iGEM 2016 — Team Hamburg

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# Team:Hamburg/Notebook

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## 10 mL SOC Medium

* 2 g Bacto-Tryptone
* 0.019 g KCl
* 0.5 g Bacto-Yeast extract
* 0.05 g NaCl
* 0.207 g MgCl2 · 6 H2O
* 0.036 g Glucose

were dissolved in 10 mL H2O and stored at 4°C in 1 mL aliquots.

## 100 mL TB PIPES Buffer

* 1.87 g KCl
* 0.17 g CaCl2
* 15.1 g PIPES

were dissolved in 100 mL H2O. NaOH was added to adjust the pH to 6.7.

## 1 L LB Medium

* 20.3 g Bacto-Tryptone
* 5 g Bacto-Yeast extract
* 10.1 g NaCl

were dissolved in 1 L H2O and autoclaved for 20 min at 120°C 15 pound-force per square inch (psi), to neutralize any bacterial contaminants.

## 15 LB-Agar / CAmp Plates

* 10 g Bacto-Tryptone
* 10 g NaCl
* 15 g Agar
* 5 g Bacto-Yeast extract

Were dissolved in 950 mL H2O and autoclaved for 20 min at 120°C 15 psi.

* 1 mL Chloramphenicol (25 mg/mL)

was added to the LB-Agar at 55°C and poured into 15 sterile petri dishes. The plates were stored at 4°C.

## 1 L 100 mM CaCl2 Buffer

* 11.1 g CaCl2

was dissolved in 1 L H2O and autoclaved for 20 min at 120°C 15 psi.

## 100 mL 85 mM CaCl2, 15% Glycerol Buffer

* 85 mL 100mM CaCl2
* 15 mL Glycerol

were mixed and autoclaved for 20 min at 120°C 15 psi.

## 1 L 100 mM MgCl2

* 20.33 g MgCl2 · 6 H2O

was dissolved in 1 L H2O and autoclaved for 20 min at 120°C 15 psi.

## 10 LB Agar / Kanamycin plates

* 20 g LB-Agar

Was dissolved in 500 mL H2O and autoclaved at 120°C 15 psi.

* 1 mL Kanamycin (25 mg/mL)

was added to the LB-Agar at 55°C and poured into 10 sterile petri dishes. The plates were stored at 4°C.

## 500 mL LB-Agar

* 25.0 g, 25.1 g, 24.99 g and 25.13 g LB Medium

were dissolved in 4x 1 L H2O and autoclaved at 120°C 15 psi.

## Transformation of pcDNA3 and pBAD/Rluc

150 µL competent JW 3367-3 cells were split into 3x50 µL. 57 ng pcDNA3 and 80 ng pBAD/Rluc were added to 1 competent cell tube each and mixed. The cells were incubated on ice for 1.5 h, heat shock at 42°C for 1 min and chilled on ice for 3 min. 950 µL pre-warmed LB medium was added and the cells were incubated at 37°C and 900 rpm for 1h.

The cells were harvested by centrifugation at 3000g for 1 min, resuspended in 50 µL LB medium and 40 µL were plated onto LB Agar/Amp plates. The remaining 10 µL were plated onto different plates. All plates were incubated overnight at 37°C.

### Transformation Efficiency

| plasmid | number of colonies |
| --- | --- |
| pcDNA3 | > 1000 |
| pcDNA3 (1:5) | 408 |
| pBAD/Rluc | 350 |
| pBAD/Rluc | 75 |

The negative controls did not show cell growth.

## Preparation of 0.9 % NaCl Solution

1. 1.8 g NaCl was solved in 200 mL water.
2. NaCl solution was mixed with a magnetic stirrer.
3. NaCl solution was filtrated over a 0.22 μm sterile filter into a glass flask.

## Preparation of 2.5 % Alginate Solution

1. 2.5 g Alginic Acid was slowly dissolved in 100 mL of the 0.9 % NaCl solution
2. The solution was mixed with a magnetic stirrer.
3. The solution was autoclaved for 20 min at 121 °C.

## Preparation of a CaCl2 Solution (100 mM)

1. 1.1098 g CaCl2 was solved in 100 mL water.
2. CaCl2 solution was filtrated over a 0.22 μm sterile filter into a glass flask.

## Preparation of a CaCl2 Solution (100 mM)

1. 1.1098 g CaCl2 was solved in 100 mL water.
2. CaCl2 solution was filtrated over a 0.22 μm sterile filter into a glass flask.

## Transformation of JW 3367-3 (160427DW01) with fluorescence reporter systems: BBa\_E0430, BBa\_E0240, BBa\_R0082

To 4 x 50 μL competent JW 3367-3 were added:

* 0.5 μL Part BBa\_E0430: enhanced yellow fluorescent protein derived from A. victoria GFP (160607DW02)
* 0.5 μL Part BBa\_E0240: GFP (160607DW02)
* 0.5 μL Part BBa\_R0082: Promoter OmpR (160505DC02) as positive control
* nothing as negative control

The cells were incubated on ice for 45 min, heat shocked at 42 °C for 1 min and put on ice for 5 minutes. 950 μL LB media was added to each tube. The cells were incubated at 37 °C, 900 RPM for 1.5 h and brought out on LB-Agar/CAmp plates.

The plates contained:

| LOT# | plate | # of colonies |
| --- | --- | --- |
| 160607DW03 | Part:BBa\_E0430 | 19 |
| 160607DW04 | Part:BBa\_E0240 | 154 |
| 160607DW05 | Par:BBa\_R0082 (positive control) | 36 |
| 160607DW06 | negative control | 0 |

## Fabrication of Alginate Beads

1. 4 mL of 2.5 % Alginic Acid solution was dyed with 2 droplets of red food color.
2. 50 mL of CaCl2 solution was filtrated over a 0.22 μm sterile filter into a beaker.
3. Alginic Acid was pipetted into the CaCl2 solution.
4. Alginate Beads formed.
5. Different sized Alginate Beads were produced using 1000 μL, 200 μL and 10 μL Eppendorf pipettes.
6. Polymerized alginate beads were separated with a coffee filter.

## Preparation of a CaCl2 Solution (100 mM)

1. 4.44 g CaCl2 was solved in 400 mL water.
2. CaCl2 solution was filtrated over a 0.22 μm sterile filter into a glass flask

## Fabrication of Alginate Beads

1. 50 mL of CaCl2 solution was filtrated over a 0.22 μm sterile filter into a beaker.
2. Alginic acid was pipetted into the CaCl2 solution.
3. Alginate beads formed.
4. Different sized alginate beads were produced using 1000 μL, 200 μL and 10 μL Eppendorf pipettes.
5. Polymerized alginate beads were separated with a coffee filter.
6. The beads were stored in different places and the stability was assessed.

   | storage | stability |
   | --- | --- |
   | RT | very stable |
   | fridge | very stable |
   | freezer | not stable (without glycerol) |
7. Images were captured with an optical muscope.
8. Beads were put in the muwave to test stability: aliginate capsules were stable.

## Minipreparation of BBa\_E0240 and BBA\_E0430

The overnight cultures from the day before were centrifuged for 3 min at 5000 x g. The supernatant was discarded. For the further steps see the protocol from the GeneJet Plasmid Miniprep Kit from Thermo Scientific. #K0503. All centrifugation steps were performed at 13000 x g. In the end the columns were eluted with 20 μL elution buffer twice. The dsDNA concentration was measured at the NanoDrop.

| LOT# | Part | DNA concentration |
| --- | --- | --- |
| 160615CL01 | BBA\_E0430 | 159.66 ng/μL |
| 160615PW01 | BBA\_E0240 | 145.56 ng/μL |

## Restriction of BBa\_E0240 (GFP), BBA\_E0430 (eYFP) and BBa\_R0082 (OmpR)

|  | BBa\_E0240 | BBa\_E0430 | BBa\_R0082 |
| --- | --- | --- | --- |
| **Volume** | 3.44 μL | 3.13 μL | 1.37 μL |
| **XbaI** | 1 μL | 1 μL | 1 μL |
| **SpeI** | 1 μL | 1 μL | 1 μL |
| **10x NEB 2.1 Buffer** | 5 μL | 5 μL | 5 μL |
| **water** | 39.76 μL | 39.87 μL | 42.63 μL |
| **LOT%** | 160615CL02 | 160615CL03 | 160615PW02 |

The restriction preparations were incubated at 37 °C for 10 min and afterwards heat-inactivated at 80 °C for 20 min.

