240 + glucose (1 g/L)
5. DH5alpha + pSB1C3-NOT\_MlcRE-BBa\_E0240 + glucose (0.5 g/L)
6. DH5alpha + pSB1C3-NOT\_MlcRE-BBa\_E0240 + glucose (0.25 g/L)
7. DH5alpha + pSB1C3-NOT\_MlcRE-BBa\_E0240 + glucose (0.125 g/L)
8. DH5alpha + pSB1C3-BBa\_I20270 (positive control)

Probes were incubated for 60, 90, 120, 180 min

1 ml was removed from each culture and stored on ice. Cells were washed and resuspended in 600 µl PBS and stored at 4°C overnight.

## Colony PCR

Colony PCRs of the transformations performed on October 8 were made.

Mastermix

|  |  |  |
| --- | --- | --- |
| Substance | 1x (volume in µl) | 20x (volume in µl) |
| 10x DreamTaq Buffer | 2 | 40 |
| dNTPs 10mM | 0.4 | 8 |
| MgCl2 50mM | 1.2 | 24 |
| Fw-Primer VF2 | 0.6 | 12 |
| Rv-Primer VR | 0.6 | 12 |
| Taq-Polymerase | 0.1 | 2 |
| ddH2O ad 20µl | 15.1 | 302 |

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Program (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 95 | 180 |
| 95 | 30 |
| 55 | 30 |
| 72 | 120 |
| 72 | 300 |

181009.png

## Minipreps

The ONCs set up on October 8 were purified by using the “GeneJET Plasmid Miniprep Kit” from Thermo Fisher.

## PSB1C3-NOT-MlcRE characterization

The probes performed on October 9 were used to measure the OD and the GFP-fluorescence using a plate reader. Since the measurements were unusable the characterization needed to be repeated.

## DNA submission

DNA was submitted following the provided iGEM protocol.

## Preparation for the oHybB characterization

Following cryostocks were used to set up ONCs.

1. pSB1C3-oHybB-BBa\_E0240
2. pSB1C3-BBa\_J45503-BBa\_E0840
3. DH5α

## Repetition of the oHybB characterization

The ONCs prepared on October 10 were used to set up 10 ml cultures. Each culture was set up three times and incubated overnight.

1. Incubation at 37°C
2. Incubation at 16°C
3. Incubation at room temperature

## ONCs

ONCs were set up for the MlcRE characterization and also the following ones

1. pGGA-pBAD-BjalT-OmpT-FLAG-HlyA
2. pSB1C3-BBa\_K206000-BBa\_B0034-ldhA

## Repetition of the NOT-MlcRE characterization

The ONCs prepared on October 11 were used to repeat the characterization as described on October 9. Since the results weren’t usable the culture was incubated overnight.

## Preparation for cell lysis of BjaIT and ldhA

The ONCs pGGA-pBAD-BjalT-OmpT-FLAG-HlyA and pSB1C3-BBa\_K206000-BBa\_B0034-ldhA were used to inoculate 750 ml cultures. A 500 ml DH5α culture was used as a negative control.  At Timepoint 0 al cultures got induced with arabinose

|  |  |
| --- | --- |
| Culture | Starting OD |
| pGGA-pBAD-BjalT-OmpT-FLAG-HlyA | 0.16 |
| pSB1C3-BBa\_K206000-BBa\_B0034-ldhA | 0.3 |

 After 6h all three probes were centrifuged at 5000rpm; 15 min; at 8°C

## Repetition of the oHybB characterization

The cultures set up on October 11 were used. After 13, 18 and 20.5 h 1 ml was taken from each culture and the OD and GFP-absorption was measured using a plate reader.

## ONCs

Following ONCs were set up:

1. pSB1C3-BBa\_K206000-BBa\_B0034-cbtA-BBa\_B0032-cspD-BBa\_B0032-mraZ
2. pSB1C3-BBa\_I20270
3. DH5α

## PSB1C3-NOT-MlcRE characterization

The culture set up on October 12 was measured again after 1140 min. Since the measurements weren’t usable the characterization needed to be repeated.

## Characterization of pSB1C3-BBa\_K206000-BBa\_B0034-cbtA-BBa\_B0032-cspD-BBa\_B0032-mraZ (growth inhibition module)

The ONCs set up on October 12 were used. 10 ml cultures were set up with a starting OD of 0.1

Measurements

|  |  |  |  |
| --- | --- | --- | --- |
| Time (h) | OD BBa\_I20270 | OD DH5α | OD *growth inhibiton module* |
| 0 | 0.01 | 0.01 | 0.01 |
| 1 | 0.025 | 0.032 | 0.021 |
| 2 | 0.084 | 0.149 | 0.058 |
| 3 | 0.380 | 0.728 | 0.260 |
| 4 | 1.09 | 1.65 | 0.81 |
| 5 | 1.58 | 1.75 | 1.16 |
| 6 | 1.88 | 2.24 | 1.67 |
| 7 | 2.33 | 2.57 | 2.12 |
| 8 | 3.27 | 3.13 | 2.58 |
| 9 | 2.98 | 3.20 | 2.18 |

## Restriction, ligation and transformation

Restriction

|  |  |  |  |
| --- | --- | --- | --- |
|  | Tube | **1** | **2** |
|  | Part | pSB1C3-E0240 | RnaG120-MlcRE |
|  | Lot# | 181006LD02 | IDT-syn |
|  | **ρ** | 53 ng/µL | 100 ng/µL |
|  | Use |  |  |
|  | target m |  |  |
|  |  |  |  |
| Volumes | DNA | 15.00 µL | 2.50 µL |
| NEBuffer 2.1 | 2.00 µL | 1.00 µL |
| EcoRI-HF | 0.50 µL | 0.50 µL |
| XbaI | 0.50 µL |  |
| SpeI |  | 0.50 µL |
| PstI |  |  |
| FastAP | 0.50 µL |  |
| H2O | 1.50 µL | 5.50 µL |
|  | ad | 20.00 µL | 10.00 µL |
|  | length Produckt |  |  |
|  |  |  |  |
|  | ρ final | 25 ng/µL | 4 ng/µL |

The probes were incubated at 37°C for 1h following another 20 min at 80 °C.

![](https://static.igem.org/mediawiki/2018/7/79/T--Hamburg--181013Restriction_NOTMlcRE-GFP.JPG)

Ligation

|  |  |  |
| --- | --- | --- |
| Tube | | **1** |
| Product | | pSB1C3-E0240-RnaG120-MlcRE |
|  | |  |
| **Vector** | Vector | pSB1C3-E0240 |
| Tube | **1** |
| ρ | 25 ng/µL |
| Länge | 2800 bp |
| V | 1.50 µL |
| **Part 1** | Part 1 | RnaG120-MlcRE |
| Tube | **2** |
| ρ | 4 ng/µL |
| Länge | 500 bp |
| V | 16.00 µL |
| **Part 2** | Part 2 |  |
| Tube |  |
| ρ |  |
| Länge |  |
| V |  |
| T4 Buffer | | 2.00 µL |
| T4 Ligase | | 0.50 µL |
| H2O | | 0.00 µL |
| ad | | 20.00 µL |

The probe was incubated at 37 °C for 1 h.

The product was transformed into DH5α.

64. 150 µl Competent cell aliquots were thawed on ice for 15-30 min.
65. 10 µl DNA were added and mixed
66. Cells were incubated for 35 min on ice
67. Heat-shock: 42°C for 45 sec, then put back on ice for 2-5 min
68. Pre-warm 850 µl LB-Medium
69. Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
70. Cetrifuge 4500 rpm 2 min to collect cells.
71. Put cells on appropriate plates or medium
72. Incubation at 37°C overnight.

## ONCs

1. DH5a,
2. pSB1C3-BBa\_I20270
3. pSB1C3-pBAD-RBS-cspD

## Characterization of cspD

The ONCs set up on October 13 were used and thinned to an OD of 0.1 in 8 ml LB-media. After one hour cspD was induced using arabinose. The OD was measured every hour and the OD thinned down to 0.1 again. After 4h the experiment was stopped since no changes were observed. Therefor, the experiment needs to be repeated.