## Ligation of Restricted BBa\_E0240 (GFP), BBA\_E0430 (eYFP) with BBa\_R0082 (OmpR)

|  | BBa\_E0240 with BBa\_R0082 | BBa\_E0430 with BBa\_R0082 |
| --- | --- | --- |
| **Volume Reporters** | 2 μL BBa\_E0240 restricted | 2 μL BBa\_E0430 restricted |
| **Volume OmpR** | 2 μL | 2 μL |
| **10x T4 DNA Ligase Buffer** | 2 μL | 2 μL |
| **T4 DNA Ligase** | 1 μL | 1 μL |
| **water** | 13 μL | 13 μL |
| **LOT%** | 160615PW04 | 160615PW03 |

The ligation preparations were incubated at RT for 10 min and afterwards heat-inactivated at 80 °C for 20 min.

## Transformation of JW 3367-3 with Ligation Preparations

14 μL BBa\_E0240 ligated with OmpR was added to 150 μL competent JW 3367-3 cells and mixed. 14 μL BBa\_E0430 ligated with OmpR was added to 150 μL competent JW 3367-3 cells and mixed. The cells were incubated on ice for 1.5 h, heat shocked at 42 °C for 1 min and chilled on ice for 3 min. 950 μL pre-warmed LB medium was added to each tube and the cells were incubated at 37 °C and 900 rpm for 1 h. The cells were brought onto LB Agar plates with Chloramphenicol (CAmp) as a selection antibiotic and stored at 37 °C overnight.

* 160615PW05: LB-Agar CAmp plates with JW 3357-3 transformed with BBa\_R0082/BBa\_E0240
* 160615CL04: LB-Agar CAmp plates with JW 3357-3 transformed with BBa\_R0082/BBa\_E0430

## Fabrication of LB-Agar-Plates with Chloramphenicol

1. 50 g  of LB-Agar (Lucia/Miller) was solved in 1000 mL water.
2. Autoclaved for 1.5 h at 121 °C.
3. Added 250 μL of chloramphenicol.
4. LB-Agar solution was prepared under sterile conditions in petri dishes and after the cooling process was it stored at 4 °C in the fridge

## Transformation of DH5α and BL21 Cells

1. Competent cells were took from the -80 °C freezer onto ice for 30 min (JW 3367-3 / 160427DW01).
2. 2.5 μL of plasmid pET\_Duet\_1 with a Hat t7 promoter and a yellow fluorescent protein (YFP) insert was prepared on ice for 1.5 h.
3. Heat-shock at 42 °C for 45 s.
4. Transformed cells chilled on ice for 3 min.
5. 850 μL LB-Medium was warmed up to 37 °C.
6. After the 3 min the LB-Medium was added to the transformed cells.
7. Incubation of the cells at 37 °C, 900 rpm for 1 h.
8. Centrifugation at 4500 rpm for 2 min, afterwards 950 μL were removed.
9. The pelleted cells resuspended with the rest (~50 μL) of the LB-Medium.
10. 50 μL of the cell solution on a LB-Agar-Plate with Ampicillin was plated out.
11. Plates was stored overnight at 37 °C.
12. * 160615ST01 => BL21 *E. coli* Strain
    * 160615ST02 => BL21 *E. coli* Strain
    * 160615ST03 => DH5α *E. coli* Strain

## Preparation of Bacteria for Alginate Encapsulation

1. Cultures (160615ST01, 160615ST02, 160615ST03) were picked from the incubator.
2. 1 mL LB-Medium with bacteria was pipetted in a cuvette and OD600 was measured versus a blank measurement of LB-Medium.
3. * OD600(DH5α)=2.422
   * OD600(BL21)=1.224
4. Cells were pipetted from the liquid culture to 1 mL into 1.5 mL Eppendorf tubes.

## Isolation of Plasmid pSB1C3-LacZα (160616DC01)

1. Isolation using the Thermo Scientific Purification Kit.
2. Measurement of plasmid concentrations.
3. * 160620CSW01: 45,62 ng/μl
   * 160620CSW02: 63,97 ng/μl

## Restriction of OmpR-ColA and BBa\_I732018 (LacZα)

1. Restriction of OmpR-ColA with SpeI and PstI.
2. Restriction of BBa-I732018 with PstI and XbaI.

|  | BBA\_I732018 | OmpR |
| --- | --- | --- |
| **Volume Plasmid DNA** | 7.8 μL | 2.24 μL |
| **XbaI** | 1 μL | 1 μL |
| **PstI** | 1 μL | 1 μL |
| **10x NEB 2.1 Buffer** | 5 μL | 5 μL |
| **water** | 35.2 μL | 40.5 μL |

The restriction preparations were incubated at 37 °C for 10 min and afterwards heat-inactivated at 80 °C for 20 min.

## Purification of Restriction Reactions

Preparation of 1.75% agarose gel. 10 μL restriction reaction with 2 μL loading dye were added.

| Lane 1 | Lane 2 | Lane 3 |
| --- | --- | --- |
| 2 Log DNA Ladder | BBa\_I732018 (insert) | OmpR (vector) |
| 10 μL | 12 μL | 12 μL |

## Restriction of BBa\_E0240 (GFP) and BBa\_R0082 (OmpR)

|  | BBa\_E0240 | BBa\_R0082 Colony B |
| --- | --- | --- |
| 500 ng Plasmid | 3.4 μL | 3.2 μL |
| XbaI | 1 μL | - |
| SpeI | 1 μL | 1 μL |
| 10X Cutsmart Buffer | 5 μL | 2 μL |
| water | 19.6 μL | 13.8 μL |

The restriction preparations were incubated at 37 °C for 30 min and afterwards heat-inactivated at 80 °C for 20 min.

## Restriction of BBa\_R0082 (OmpR) with BcuI

In the end the plasmid with OmpR should be restricted with SpeI (or BcuI from Thermo Fisher) and PstI. Because the buffer from NEB and Thermo Fisher are not compatible we decided to first restrict the plasmid OmpR with BcuI and afterwards purify it to loose the buffer and enzymes. Then we would perform a second restriction.

 There were three preparations of the OmpR restriction.

|  |  |
| --- | --- |
| Plasmid | 3 x 3.2 μL |
| BcuI (SpeI) | 3 x 1 μL |
| 10X FastDigest Buffer | 3 x 2 μL || Water | 3 x 13.8 μL |

The restriction preparations were purified with the GeneJet PCR Purification Kit von Thermo Scientific. Centrifugations were carried out at 13000 x g and in the end just 20 μL elution buffer was added to the column. Afterwards the concentration were measured at the NanoDrop.

| Preparation | DNA concentration |
| --- | --- |
| I | 85.44 ng/ μL |
| II | 21.57 ng/ μL || III | 23.97 ng/ μL |

## Restriction of BBa\_I732018 (LacZ-160620CSW01) / BBa\_E0430 (eYFP-160615CL01) / BBa\_E0240 (GFP-160615PW01) / BBa\_R0082 (OmpR-160511DC02)

|  | LacZ | eYFP | GFP | OmpR Prep. I-III | OmpR Control 1 | OmpR Control 2 | OmpR Control 3 | OmpR Control 4 | OmpR Control 5 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Plasmid** | 10.9 μL | 3.1 μL | 3.4 μL | 3 x 20 μL | 3.2 μL | 3.2 μL | 3.2 μL | 3.2 μL | 3.2 μL |
| **XbaI** | 1 μL | 1 μL | 1 μL | - | - | - | - | 1 μL | - |
| **PstI** | 1 μL | 1 μL | 1 μL | 3 x 1 μL | - | - | - | - | 1 μL |
| **10X NEB 3.1** | 2.5 μL | 2.5 μL | 2.5 μL | 3 x 2.5 μL | - | 2.5 μL | 2.5 μL | 2.5 μL | 2.5 μL |
| **10X NEB 2.1** | - | - | - | - | 2.5 μL | - | - | - | - |
| **water** | 9.6 μL | 17.4 μL | 17.5 μL | 3 x 1.5 μL | 18.3 μL | 18.3 μL | 19.3 μL | 18.3 μL | 18.3 μL |
| **BcuI** | - | - | - | - | 1 μL | 1 μL | - | - | - |

The restriction preparations were incubated at 37 °C for 30 min and afterwards heat-inactivated at 80 °C for 20 min.

## Control of the restriction of BBa\_I732018, BBa\_E0430, BBa\_E0240, BBa\_R0082 and controls

A 1.7 % agarose gel was prepared with 10 μL RedSafe and loaded using the following setup:

1. 2-Log DNA Ladder (NEB)
2. BBa\_I732018
3. BBa\_E0430
4. BBa\_E0240
5. BBa\_R0082 1
6. BBa\_R0082 2
7. BBa\_R0082 3
8. BBa\_R0082 unrestricted
9. BBa\_R0082 Control XbaI
10. BBa\_R0082 Control PstI
11. BBa\_R0082 Control BcuI in 3.1 NEB
12. BBa\_R0082 Control BcuI in 2.1 NEB

![](“/img/notebook/entry-160704-1-gel.png“)

The restriction was performed to receive the BBa\_I732018, BBa\_E0430 and BBa\_E0240 genes as well as restricting the BBa\_R0082 Plasmid to ligate the Plasmid with the restricted genes of BBa\_I732018, BBa\_E0430 and BBa\_E0240. Therefore the lanes were cut out of the gel and afterwards purified with the GeneJet Gel Extraction Kit from Thermo Fisher Scientific. The centrifugation was performed at 13000 g for 1 min.

|  |  |
| --- | --- |
|  | Concentration |
| BBa\_I732018 | 22.24 ng/μL |
| BBa\_E0430 | 22.40 ng/μL |
| BBa\_E0240 | 23.57 ng/μL |
| BBa\_R0082 1 | 25.42 ng/μL |
| BBa\_R0082 2 | 32.28 ng/μL |
| BBa\_R0082 3 | 24.33 ng/μL |

## Restriction of BBa\_I732018 / BBa\_E0430 / BBa\_E0240 / BBa\_R0082

|  | BBa\_I732018 | BBa\_E0430 | BBa\_E0240 | Control BBa\_R0082 |
| --- | --- | --- | --- | --- |
| Plasmid | 10.9 μL | 3.1 μL | 3.4 μL | 1.4 μL |
| XbaI | 1 μL | 1 μL | 1 μL | - |
| PstI | 1 μL | 1 μL | 1 μL | - |
| BcuI | - | - | - | 1 μL |
| 10x NEB 3.1 | 2.5 μL | 2.5 μL | 2.5 μL | 2.5 μL |
| H2O | 9.6 μL | 17.4 μL | 17.1 μL | 19.1 μL |

Incubation for 1 h at 37 °C. Heat-inactivation for 20 min at 80 °C.

## Control of the restriction of BBa\_I732018, BBa\_E0430, BBa\_E0240, BBa\_R0082 and controls

A 2.2 % agarose gel with 10 μL RedSafe was prepared and loaded using the following setup:

1. 2-Log DNA Ladder (NEB)
2. BBa\_I732018
3. BBa\_E0430
4. BBa\_E0240
5. Control BBa\_R0082 with BcuI

![](“/img/notebook/entry-160704-3-gel.png“)

Control of the restriction of BBa\_I732018, BBa\_E0430, BBa\_E0240, BBa\_R0082 and controls

The lanes were cut out of the gel and afterwards purified with the GeneJet Gel Extraction Kit from Thermo Fisher Scientific. The centrifugation was performed at 13000 g for 1 min.

Afterwards the preparations were measured at the NanoDrop.

|  |  |
| --- | --- |
| BBa\_I732018 | c=100 ng/μL |
| BBa\_E0430 | c=160 ng/μL |
| BBa\_E0240 | c=160 ng/μL |
| Control with gel | c=110 ng/μL |

## Ligation of restricted BBa\_I732018, BBa\_E0430, BBa\_E0240-genes with BBa\_R0082

Ligation mixtures consisted of the following components:

| Amount [μ] | BBa\_E0430 | BBa\_E0240 | BBa\_I732018 |
| --- | --- | --- | --- |
| V (Insert) | 3.57 μL | 3.39 μL | 1.20 μL |
| V (Vektor) | 2 μL | 2 μL | 2 μL |
| V (T4 Ligase) | 1 μL | 1 μL | 1 μL |
| V (H20) | 11.43 μL | 11.61 μL | 13.80 μL |

The preparations were incubated at 16 °C overnight.

## Transformation of JW3367-3 with the ligated plasmids

Competent cells were split into aliquots and incubated on ice with the following DNA samples:

| Aliquot amount | DNA | DNA amount [μL] |
| --- | --- | --- |
| 150 μL | BBa\_R0082/BBa\_I732018 ligation | 10 |
| 150 μL | BBa\_R0082/BBa\_E0430 ligation | 10 |
| 150 μL | BBa\_R0082/BBa\_E0240 ligation | 10 |
| 50 μL | pcDNA | 0.5 |
| 50 μL | pSB1C3/BBa\_R0082 | 1 |
| 50 μL | pSB1C3/BBa\_E0430 | 1 |
| 50 μL | pSB1C3/BBa\_E0240 | 1 |
| 50 μL | pSB1C3/BBa\_I732018 | 1 |

The cells were incubated on ice for 1 h, heat-shocked at 42 °C for 1 min and put back on ice for another 5 min. Ad 1 mL pre-warmed LB medium was added and the cells were incubated at 37 °C / 900 rpm for 1 h on the ThermoMix. The cells were brought onto LB/Agar plates according to the resistances coded in the transformed plasmids in following order:

| Plasmid | Amount [μL] |
| --- | --- |
| pcDNA3 | 150 |
| pSB1C3/BBa\_R0082/BBa\_E0240 | 100  50 |
| pSB1C3/BBa\_R0082/BBa\_E0430 | 100  50 |
| pSB1C3/BBa\_R0082/BBa\_I732018 | 100  50 |
| pSB1C3/BBa\_E0240 | 100  50 |
| pSB1C3/BBa\_E0430 | 100  50 |
| pSB1C3/BBa\_I732018 | 100  50 |
| pSB1C3/BBa\_R0082 | 100  50 |
| negativ control | 100  50 |

## Colony PCR with pSB1C3/BBa\_R0082/BBa\_I732018, pSB1C3/BBa\_R0082/BBa\_E0430 and pSB1C3/BBa\_R0082/BBa\_E0240

(All of the transformed cells (except for the negative control preparations) formed single colonies on the according plates. There was not one cell colony on the negative control plates.)

The mixture of the Colony PCR tubes was done as follows:

|  | BBa\_E0240 | BBa\_E0430 | BBa\_I732018 |
| --- | --- | --- | --- |
| V (DreamTaq-Buffer) | 1.5 μL | 1.5 μL | 1.5 μL |
| V (dNTPs) | 0.5 μL | 0.5 μL | 0.5 μL |
| V (H20) | 5.9 μL | 5.9 μL | 5.9 μL |
| V (VF2) | 1 μL | 1 μL | 1 μL |
| V (VR) | 1 μL | 1 μL | 1 μL |
| Taq | 0.1 μL | 0.1 μL | 0.1 μL |

Assignment of the plates with the colonies that were picked for the PCR and the PCR tubes was as described in the following table:

| Tubes | Plate |
| --- | --- |
| 1, 2, 3, 4 | pSB1C3/BBa\_R0082/BBa\_E0240 |
| 5, 6, 7, 8 | pSB1C3/BBa\_R0082/BBa\_E0430 |
| 9, 10, 11, 12 | pSB1C3/BBa\_R0082/BBa\_I732018 |

## Glycerol Stock of pSB1C3/BBa\_R0082/BBa\_E0430 and pSB1C3/BBa\_R0082/BBa\_E0240

Made a Glycerol Stock of the overnight cultures from the day before. Therefore mixed 250 μL Glycerol with 250 μL water and 500 μL culture. The stocks were stored at -80 °C.

* Stock of BBa\_R0082/BBa\_E0240
* Stock of BBa\_R0082/BBa\_E0430 A
* Stock of BBa\_R0082/BBa\_E0430 B
* Stock of BBa\_R0082/BBa\_E0430 C
* Stock of BBa\_R0082/BBa\_E0430 D

## Minipreparation of pSB1C3/BBa\_R0082/BBa\_E0430 and pSB1C3/BBa\_R0082/BBa\_E0240

The over night cultures from the day before were centrifuged for 3 min at 5000 g. The supernatant was discarded. For the further steps see the protocol from the GeneJet Plasmid Miniprep Kit from Thermo Scientific. #K0503. All centrifugation steps were performed at 13000 g. In the end the columns were eluted with 20 μL Elutionbuffer twice.