## Colony PCR of pSB1C3-NOT-MlcRE-BBa\_E0240

MasterMix

|  |  |  |
| --- | --- | --- |
| Substance | 1x (volume in µl) | 20x (volume in µl) |
| 10x DreamTaq Buffer | 2 | 40 |
| dNTPs 10mM | 0.4 | 8 |
| MgCl2 50mM | 0.6 | 12 |
| Fw-Primer VF2 | 0.6 | 12 |
| Rv-Primer VR | 0.6 | 12 |
| Taq-Polymerase | 0.1 | 2 |
| ddH2O ad 20µl | 15.7 | 314 |

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Program (30 cycles)

|  |  |
| --- | --- |
| Temperature (°C) | Time (s) |
| 95 | 180 |
| 95 | 30 |
| 54 | 30 |
| 72 | 120 |
| 72 | 300 |

The probes were applied to a 1% agarose gel

![](https://static.igem.org/mediawiki/2018/1/17/T--Hamburg--181014.jpg)

Colony one was characterized, as it showed fluorescence after incubation with glucose.

## ONCs

DH5α and pSB1C3-BBa\_K206000-BBa\_B0034-AOX1 were set up as ONCs for the AOX1 characterization on the following day.

DH5α, pSB1C3-BBa\_I20270 and pSB1C3-pBAD-RBS-cspD were set up as ONCs for the cspD characterization

## AOX1 characterization

Two 8 ml cultures were inoculated with the ONCs set up on October 14 to an OD600 = 0.1 and incubated at room temperature; 120 rpm. At OD600 = 0.2 the pSB1C3-BBa\_K206000-BBa\_B0034-AOX1 culture was induced with 10 µM arabinose.

Measurements

|  |  |  |
| --- | --- | --- |
| Time after induction [min] | Temperature DH5α | Temperature AOX1 |
| 0 | 23.8 | 23.8 |
| 60 | 24.4 | 24.5 |
| 120 | 24.5 | 24.5 |
| 150 | 25.5 | 25.5 |
| 180 | 25.8 | 25.8 |
| 210 | 25.8 | 25.7 |
| 240 | 25.4 | 25.3 |
| 270 | 25.3 | 25.2 |
| 300 | 25.1 | 25.0 |
| 330 | 25.0 | 24.9 |
| 390 | 23.7 | 23.7 |
| 430 | 24.2 | 24.2 |
| 470 | 24.3 | 24.3 |

Since the measurements didn’t show any significant differences the characterization needed to be repeated.

## Cell lysis of BjaIT and ldhA

The pellets prepared on October 12 were lysed with 2mL  lysis buffer (5 mM EDTA and 1 mg/mL lysozyme) per pellet

1. incubation 1h on ice
2. freezing and unfreezing 6 times with liquid nitrogen and 37°C water bath
3. addition of 5 µL benzonase per 6 mL cell lysate → incubation 30 min on ice
4. centrifugation 4°C, 5000 rpm. 30 min
5. supernatant was transferred into new falcons

## Preparation of solutions containing our odour baits

Lactate, Myristic acid and 3-Methyl-1-butanol each diluted in ethanol

1. 10%
2. 1%
3. 1%
4. 01%
5. 001%
6. 0001%

## CspD characterization

ONCs were thinned down to a starting OD of 0.1 in 8 ml LB-media. The pSB1C3-pBAD-RBS-cspD culture was induced using 0.1 M arabinose. The OD was measured every hour. After every OD measurement the cultures were thinned down to OD 0.1 including the addition of Camp and arabinose. After 5 hours the experiment was stopped because of unrealistic ODs.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | t=0 | t=2h | t=3h | t=4h | t=5h | t=5.5h |
| DH5a | 0.1 | 1.408 | 0.96 | 1.038 | 1.576 | 1.778 |
| BBa\_I20270 | 0.1 | 0.734 | 0.46 | 0.548 | 1.18 | 1.348 |
| cspD | 0.1 | 1.078 | 0.734 | 0.694 | 1.894 | 2.328 |

## Characterization of pSB1C3-NOT-MlcRE

The characterization was done as described on October 11. Since the results weren’t usable the characterization had to be repeated.