* Miniprep of BBa\_R0082/BBa\_E0240
* Miniprep of BBa\_R0082/BBa\_E0430 A
* Miniprep of BBa\_R0082/BBa\_E0430 B
* Miniprep of BBa\_R0082/BBa\_E0430 C
* Miniprep of BBa\_R0082/BBa\_E0430 D

The dsDNA concentration was measured at the NanoDrop.

|  |  |
| --- | --- |
| BBa\_R0082/BBa\_E0240 | 188.46 μL |
| BBa\_R0082/BBa\_E0430 A | 219.83 μL |
| BBa\_R0082/BBa\_E0430 B | 321.86 μL |
| BBa\_R0082/BBa\_E0430 C | 223.68 μL |
| BBa\_R0082/BBa\_E0430 D | 339.88 μL |

## Colony PCR with pSB1C3/BBa\_R0082/BBa\_I732018

The PCR was performed with 4 colonies. The mixture of the Colony PCR tubes was done as follows:

The PCR Program TAQSCREEN was used.

|  | BBa\_I732018 |
| --- | --- |
| V (DreamTaq Buffer) | 1.0 μL |
| V (dNTPs) | 0.5 μL |
| V (H2O) | 6.4 μL |
| V (VF2) | 1.0 μL |
| V (VR) | 1.0 μL |
| Taq | 0.1 μL |

| Tubes | Plates |
| --- | --- |
| 1, 2 | pSB1C3/BBa\_R0082/BBa\_I732018 (colonies 1 and 2) |
| 3, 4 | pSB1C3/BBa\_R0082/BBa\_I732018 (colonies 3 and 4) |

## Gel electrophoresis in 2.2 % agarose gel and 1:10000 RedSafe with the samples from the colony PCR

Potential difference: 100 V

Duration: ˜45 min

![](“/img/notebook/entry-160707-4-gel.png“)

Control of the Colony PCR of pSB1C3/BBa\_R0082/BBa\_I732018

## Sequencing ofpSB1C3/BBa\_R0082/BBa\_E0430 and pSB1C3/BBa\_R0082/BBa\_E0240

* 2.4 μL of pSB1C3/BBa\_R0082/BBa\_E0240 once with 5 μL VF2 and once with 5 μL VR
* 2.2 μL of pSB1C3/BBa\_R0082/BBa\_E0430 1 once with 5 μL VF2 and once with 5 μL VR
* 1.6 μL of pSB1C3/BBa\_R0082/BBa\_E0430 2 once with 5 μL VF2 and once with 5 μL VR
* 2.2 μL of pSB1C3/BBa\_R0082/BBa\_E0430 3 once with 5 μL VF2 and once with 5 μL VR
* 1.5 μL of pSB1C3/BBa\_R0082/BBa\_E0430 4 once with 5 μL VF2 and once with 5 μL VR

## Preparation of overnight cultures

Plates from the 5.07.2016 were picked and put into overnight culture

* pcDNA
* pSB1C3/BBa\_E0240
* pSB1C3/BBa\_E0430
* pSB1C3/BBa\_I732018
* pSB1C3/BBa\_R0082

## LB-Agar Plates

Each 35 CAmp and Amp plates were prepared, with 100 μL Ampicillin and 33 μg/L Chloramphenicol, using Roth: LB-Broth chemical mix.

Plates were stored in 4 °C freezer until needed.

## 1 L LB-Medium

Was prepared as well, using Roth: LB-medium chemical mix and stored in 4 °C freezer.

## Miniprep of overnight cultures

| **Liquid culture from JW 3367-3** |
| --- |
| Colony of pSB1C3/BBa\_E0240 GFP plate |
| Colony from pSB1C3/BBa\_E0430 YFP plate |
| Colony from pSB1C3/BBa\_I732018 LacZΑ plate |
| Colony from pSB1C3/BBa\_I732018 LacZΑ plate |
| Colony from pSB1C3/BBa\_R0082 |
| Colony from pBAD/Rluc plate |
| Colony from pcDNA3 plate |

Miniprep was performed according to the GeneJET Miniprep Kit Protocol by ThermoScientific. Storage of plasmid eluates at -20°C.

## Restriction of EnvZ-LRR, pSB1C3, BBa\_I732018 and pSB1C3/BBa\_R0082

**First restriction**

|  | **EnvZ-LRR 3'** | **EnvZ-LRR 5'** | **pSB1C3** | **pSB1C3/BBa\_R0082** | **pSB1C3/BBa\_I732018** |
| --- | --- | --- | --- | --- | --- |
| **Plasmid/DNA** | 10  μL | 10  μL | 2  μL | 20  μL | 11  μL |
| **PvuI** | 1  μL | 1  μL | - | - | - |
| **BcuI** | 1  μL | - | 1  μL | - | - |
| **XbaI** | - | - | - | 1  μL | 1  μL |
| **Buffer** | 2  μL FastDigest | 2  μL FastDigest | 2  μL FastDigest | 2  μL NEB2.1 Buffer | 2  μL NEB2.1 Buffer |
| **water** | 16  μL | 17  μL | 15  μL | 6  μL | 6  μL |
| **Total** | **30  μL** | **30  μL** | **20  μL** | **30  μL** | **21  μL** |

## Purification of restricted DNA EnvZ-LRR 5’ and pSB1C3

* Protocol see manufacturer's instructions of Thermo Scientific Purification Kit

## Restriction of EnvZ-LRR 5’ and pSB1C3 with EcoRI

Second restriction of the two samples was needed because EcoRI needs a different buffer than PvuI and BcuI.

|  | **EnvZ-LRR 5'** | **pSB1C3** |
| --- | --- | --- |
| **Plasmid/DNA** | 50  μL | 50  μL |
| **EcoRI** | 2  μL | 2  μL |
| **NEB2.1 Buffer** | 6  μL | 6  μL |
| **water** | 2  μL | 2  μL |
| **Total:** | **60  μL** | **60  μL** |

## Ligation of pSB1C3 with EnvZ-LRR3’ and EnvZ-LRR5’

* 1:10 (Vector:Insert) Ligation

|  | **Amount [ μL]** |
| --- | --- |
| **EnvZ-LRR3’ (Insert)** | 5.7 |
| **EnvZ-LRR5’ (Insert)** | 5.7 |
| **pSB1C3 (Vector)** | 5.7 || **T4 Ligase** | 1 || **DNA Ligase Buffer** | 2 |

* 1:5 (Vector:Insert) Ligation

|  | **Amount [ μL]** |
| --- | --- |
| **EnvZ-LRR3’ (Insert)** | 4.25 |
| **EnvZ-LRR5’ (Insert)** | 4.25 |
| **pSB1C3 (Vector)** | 8.5 || **T4 Ligase** | 1 || **DNA Ligase Buffer** | 2 |

All samples were left for incubation at 12° C overnight.

## Ligation of pSB1C3/BBa\_R0082 with pSB1C3/BBa\_I732018

* 1:10 (Vector:Insert) Ligation

|  | **Amount [ μL]** |
| --- | --- |
| **pSB1C3/BBa\_I732018** | 8.5 |
| **pSB1C3/BBa\_R0082** | 8.5 |
| **T4 Ligase** | 1 |
| **DNA Ligase Buffer** | 2 |

All samples were left for incubation at 12° C overnight.

## Transformation of competent JW-33673 cells with pSB1C3-EnvZ-LRR 1:10 and pSB1C3-EnvZ-LRR 1:5

| **Amount JW-33673 cells** | **Plasmid** | **Amount plasmid** |
| --- | --- | --- |
| 150  μL | pSB1C3-EnvZ-LRR 1:10 | 10  μL |
| 150  μL | pSB1C3-EnvZ-LRR 1:5 | 10  μL |
| 75  μL | pSB1C3 1:4 (positive control) | 1  μL |
| 75  μL | - (negative control) | - |

All plates were put in incubator at 37° C overnight.

## Colony PCR of pSB1C3/EnvZ-LRR 3’

The ligation preparations of EnvZ-LRR and pSB1C3 were amplified by colony PCR after preparing the following master mix:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | **1x** | **7x** || **Dreamtaq-Buffer** | 1  μL | 7  μL |
| **dNTPs (10 mM)** | 0.5  μL | 3.5  μL |
| **water** | 6.4  μL | 44.8  μL |
| **VF2 primer (10 mM)** | 1  μL | 7  μL |
| **VR primer (10 mM)** | 1  μL | 7  μL |
| **DreamTaq-Polymerase** | 0.1  μL | 0.7  μL |

4 colonies from the 1:10 and 2 colonies from the 1:5 ligation were picked and LB cultures were prepared in parallel.

The following PCR setup was used:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1. | 98° C | 2:00 || 2. | 98° C | 0:10 |
| 3. | 55° C | 0:45 |
| 4. | 72° C | 3:15 |
| 5. GOTO 2 repeat 34x |
| 6. HOLD at 4° C |

## Restriction of BBa\_R0082 and BBa\_I732018

Because of buffer difficulties, Omp had to be restricked sequentielly with two restriction enzyms BcuI (ThermoF.) and PstI (NEB). After the first restriction, the restricted vector was purified via the PCR Purification Kit, following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-PCR-Purification-Kit). Pippeting of the samples was done, as described in the following table:

* 1. Restriction of BBa\_R0082

| Intredient | Volume |
| --- | --- |
| 1:10 diluted pSB1C3/BBa\_R0082 (363.67 ng/μL) | 1.3 μL |
| BcuI | 1.0 μL |
| Fast Digest Buffer (T.F.) | 5.0 μL |
| H2O | 42.8 μL |

* 2. Restriction of BBa\_I732018

| Intredient | Volume |
| --- | --- |
| BBa\_I732018 (33.89 ng/μL) | 8.0 μL |
| XbaI | 1.0 μL |
| PstI | 1.0 μL |
| NEBuffer 3.1 | 5.0 μL |
| H2O | 35.0 μL |

* 3. Second Restriction of BBa\_R0082

| Ingredient | Volume |
| --- | --- |
| BBa\_R0082 (Restricded with BcuI) | 10 μL |
| PstI | 1.0 μL |
| NEBuffer 3.1 | 5.0 μL |
| H2O | 34.0 μL |

## Minipreparation of the Over-Night-Cultures from pSB1C3-EnvZ-LRR

From each sample a cryostock was prepared, following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#glycerol-stocks).

The rest of the samples were miniprapareted, following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-Plasmid-Miniprep-Kit).

## Gel control of the mini-prep, the restriction products BBa\_R0082 and BBa\_I732018

200 mL gel with 2.0 μL redsafe at 150 V, t:0.75 h

1. Ladder
2. Mini-prep product 1
3. Mini-prep product 2
4. Mini-prep product 3
5. Mini-prep product 4
6. Mini-prep product 5
7. Mini-prep product 6
8. Mini-prep product 7
9. Restrection product BBa\_R0082
10. Restricton product BBa\_I732018

![](“/img/notebook/entry-160715-3-gel.png“)

Control of the mini-prep, the restriction products BBa\_R0082 and BBa\_I732018

## Restriction of BBa\_I732018 and pSB1A3 and sequential restriction of BBa\_R0082 with BcuI and EcoRI

The BBa\_I732018 and BBa\_R0082 genes should be inserted into pSB1A3. Therefore BBa\_I732018 and pSB1A3 were restricted like shown in the table below:

|  |  |  |
| --- | --- | --- |
|  | BBa\_I732018 | BBa\_R0082 |
| Plasmid DNA | 2.0 μL (700 ng) | 4.0 μL (100 ng) |
| XbaI | 1.0 μL | 0.5 μL |
| PstI | 1.0 μL | 0.5 μL |
| 10x NEB 2.1 Buffer | 2.0 μL | 2.0 μL |
| H2O | 14.0 μL | 13.0 μL |

The Plasmid with BBa\_R0082 should be restricted with SpeI (or BcuI from Thermo Fisher) and EcoRI. Because the buffer from NEB and Thermo Fisher are not compatible. The BBa\_R0082 plasmid is first restricted with BcuI and afterwards purify it to loose the Buffer and Enzyme. Only then the second restriction.

First restriction:

|  | BBa\_R0082 |
| --- | --- |
| Plasmid DNA | 2.0 μL (720 ng) |
| BcuI | 1.0 μL |
| 10x Fast Digest Buffer | 2.0 μL |
| H2O | 15 μL |

There were two preparations of the BBa\_R0082 restriction.

The restriction products were purified with the GeneJet PCR Purification Kit, following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-PCR-Purification-Kit). Centrifugations were carried out at 12000 g. For preperation I 50 μL of Elutions buffer was added to the column. For Preperation II 15 μL of Elutions buffer was added to the column. Afterwards the concentration was measured at the NanoDrop.

|  |  |
| --- | --- |
| Preparation I | 8.34 ng/μL |
| Preparation II | 23.61 ng/μL |

Second Restriction

|  | Preparation I | Preparatin II |
| --- | --- | --- |
| Plasmid DNA | 29 μL | 29 μL |
| EcoRI | 1 μL | 1 μL |
| 10x NEBuffer 2.1 | 2.0 μL | 2.0 μL |
| H2O | 18 μL | 32 μL |

Heat-inactivation: 80 °C, 20 min

## Sequential restriction of BBa\_R0082 with BcuI and PstI

The Plasmid with BBa\_R0082 should be restricted with SpeI (or BcuI from Thermo Fisher) and PstI. Because the buffer from NEB and Thermo Fisher are not compatible we decided to first restrict the Plasmid BBa\_R0082 with BcuI and afterwards purify it to loose the Buffer and Enzyme. Then we would perform a second restriction.

First restriction:

|  | BBa\_R0082 |
| --- | --- |
| Plasmid DNA | 4.0 μL |
| BcuI | 1.0 μL |
| 10x Fast Digest Buffer | 2.0 μL |
| H2O | 13 μL |

The restriction product was purified with the GeneJet PCR Purification Kit von Thermo Scientific, following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-PCR-Purification-Kit). Centrifugations were carried out at 12000 g. Washing was performed with using only 50 μL Wash buffer and for elution only 50 μL of Elutions buffer was added to the column. The concentration was measured at the NanoDrop.

Purified extract (BBa\_R0082) are c=15.62 ng/μL

Second Restriction:

|  | Purified extract (BBa\_R0082) |
| --- | --- |
| Plasmid DNA | 40.0 μL |
| PstI | 1.0 μL |
| 10x NEBuffer 2.1 | 5.0 μL |
| H2O | 4.0 μL |

Heat-inactivation: 80 °C, 20 min

## Ligation of restricted BBa\_I732018-gene with BBa\_R0082

Ligation mixtures consisted of the following components:

|  | Preparation I | Preparation II | Praparation III |
| --- | --- | --- | --- |
| BBa\_I732018 | 2.74 μL  (Insert:Vector=3:1) | 3.706 μL  (Insert:Vector=5:1) | 5.78 μL  (Insert:Vector=7:1) |
| BBa\_R0082 | 2.0 μL (31.24 ng) | 2.0 μL (31.24 ng) | 2.0 μL (31.24 ng) |
| T4 Ligase | 1.0 μL (31.24 ng) | 1.0 μL (31.24 ng) | 1.0 μL (31.24 ng) |
| H2O | 12.26 μL | 11.29 μL (31.24 ng) | 9.22 μL (31.24 ng) |

The preparations were incubated at 16 °C overnight.

## Transformation of the Ligation preparations of pSB1C3/BBa\_R0082/BBa\_I732018 and the Parts BBa\_K081005, BBa\_K608006 and BBa\_K608007

The parts BBa\_K608006 (Plate 1, 5E), BBa\_K081005 (Plate 3, 16E) and BBa\_K608007 (Plate 1, EG) were resuspended in 10 μL dest. H2O (DNAse free).

10 μL of the Ligation Preparations pSB1C3/BBa\_R0082/BBa\_I732018 with Insert:Vector of 3:1, 5:1 and 7:1 were added to 150 μL competent cells and mixed. 150 μL competent JW3367-3 cells were split into 3x 50 μL. 1 μL of the resuspended Parts were added to 1 competent cell tube each and mixed. The cells were incubated on ice for 1 h, heat shock at 42 °C for 1 min and chilled on ice for 3 min. 850 μL pre-warmed LB medium was added and the cells were incubated at 37 °C 900 rpm for 1 h.

The cells were harvested by centrifugation at 4500 rpm for 1 min, resuspended in 50 μL LB medium and 50 μL were plated onto LB Agar/Camp plates. All plates were incubated over night at 37 °C.