## Funding

![](https://static.igem.org/mediawiki/2018/b/b8/T--Hamburg--Sponsors_CSS.svg)

![](https://static.igem.org/mediawiki/2018/a/a5/T--Hamburg--Sponsors_AltonaDiagnostics.svg)

![](https://static.igem.org/mediawiki/2018/d/d2/T--Hamburg--Sponsors_SnapGene.svg)

![](https://static.igem.org/mediawiki/2018/e/e8/T--Hamburg--Sponsors_biomol.svg)

![](https://static.igem.org/mediawiki/2018/b/be/T--Hamburg--Sponsors_DeutscheMalaria.svg)

![](https://static.igem.org/mediawiki/2018/1/13/T--Hamburg--Sponsors_NEB.svg)

![](https://static.igem.org/mediawiki/2018/b/b8/T--Hamburg--Sponsors_BioGlobe.svg)

![](https://static.igem.org/mediawiki/2018/7/73/T--Hamburg--Sponsors_IDT.svg)

![](https://static.igem.org/mediawiki/2018/3/32/T--Hamburg--Sponsors_Eurofins.svg)

Ⓒ 2018 iGEM Hamburg


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Hamburg/Experiments

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* [Home(current)](https://2018.igem.org/Team%3AHamburg)
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  [Description](https://2018.igem.org/Team%3AHamburg/Description#main-content)
  [Design](https://2018.igem.org/Team%3AHamburg/Design#main-content)
  [Protocols](https://2018.igem.org/Team%3AHamburg/Experiments#main-content)
  [Notebook](https://2018.igem.org/Team%3AHamburg/Notebook#main-content)
  [InterLab](https://2018.igem.org/Team%3AHamburg/InterLab#main-content)
  [Model](https://2018.igem.org/Team%3AHamburg/Model#main-content)
  [Results](https://2018.igem.org/Team%3AHamburg/Results#main-content)
  [Demonstrate](https://2018.igem.org/Team%3AHamburg/Demonstrate#main-content)
  [Improve](https://2018.igem.org/Team%3AHamburg/Improve#main-content)
  [Chemistry](https://2018.igem.org/Team%3AHamburg/Chemistry#main-content)
  [Attributions](https://2018.igem.org/Team%3AHamburg/Attributions#main-content)
* Parts

  [Overview](https://2018.igem.org/Team%3AHamburg/Parts#main-content)
  [Basic Parts](https://2018.igem.org/Team%3AHamburg/Basic_Part#main-content)
  [Composite Parts](https://2018.igem.org/Team%3AHamburg/Composite_Part#main-content)
  [Part Collection](https://2018.igem.org/Team%3AHamburg/Part_Collection#main-content)
* [Safety](https://2018.igem.org/Team%3AHamburg/Safety#main-content)
* [Human Practices](https://2018.igem.org/Team%3AHamburg/Human_Practices#main-content)
* Awards

  [Entrepreneurship](https://2018.igem.org/Team%3AHamburg/Entrepreneurship#main-content)
  [Hardware](https://2018.igem.org/Team%3AHamburg/Hardware#main-content)
  [Model](https://2018.igem.org/Team%3AHamburg/Model#main-content)

# Experiments

On this page we only display our protocols. If you are looking for detailed information on important experiments, visit our [Results](https://2018.igem.org/Team%3AHamburg/Results#main-content) page and the registry entries of our [Parts](https://2018.igem.org/Team%3AHamburg/Parts#main-content). If you are looking for detailed information on any experiment not mentioned in Results, visit our [Notebook](https://2018.igem.org/Team%3AHamburg/Notebook#main-content).

##### Agarose gel

Ignatova Lab

* 0.5 g Agarose
* 50 mL TAE-buffer (gel room)

1. Heat in microwave until agarose is dissolved
2. Transfer into gel slide, tip pipet into EtBr, then into gel
3. Cool for 30 minutes

##### Chloramphenicol Stock

Ignatova Lab

34 mg/mL preparation of Choramphenicol Stock (3 in EtOH)

1. dissolve 34 mg of Chloramphenicol in 1 mL 100% ethanol
2. filter through a 0.22 μL filter to sterilize
3. use at 1:1000 dilution in LB or LB-agar
4. mark as CAmp (top) and CAmp/dd.mm.yy/34 mg/mL (side) in Antibiotics
5. store at -20 °C

##### Competent Cells

Zhang Gong

Buffers and solutions:

* **Ca/glycerol buffer:** 60mM CaCl2, 10mM PIPES, 150mL glycerol, Fill water up to 1L, pH=7.0
  Filter sterilization. Avoid autoclaving.