Transformation following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#transformation)

## Transformation of DH5a and JW 3367-3 with pSB1C3/EnvZ-NOD1 and pSB1C3/BBa\_R0082/BBa\_I732018

Competent DH5a and JW 3367-3 were thawed at 4° C, split and inoculated with DNA as following:

| Volume of cells | Cell line | Transfected with | Amount of DNA |
| --- | --- | --- | --- |
| 50 µL | DH5α | pSB1C3/BBa\_R0082/BBa\_I732018 | 50 ng |
| 50 µL | JW 3367-3 | pSB1C3/BBa\_R0082/BBa\_I732018 | 50 ng |
| 50 µL | JW 3367-3 | pSB1C3/BBa\_R0082/BBa\_I732018 | 50 ng |
| 150 µL | JW 3367-3 | pSB1C3/EnvZ-NOD1 1:10 | 10 µL |
| 150 µL | JW 3367-3 | pSB1C3/EnvZ-NOD1 1:5 | 10 µL |
| 150 µL | JW 3367-3 | pSB1C3/EnvZ-NOD1 1:5 | 2 µL |
| 50 µL | DH5α | none | none |
| 50 µL | JW3367-3 | none | none |
| 50 µL | JW3367-3 | none | none |

Cells and DNA were incubated at 4° C for 1 h, heat shocked at 42 ° C for 1 minute and put on ice at 4°C for 5 more minutes. Add 1 mL of 37° C pre-warmed.

## Transformation of DH5α with eYFP (pSB1C3/BBa\_R0082/BBa\_E0430) and GFP (pSB1C3/BBa\_R0082/BBa\_E0240) reporter constructs

150 µL competent DH5ɑ cells were thawed at 4°C split into three 50 µL tubes. Plasmids were added:

| cells | Plasmid | amount |
| --- | --- | --- |
| DH5α | pSB1C3/BBa\_R0082/BBa\_E0430 | 50 ng |
| DH5α | pSB1C3/BBa\_R0082/BBa\_E0240 | 50 ng |
| DH5α (negative control) | none | none |

The cells were incubated at 4°C for 1h, heat shocked at 42°C for 1 min and put back on ice at 4°C for 5 minutes. 950 µL 37°C warm LB broth was added to each tube and the cells were incubated at 37°C / 900 RPM on the thermomix for 1 h.

Cells were centrifuged, resuspended in 50 µL LB broth, brought onto LB-Agar/CAmp Plates and incubated at 37°C overnight.

## Fabrication of ED01 SU-8 Master (1st attempt)

* A 4-inch Si-wafer was used to fabricate a master(stamp) for polydimethylsiloxane (PDMS) printing. Channels of the final microfluidic (MF) chip have 125 μm width according to CAD file.
* SU-8 photoresist was chosen to serve as the imprinting material
* To acquire square sections of the MF-chip channels, the Master was processed according to the datasheet for typical processes with 100 μm resist thickness
* The 4-inch Si-wafer was baked in an oven at 130 °C for 10 minutes
* SU-8 (Gersteltec GM1075) was spin coated on the Si-wafer directly after baking using the following parameters:
  1) 100 rpm/second acceleration to 1700 rpm (for 17 seconds)
  2) 100 seconds spinning at 1700 rpm
  3) spin peak with 400 rpm/second acceleration to 2100 rpm (for 1 second)
  4) -100 rpm/second deceleration to 0 rpm (for 21 seconds)
* The Si-wafer was kept at 40 °C for 30 minutes to allow for relaxation of the resis edges
* appearing gas bubbles in the resist were popped with a sterile syringe tip
* The wafer was soft baked for 2 minutes at 120 °C with temperature ramps of 4 °C/minute up and down
* The wafer was illuminated for 16 seconds with an exposure dose of 13 mW/cm2
  (208 mJ/cm2 - instead of recommended 200 mJ/cm2)
* After exposure the wafer was stored for a delay time of 1 hour to let the created initiator molecules diffuse until homogeneity was reached
* The wafer was PEBd for 30 minutes at 95 °C with 4 °C/minute temperature ramps up and down from room temperature
* After RT was reached, the wafer was put into used propylene glycol methyl ether acetate (PGMEA) for 3 minutes for development of the SU-8 resist. Slight detachment of the SU-8 from the Si-wafer was observed
* Development was cancelled by rinsing with isopropanol after removing the wafer from PGMEA. Strong white liquid formation occurred whilst rinsing with isopropanol
* SU-8 was undeveloped
* The wafer was kept in isopropanol for about 1 hour
* After removal from the isopropanol bath the wafer remained underdeveloped
* The wafer was put back in used PGMEA for 3 minutes. Partial detachment of the remaining structures after completed development became clearly visible. The structures remained intact.
* Rinsing with isopropanol showed slight to no white traces, indicating completed development
* The imprint structures were clearly visible
* The smallest features of the SU-8 Master detached completely and eventually rested randomly across the Master’s Si-wafer surface
* Examination of the shape of the structures with a profilometer (DektakXT; Bruker) showed a SU-8 thickness of 65 +/- 1 μm (as opposed to 100 μm according to the datasheet)
* SU-8 Master for the Encapsulation Device (ED01-Master01) was not suitable for channel formation with square-like sections

## Fabrication of ED01 SU-8 Master (2nd attempt)

* A 4-inch Si-wafer was put in an oven at 130 °C overnight from the seventh to the eighth of August
* GM1060 photoresist (SU-8 thin application; Gersteltec) was used as a base adhesion layer for the GM1075 photoresist (SU-8 thick application; Gersteltec) to increase structure layer attachment to the substrate
* The parameters used for the GM1060-SU-8 base layer were derived from the GM1060 datasheet typical processes for 6 μm layer thickness except for an adjusted spin speed of 6000 rpm and the cancellation of the process after the post-exposure bake (PEB) due to the lack of need for structuring
* After dehydration of the Si-wafer in the oven the base layer was spinned on the Si-wafer with the following parameters:

  Spinning:
  + 100 rpm/second to 6000 rpm (for 60 seconds)
  + 6000 rpm for 40 seconds
  + -100 rpm/second to 0 rpm (for 60 seconds)Relaxation:
  + 5 minutes at RTSoft bake:
  + 2 °C/minute from RT to 65 °C
  + 5 minutes at 65 °C
  + 2 °C/minute from 65 °C to 95 °C
  + 5 minutes at 95 °C
  + coli down to 50 °CExposure:
  + 20 seconds with 13 mW/cm2 (260 mJ/cm2 instead of recommended 200 mJ/cm2) on i-line (365 nm)Delay:
  + 10 minutesPEB:
  + Same as soft bake except 15 minutes at 95 °C
* After the soft bake small white dots (bubbles) were visible in or on the SU-8 layer, but they were too small to pop open, as well was too many; temperature profile may be irrelevant or relaxation time should be longer to allow for more gentle solvent evaporation
* After the PEB the structural GM1075-SU-8 layer was applied and processed
* The fabrication of the structural layer of ED01-Master02 was performed similiar to ED01-Master01, but according to the datasheet parameters for the typical process for 150 μm layer thickness with slight modifications to reach the desired 100 μm thickness. Following parameters were used:

  Spinning:
  1. 100 rpm/second to 1200 rpm (for 12 seconds)
  2. 1200 rpm for 100 seconds
  3. 400 rpm/second to 1600 rpm (for 1 second)
  4. -100 rpm/second to 0 rpm (for 16 seconds)Relaxation:
  1. 30 minutes at 40 °C
  Soft bake:2. 4 °C/minute from 40 to 120 °C
  3. 5 minutes at 120 °C
  4. -4 °C/minute from 120 °C to RT
  Exposure:5. 20 seconds with 13 mW/cm2 (260 mJ/cm2 instead of recommended 200 mJ/cm2 for 100 μm) on i-line (365 nm)
  Delay:6. 1 hour
  PEB:7. 4 °C/minute from 40 to 95 °C
  8. 40 minutes at 95 °C; instead of 30 minutes recommended for 100 μm
  9. -4 °C/minute from 95 °C to RT
* Slight to no white traces were observed during post-development isopropanol rinse; structures remained attached and intact remaining clear
* Examination of ED01-Master02 and the shape of the structures with a profilometer (DektakXT; Bruker) showed a SU-8 thickness of 87 +/- 1 μm (as opposed to 150 μm according to the datasheet); structures appeared clear and the edges sharp; the measured properties are sufficient to serve as a stamp for the ED01-chips
* To create castings of PDMS from the ED01-Master02 72.37 g of PDMS oligomer (Sylgard 184 silicone elastomer base) and 7.26 g of initiator (Sylgard 184 elastomer curing agent) were thoroughly mixed
* The ED01-Master02 was put into a glass petri dish which was covered with aluminium foil
* The PDMS mixture was poured over the ED01-Master02 and the dish was put into a desiccator to remove air bubbles from inside the PDMS
* Subsequently the dish including the mixture was covered to prevent dust from entering and incubated in an oven at 65 °C overnight to allow it to polymerize completely

## LB - medium

* 25 g LB - medium
* add to 1 L H2O
* -> autoclave

## LB - agar

* 10 g LB - agar
* add 250 mL H2O
* -> autoclave

## LB agar plates

* heat 250 mL LB - agar (is hot from the autoclave)
* Cool down to approx. 50 °C
* add 250 µL chloramphenicol
* Preparation of 13 plates under steril conditions
* -> store at 4 °C
* Mark as LB CAmp Plates 160809

## Plating of JW 3367-3 and DH5α with fluorescence reporters

To use

* 3 plates (LB CAmp Plates iGEM 09.08.2015)
* Cells LOT# 190707CL03 (JW 3367-3 with pSB1C3/BBa\_R0082/BBa\_E0430) (Omp/eYFP)
* Cells LOT# 160803KP03 (DH5&alpha with pSB1C3/BBa\_R082/BBa\_E0240) (Omp/GFP)
* Cells LOT# 160803KP02 (DH5&alpha with pSB1C3/BBa\_R082/BBa\_E0430) (Omp/eYFP)

Now work under sterile conditions:

* For DH5α/eYFP: pick one colony from the plate 160803KP02, put it in 50 µL LB medium, put the 50 µL LB medium with cells on the LB - Agar Plate and grease it -> stored at 37 °C overnight
* For DH5α/GFP: the same as the DH5α/eYFP
* For JW 3367-3/eYFP: warm up the cryostock, than pick 5 µL up and put it on 45 µL LB medium, put the 50 µL to the plate and grease it -> stored at 37 °C overnight

## Fabrication of ED01 MF-Chips

* Petri dish with cured PDMS on ED01-Master02 was removed from the oven (65 °C) after overnight incubation
* PDMS was removed from the Master by fragile pulling
* PDMS was cut with scalpel and broken by hand to retrieve three replicated channel systems in one PDMS piece
* Access channels for syringe tubes (input and output) were cut with a biopsy cutter / hole punch (0.75 mm diameter) and rinsed with isopropanol and dried with a N2-gun
* 3 glass object slides and 3 triple-device PDMS pieces were plasma treated in 02-plasma for 100 seconds at approx. 10 W and 0.38 mbar
* The glass object slides and PDMS pieces were brought into contact with their plasma treated surfaces by pressing them together by hand, forming the ED01 MF-Chips
* The ED01 MF-chips were kept still for 3 hours until stable binding of glass and PDMS was tested
* One of the three created ED01 MF-chips bonded completely with all three devices appearing intact

## Fluorescence microscopy of plates

Fluorescence microscopy of bacteria on plates:

![](/img/Notebook/Entry-160813-1-1.tif)
**DH5α/pSB1C3/BBa\_R0082/BBa\_E0240 (Omp/GFP).** Magnification factor: 40x; exposure: 75,31 ms.

![](/img/Notebook/Entry-160813-1-2.tif)
**JW3367-3/pSB1C3/BBa\_R0082/BBa\_E0240 (Omp/GFP).** Magnification factor: 40x; exposure: 75,31 ms.

![](/img/Notebook/Entry-160813-1-3.tif)
**JW3367-3/pSB1C3/BBa\_R0082/BBa\_E0240 (Omp/GFP).** Magnification factor: 40x; exposure: 1 s.

Note: As expected, DH5α cells showed higher fluorescence than JW3367-3, which are lacking the EnvZ receptor required for activation of ompR.

## FACS

Following cell lines were measured with FACS:

* DH5α/pSB1C3/BBa\_R0082/BBa\_E0240 (Omp/GFP)
* DH5α/pSB1C3/BBa\_R0082/BBa\_E0430 (Omp/eYFP)
* JW3367-3/pSB1C3/BBa\_R0082/BBa\_E0240 (Omp/GFP)
* JW3367-3/pSB1C3/BBa\_R0082/BBa\_E0430 (Omp/eYFP)
* DH5α (negative control)
* JW3367-3 (negative control)

As buffer 100 mM MgCL2 was used and the cells were diluted 1:100.

Setup: Cytometry was conducted with the *FACSCalibur* System configured as follows.

| Detector | Voltage | Mode |
| --- | --- | --- |
| FSC | E02 | Lin |
| SSC | 628 | Lin |
| FL1 | 705 | Log |

Threshold FSC: 0
Mode: Hi

## Preparation of Alginic Acid Solution

* Second creation of alginate solution 2.5 % ; soluted for about 12 hours

## Preparation encapsulated JW cells from 22.08.16

* The OD600 of the overnight culture was measured and amounts to 4.770
* Due to the encapsulation protocol which requires an OD of about 1.5, 2 mL of the main culture were diluted with 4 mL of fresh LB medium without any antibiotics
* OD600 of the diluted culture equaled to 2.00
* The 6 mL of bacterial solution were centrifuged in 2 mL tubes separately for 5 minutes at 4500 rpm. Due to unexpected waiting time the mixtures were centrifuged for another 2 minutes before separating the pellet from the fluid
* The 3 tubes with bacterial pellets were resuspended with 333 μL 0.9 % NaCl each and thereafter reunited
* 1 mL of diluted bacteria were mixed with 2 mL of 2.5 % alginate solution
* Encapsulation was managed through pipetting the alginate-bacteria solution in 100 mL BaCl2
* The BaCl2 was sterile filtered for reuse
* The capsules were washed with 100 mL dist. water
* About 10 capsules were crushed immediately and set into culture with 5 mL LB cAmp
* Mistake: JW do not have cAmp resistance, therefore no growth was possible
* Mistake was identified after first measurement (1.5 hours)
* Measurements of the intact capsules in LB cAmp as well did not show any growth after 1.5 hours and 2 hours
* Analogous to the BaCl2 encapsulation, CaCl2 encapsulation was performed; a bacterial solution of OD600 = 1.83 was used
* Incubation with LB medium (antibiotics free) did not show growth
* Encapsulated bacteria could be seen using a light microscope; bacteria supposedly strongly bound; new dilution/capsule-crushing methods needed
* Overnight culture of crushed parts was fixed

## OD600 Measurement of Overnight Cultures from 23.08.16

* No visible indication for bacterial growth were observed in the overnight cultures

## Restriction of BBa\_C0082 and the Anderson Promoters for 3A Assembly

| Part | BBa\_C0082 | BBa\_K081005 | BBa\_K081006 | BBa\_K081007 |
| --- | --- | --- | --- | --- |
| DNA | 48 µL | 16 µL | 10.3 µL | 14.1 µL |
| NEB Buffer 2.1 | 6 µL | 2 µL | 2 µL | 2 µL |
| XbaI | 2 µL | - | - | - |
| PstI | 2 µL | 1 µL | 1 µL | 1 µL |
| H2O | 2 µL | 1 µL | 6.7 µL | 2.9 µL |
| Total | 60 µL | 20 µL | 20 µL | 20 µL |

The DNA was restricted at 37 °C for 45 minutes. Enzymes were heat-inactivated at 65 °C for 20 minutes. DNA was purified using the *GeneJET PCR Purification Kit*, eluting the DNA with 8 µL elution buffer for the promoters and 27 µL for the BBa\_C0082.

A second restriction was prepared:

| Part | BBa\_K081005 | BBa\_K081006 | BBa\_K081007 |
| --- | --- | --- | --- |
| DNA | 8 µL | 8 µL | 8 µL |
| FastDigest Buffer | 1 µL | 1 µL | 1 µL |
| BcuI | 1 µL | 1 µL | 1 µL |
| Total | 10 µL | 10 µL | 10 µL |

The DNA was restricted at 37 °C for 30 minutes and purified using the GeneJET PCR Purification Kit, eluting with 8 µL.

## Ligation of BBa\_C0082 with the Anderson Promoters

Three ligation reactions were set up as following:

| Part | BBa\_K081005 | BBa\_K081006 | BBa\_K081007 |
| --- | --- | --- | --- |
| Anderson Promoter | 8 µL | 8 µL | 8 µL |
| BBa\_C0082 | 9 µL | 9 µL | 9 µL |
| T4 DNA Ligase | 1 µL | 1 µL | 1 µL |
| Ligation Buffer | 2 µL | 2 µL | 2 µL |
| Total | 20 µL | 20 µL | 20 µL |

The ligation was conducted overnight at 16 °C.