Protocol:

1. Grow the Cells at 37°C till OD=0.2-0.4
2. Chill the culture on ice for 5min.
3. Cllect cells by centrifugation, 6000rpm 10min 4°C
4. Gently resuspend the cells from 500ml LB in 40ml cold Ca/glycerol buffer on ice.
5. Incubate the cells on ice for 5~30min.
6. Repeat step 3 and 4.
7. Incubate the resuspended cells in Ca/glycerol buffer on ice for 30min. (The longer, the better)
8. Collect cells by centrifugation, 6000rpm 10min 4°C
9. Resuspend cells in 6ml Ca/glycerol buffer.
10. Aliquote 150µl/tube. Freeze in liquid N2 and store at -80°C.

##### GeneJET PCR Purification Kit

Thermo SCIENTIFIC Product Information

### PURIFICATION PROTOCOLS

**Note**

* Read IMPORTANT NOTES on p.3 before starting.
* All purification steps ahould be carried out at **room temperature.**
* All centrifugations should be carried out in a table-top microcentrifuge at **>12000 x g** (10000-14000 rpm, depending on the rotor type).

**Protocol A. DNA purification using centrifuge**

| Step | Procedure |
| --- | --- |
| 1 | Add a **1:1 volume** of **Binding Buffer** to completed PCR mixture (e.g. for every 100 µL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow. |
| 2   for DNA ≥500 bp | *Optional:* if the DNA fragment is ≥ 500 bp, add a 1:2 volume of 100% isopropanol (e.g., 100 µL of isopropanol should be added to 100 µL of PCR mixture combined with 100 µL of Binding Buffer). Mix thoroughly.    **Note.** If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower. |
| 3 | Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through.    **Note.** If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane.   **Close the bag with GeneJET Purification Columns tightly after each use!** |
| 4 | Add a **700 µL** of **Wash Buffer** (diluted with the ethanol as described on p.3) to the GeneJET purification column. Centrifuge for 30-60 s.   Discard the flow-through and place the purification column back into the collection tube. |
| 5 | Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.   **Note.** This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions. |
| 6 | Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included). |
| 7 | Discard the GeneJET purification column and store the purified DNA at -20 °C. |

##### GeneJET Plasmid Miniprep Kit

Thermo SCIENTIFIC Product Information

### Growth of Bacterial Cultures

* Pick a single colony from a freshly streaked selective plate to inoculate 1-5 mL of LB medium supplemented with the appropriate selection antibiotic. Incubate for 12-16 hours at 37 °C while shaking at 200-250 rpm. Use a tube or flask with a volume of least 4 times the culture volume.
* Havest the bacterial culture by centrifugation at 8000 rpm (6800 x g) in a microcentrifuge for 2 min at room temperature. Decant the supernetant and remove all remaining medium.

Do not overload the column:

For **high-copy-number plasmids** (*see* Table 1), do not process more than **5 mL** of bacterial culture. If more than 5 mL of such a culture are processed, the GeneJET spin column capacity (20 μL of dsDNA) will be exceeded and no increase in plasmid yield will be obtained.

For **low-copy-number plasmids** (*see* Table 1), it may be necessary to process larger volumes of bacterial culture (up to **10 mL**) to recover a sufficient quantity of DNA.

**Table 1.** Copy numbers of various vectors

|  |  |  |
| --- | --- | --- |
| **High-copy**  300-700 copies per cell | **Low-copy**   10-50 copies per cell | **Very low-copy**  up to 5 copies per cell |
| pUC vectors   pBluescript vectors  pGEM vectors  pTZ vectors  pJET vectors | pBR322 and derivatives  pACYC and derivatives | pSC101 and derivatives |

### PURIFICATION PROTOCOLS

**Note**

* Read IMPORTANT NOTES on p.3 before starting.
* All purification steps should be carried out at **room temperature.**
* All centrifugations should be carried out in a table-top microcentrifuge at **>12000 x g** (10000-14000 rpm, depending on the rotor type).

Use 1-5 mL of *E. coli* culture in LB media for purification of **higt-copy** plasmids.

For **low-copy** plasmids use up to 10 mL of culture.

**Protocol A. Plasmid DNA purification using centrifuges**

| **Step** | **Procedure** |
| --- | --- |
| 1 | Resuspend the pelleted cells in **250 μL of the Resuspension Solution**. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.   **Note.** Ensure RNase A has been added to the Resuspension Solution (as describe on p.3) |
| 2 | Add **250 μL of the Lysis Solution** and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.   **Note.** Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA. |
| 3 | Add **350 μL of the Neutralization Solution** and mix immediately and thoroughly by inverting the tube 4-6 times.    **Note.** It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris.  The neutralized bacterial lysate should become cloudy. |
| 4 | Centrifuge for 5 min to pellet cell debris and chromosomal DNA. |
| 5 | Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white prepipitate.  **Note. Close the bag with GeneJET Spin Columns tightly after each use!** |
| 6 | Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.   **Note.** Do not add bleach to the flow-through, see p.8 for Safety Information. |
| 7   for EndA+ strains only | *Optional:* use this preliminary washing step only if EndA+ strains which have high level of nuclease activity are used.  Wash the GeneJET spin column by adding 500 μL of Wash Solution I (#R1611, diluted) |
| 8 | Add **500 μL of Wash Solution** (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centriifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube. |
| 9 | Repeat the wash procedure (step 8) using **500 μL of the Wash Solution**. |
| 10 | Discard the flow-trough and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps. |
| 11 | Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add **50 μL of the Elution Buffer** to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.    **Note.** An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%  For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70 °C before applying to silica membrane. |
| 12 | Discard the column and store the purified plasmid DNA at -20 °C. |

##### Glycerol stocks

Ignatova Lab

from liquid culture

* 500 μL (50% Glycerol, 50% H2O)
* 500 μL Cells
* Store at -80 °C

##### Preparation for sequencing

Ignatova Lab

* Primer concentration: 100 μM → Concentration needed: 5 μM (dilute at 1:20)
* take 50 nm of sample mix with 5 μL of (5 μM primer, only forward)

##### Standard Assembly

Daniel Wedemyer

Choose enzymes and DNA amount:

|  |  |  |
| --- | --- | --- |
| Fragment | Enzymes | DNA amount |
| Vector | EcoRI, PstI, FastAP | 1000 ng |
| Single insert (e.g. PCR product) | EcoRI, PstI | 500 ng |
| Assembly insert 1 | EcoRI, SpeI | 500 ng |
| Assembly insert 2 | XbaI, PstI | 500 ng |

Set up digestion reactions with DNA volume according to needed amount, 1 µL
of each chosen enzyme, NEBuffer 2.1 to match one tenth of the total
reaction volume, and H2O to fill up to desired reaction volume.

Incubate digestion at 37°C for an hour prior to heat inactivation at 80°C
for 15 minutes. Run a 1% agarose gel with all samples and extract desired
bands using GeneJET Gel Extraction Kit (ThermoFisher).

Set up ligations with 10-fold molar excess of inserts over vector, with
total DNA per ligation to match 200 ng. Add 1 µL T4 DNA Ligase and T4
ligase buffer (ThermoFisher) to match one tenth of the total reaction
volume. Add H2O to fill up to desired reaction volume. Incubate
ligation at 37°C for 1 h prior to heat inactivation at 80°C for 15 minutes.
Transform competent DH5α with all the DNA according to Zhang’s
transformation protocol.

##### Transformation

Zhang Gong

Thaw competent DH5α at 4°C on ice for 5 minutes. Inoculate cells with up to
200 ng DNA, and incubate cells at 4°C on ice for 1 h. Heat shock cells at
42°C for 45 s, and immediately put them back at 4°C on ice for 5 min. Add
850 µL LB medium and incubate at 37°C, 900 RPM on the thermomix for 1h.
Spin down cells, discard 800 µL supernatant and resuspend cells in the
remaining medium. Bring cells onto LB-Agar plates with appropriate
antibiotics and incubate at 37°C overnight.

## Funding

![](https://static.igem.org/mediawiki/2018/b/b8/T--Hamburg--Sponsors_CSS.svg)

![](https://static.igem.org/mediawiki/2018/a/a5/T--Hamburg--Sponsors_AltonaDiagnostics.svg)

![](https://static.igem.org/mediawiki/2018/d/d2/T--Hamburg--Sponsors_SnapGene.svg)

![](https://static.igem.org/mediawiki/2018/e/e8/T--Hamburg--Sponsors_biomol.svg)

![](https://static.igem.org/mediawiki/2018/b/be/T--Hamburg--Sponsors_DeutscheMalaria.svg)

![](https://static.igem.org/mediawiki/2018/1/13/T--Hamburg--Sponsors_NEB.svg)

![](https://static.igem.org/mediawiki/2018/b/b8/T--Hamburg--Sponsors_BioGlobe.svg)

![](https://static.igem.org/mediawiki/2018/7/73/T--Hamburg--Sponsors_IDT.svg)

![](https://static.igem.org/mediawiki/2018/3/32/T--Hamburg--Sponsors_Eurofins.svg)

Ⓒ 2018 iGEM Hamburg

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Hamburg/Results

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* [Home(current)](https://2018.igem.org/Team%3AHamburg)
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* [Project](https://2018.igem.org/Team%3AHamburg/Description#main-content)

  [Description](https://2018.igem.org/Team%3AHamburg/Description#main-content)
  [Design](https://2018.igem.org/Team%3AHamburg/Design#main-content)
  [Protocols](https://2018.igem.org/Team%3AHamburg/Experiments#main-content)
  [Notebook](https://2018.igem.org/Team%3AHamburg/Notebook#main-content)
  [InterLab](https://2018.igem.org/Team%3AHamburg/InterLab#main-content)
  [Model](https://2018.igem.org/Team%3AHamburg/Model#main-content)
  [Results](https://2018.igem.org/Team%3AHamburg/Results#main-content)
  [Demonstrate](https://2018.igem.org/Team%3AHamburg/Demonstrate#main-content)
  [Improve](https://2018.igem.org/Team%3AHamburg/Improve#main-content)
  [Chemistry](https://2018.igem.org/Team%3AHamburg/Chemistry#main-content)
  [Attributions](https://2018.igem.org/Team%3AHamburg/Attributions#main-content)
* Parts

  [Overview](https://2018.igem.org/Team%3AHamburg/Parts#main-content)
  [Basic Parts](https://2018.igem.org/Team%3AHamburg/Basic_Part#main-content)
  [Composite Parts](https://2018.igem.org/Team%3AHamburg/Composite_Part#main-content)
  [Part Collection](https://2018.igem.org/Team%3AHamburg/Part_Collection#main-content)
* [Safety](https://2018.igem.org/Team%3AHamburg/Safety#main-content)
* [Human Practices](https://2018.igem.org/Team%3AHamburg/Human_Practices#main-content)
* Awards

  [Entrepreneurship](https://2018.igem.org/Team%3AHamburg/Entrepreneurship#main-content)
  [Hardware](https://2018.igem.org/Team%3AHamburg/Hardware#main-content)
  [Model](https://2018.igem.org/Team%3AHamburg/Model#main-content)

# Results

## 1. BioBrick Design

We registered 51 BioBricks, of which we sent in 41. Our cloning work was either performed by Standard Assembly according to RFC10 standards or by Golden Gate Assembly with subsequent cloning into pSB1C3. Our parts bring sustainability to synthetic biology. We designed parts which both detect and respond changes to the environment, and help maintaining *E. coli* cultures over extended periods of time. Additionally, we created Parts to lure and kill mosquitoes, and make sustainable, no-maintenance malaria prevention an emerging reality.

## 2. Growth Inhibition Module

To create a Sustainable H.I.E.L.D. we needed the bacteria not to grow and divide, but to maintain a stable cell density. Our growth inhibition module BBa\_K2588021 was confirmed by characterization. It inhibits *E. coli* cell division and growth to a third of its natural growth within only 6 hours after induction. BBa\_K2588021 induces expression of growth inhibiting proteins cbtA, cspD and mraZ in absence of diaminopimelic acid (DAP). This allows to grow bacteria wearing this construct by addition of DAP, but becomes active in natural environments without high amounts of DAP.

## 3. Lure Modules

To bait mosquitoes in the environment of our trap, we designed three Composite Parts:

### 3.1 Lactate

Lactate dehydrogenase A (ldhA) from *E. coli*, which converts pyruvate to lactate. We amplified this gene from *E. coli* genomic DNA by PCR. ldhA was cloned downstream of pBAD promoter BBa\_K206000 and BBa\_0034 ribosome binding site.

We confirmed the overexpression of ldhA by SDS-PAGE with subsequently Coomassie Blue staining.

Furthermore, the reaction rate of ldhA in cell lysates was determined by an activity assay using a coupled test system. The reaction rate of lactate dehydrogenase was 2.1 nmol/(mL\*min).

### 3.2 Myristic Acid and 3-Methyl-1-butanol

Myristic acid and 3-methyl-1-butanol are well-known odor baits to attract *Anopheles gambiae* and can be synthesized in *E. coli*. We designed and submitted all basic parts required to construct both synthetic metabolic pathways, but we were not able to assemble them to their Composites in time.

### 3.3. Thermogenesis Module

Mosquitoes are attracted by heat. Registry Part BBa\_K410000 was described to generate heat at low temperatures. It is regulated by cold-shock promoter HybB (BBa\_J45503). We analysed BBa\_J45503 sequence and found room for major improvements: The original Part does not only contain the promoter sequence, but also the 5’ UTR and parts of the coding sequence of its natural downstream gene, interfering with expression of genes of interest cloned downstream of BBa\_J45503. We improved it by reducing it to its essential sequence. Improved HybB promoter, which we sent in as BBa\_K2588001, showed an over two-fold improved expression and a tighter activation pattern. To complete the thermogenesis module, we also sent in *Nelumbo nucifera* Alternative Oxidase 1a (NnAOX1a, BBa\_K2588018), taken directly from BBa\_K410000. Even after attempting to recreate results written on BBa\_K410000 five times, we could not confirm that NnAOX1a increases temperature, contradicting characterizations by previous iGEM Teams.

## 5. Hydrogel Synthesis

We synthesized a poly ammonium salt hydrogel, providing a surface for mosquitoes to land on, by polymerisation of the monomers ODA (4,4-´oxydianiline) and PMDA (pyromellitic dianhydride) and subsequently treatment with triethylamine.

## 6. Alginate Beads Synthesis

Gel beads consisting of alginate were synthesized and their decomposition in lactic acid containing environment examined. The alginate beads are stable in different for *E. coli* and *S. elongatus* physiological concentrations of lactic acid present and can therefore be used for 3D-embedded culture of *S. elongatus*.

## 7. Human Practices

We talked with various experts including Dr. Mirko Himmel, Dr. David Hutchinson, Dr. Andrew Hammond and Dr. Thomas Jacobs, gaining valuable insights to integrate into our project. We significantly improving our project’s Biosafety and -security, and environmental feasibility based on their advice, and learned much about the fight against malaria.

## 8. Hardware

We designed the S.H.I.E.L.D. to withstand harsh environmental conditions while still maintaining utility using simple yet multifunctional 3D-printed parts. We employ novel techniques like 3D gel-embedded culture and spatially separated co-culture to create an application that redefines standards of environmental synthetic biology.

## 9. Model

We designed Part BBa\_K2588003 as RNA-based transcriptional inverter to create the energy-efficient glucose-induced regulator BBa\_K2588002. In BBa\_K2588002, transcription induced by a constitutive promoter is terminated using short noncoding RNA RnaG120. We built a novel kinetic circuit model to select the constitutive promoter with the best predicted signal to noise ratio of the resulting construct. Despite we were unable to confirm BBa\_K2588002 functionality in the lab, we are confident in our modelling, both for its results, and for its potential use for future applications.

## 10. InterLab

For these years InterLab study we followed the standard protocol by transforming and measuring the GFP expressing plasmids.

## Funding

![](https://static.igem.org/mediawiki/2018/b/b8/T--Hamburg--Sponsors_CSS.svg)

![](https://static.igem.org/mediawiki/2018/a/a5/T--Hamburg--Sponsors_AltonaDiagnostics.svg)

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Ⓒ 2018 iGEM Hamburg