## Transformation of JW3367-3 with BBa\_C0082/Anderson Promotor ligation product and BBa\_B0015

Transformation was conducted according to the Zhang Gong Protocol for Transformation. 2  µL of DNA were added to the competent cells of each BBa\_B0015 and BBa\_C0082/Anderson plasmid transformation.

## New colony PCRs of Anderson Promoters

All Colony PCRs of the Anderson promoters were negative. Screen of 15 colonies from each plate.

* A: pSB1C3/BBa\_K081005 → Colony  1 - 15
* B: pSB1C3/BBa\_K608006 → Colony 16 - 30
* C: pSB1C3/BBa\_K608007 → Colony 31 - 45

TAQSCREEN:

1. 98 °C → 5 min
2. 98 °C → 10 s
3. 56 °C → 30 s
4. 72 °C → 2:15 min

## Miniprep of Terminator (BBa\_B0015)

The overnight culture of JW 3367-3 cells containing pSB1C3/BBa\_B0015 was prepared and prepped using the *ThermoScientific GeneJET Plasmid Miniprep Kit*, following the
[respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-Plasmid-Miniprep-Kit), eluting with 100 μL each. The resulting sample contained:

| Plasmid | DNA concentration [ng/μL] |
| --- | --- |
| pSB1C3/BBa\_B0015 | 46.42 |

## Gel electrophoresis of colony PCR of BBa\_K081005, BBa\_K608006 and BBa\_K608007

![](“/img/notebook/entry-160906-3-gel.png“)

Control of the colony PCR of BBa\_K081005, BBa\_K608006 and BBa\_K608007

Positive results for A16, B16, C9, C10, C11

Overnight cultures for positive colonies were produced and incubated overnight at 37 °C.

## Restriction of pSB1C3/BBa\_R0082/BBa\_I732018 and BBa\_B0015

First restriction

| Components | Volume [μL] |
| --- | --- |
| BBa\_R0082/BBa\_I732018 | 14.5 |
| PstI | 1.0 |
| NEB Buffer 3.1 | 5.0 |
| water | 29.5 |
| Total: | 50.0 |

Second restriction

|  |  |
| --- | --- |
| Components | Volume [μL] |
| BBa\_B0015 | 21.5 |
| PstI | 1.0 |
| XbaI | 1.0 |
| NEB 3.1 Buffer | 5.0 |
| water | 21.5 |
| Total: | 50.0 |

Incubation for 10 minutes at 37 °C each and heat inactivation of the first restriction at 80 °C for 20 minutes. Purification of BBa\_B0015 fragment using the GeneJET Gel Extraction Kit, with concentrations of 89.55 ng/µL and 91.46 ng/μL. A cryostock of JW3367-3/pSB1C3/BBa\_B0015 was prepared.

## Encapsulation of JW cells in alginate

* Preparations:
  1. -Sodium citrate 55 mM
  2. -JW cell culture
* Encapsulation:
  1. -OD600 = 1.6 of JW cell culture
  2. -3 tubes with each 1 mL of culture centrifuged 5 minutes at 4500 rpm
  3. -Pellet was resuspended with 1 mL 0.9 % NaCl each
  4. -Encapsulation was performed by pipetting of 1 mL NaCl-cells suspension with 2 mL alginate into 100 mL CaCl2
* Solution of Beads:
  1. -10 mL Na-citrate were incubated with beads for approx. 1 hour
  2. -Na-citrate-beads sliution was centrifuged for 5 minutes at 4500 rpm
  3. -Pellet was resuspended in LB / NaCl medium (several tubes - several pellets)
* Cultivation:
  1. -beads were cultivated and thereafter lysed in Na-citrate (up) - cultivated again (37 °C LB/CaCl2)
  2. -OD measurement revealed growth in the JWs cultivated in LB medium
  3. -One culture was cultivated overnight. Another one was lysed overnight

## Evaluation of Encapsulated JW cells from 06.09.16

* OD6600 measurement:
  1. -LB-JW (marked red): OD600 = 4.918
  2. -LB-JW (marked green) diluted ½ : OD600 = 3.550; undiluted around 7
  3. -Na-citrate-JW (marked red): OD600 = 0.002
  4. -Na-citrate-JW (marked green): OD600 = 0.006
* The vitality of the cells in the mixture with sodium citrate and LB has been confirmed via microscopy

## Miniprep of BBa\_K081007/BBa\_C0082

The overnight culture of JW3367-3 cells containing pSB1C3/BBa\_K081007/BBa\_C0082 was prepared and prepped using the *ThermoScientific GeneJET Plasmid Miniprep Kit*, following the
[respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-Plasmid-Miniprep-Kit), eluting two times.

| Colony | Concentration of first elution [ng/μL] | Concentration of second elution [ng/μL] |
| --- | --- | --- |
| 9c | 148.73 | 22.79 |
| 10c | 185.27 | 13.55 |
| 11c | 161.75 | 23.91 |

## Restriction of pSB1C3/BBa\_K081007/BBa\_C0082

Restriction following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#restriction).

|  | pSB1C3/BBa\_K081007/BBa\_C0082  Colony 9c | pSB1C3/BBa\_K081007/BBa\_C0082  Colony 10c | pSB1C3/BBa\_K081007/BBa\_C0082  Colony 11c |
| --- | --- | --- | --- |
| Plasmid [in μL] | 13.5 | 10.8 | 12.4 |
| EcoRI [in μL] | 2.0 | 2.0 | 2.0 |
| 10x CutSmart [in μL] | 5.0 | 5.0 | 5.0 |
| H2O [in μL] | 29.5 | 32.2 | 28.6 |

Incubation was performed at 37 °C for 15 minutes and then loaded on an agarose gel (1 %) for 1 hour at 120 V.
![](“/img/notebook/entry-160909-1-gel.png“)

Control of the restriction of BBa\_K608007/BBa\_C0082

Upper and two of the lower bands were cut out and extracted, using the GeneJET Gel Extraction Kit, following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments).

## Restriction of pSB1C3/BBa\_R0082/BBa\_1732018

Restriction following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#restriction).

|  | pSB1C3/BBa\_R0082/BBa\_I732018 |
| --- | --- |
| Plasmid | 15.0 μL |
| EcoRI | 1.0 μL |
| 10x CutSmart | 5.0 L |
| H2O | 29.0 μL |

Heat inactivation for 20 min (65 °C).

+ 1 μL BcuI

PCR Purification with the *ThermoScientific GeneJET PCR Purification Kit*, following the
[respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-PCR-Purification-Kit)

## Restriction of pSB1C3/BBa\_B0015

Restriction following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#restriction).

|  | pSB1C3/BBa\_B0015 |
| --- | --- |
| Plasmid | 11.2 μL |
| EcoRI | 1.0 μL |
| XbaI | 1.0 μL |
| 10x CutSmart | 5.0 μL |
| H2O | 31.8 μL |

Heat inactivation for 20 min (65 °C).

## Ligation

The consolidated extracted bands of plasmid pSB1C3/BBa\_K081007/BBa\_C0082 were self-ligated with the following reaction:

| Components | Volume [μL] |
| --- | --- |
| pSB1C3/BBa\_K081007/BBa\_C0082 | 15.0 μL (100 ng) |
| Ligase T4 | 1.0 μL |
| Ligase Buffer | 2.0 μL |
| H2O | Add 20μL |

Incubation at RT for 25 minutes. Heat inactivation at 65 °C for 10 minutes. Storage at -20 °C over weekend.

## Cryostock and Miniprep of pSB2K3/BBa\_C0082

Cryostock: 500 μL of the ONC and 500 μL of 1:1 Glycerol-water mixture. Storage at -80 °C.

Miniprep was conducted according to the *GeneJET Miniprep Kit Protocol*, following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-Plasmid-Miniprep-Kit).

Concentrations in first elution 156.66 ng/μL and 26.15 ng/μL.

## Restriction of Anderson Promoters (BBa\_K081005, BBa\_K081006, BBa\_K081007) with Taz (BBa\_C0082)

| Part | BBa\_C0082 | BBa\_K081005 | BBa\_K081006 | BBa\_K081007 |
| --- | --- | --- | --- | --- |
| Concentration [ng/µL] | 156.66 | 73.35 | 112.41 | 81.74 |
| Volume [µL for 500 ng] | 3.6 | 6.9 | 4.5 | 6.2 |
| NEB Buffer 3.1 [µL] | 6 | 2 | 2 | 2 |
| XbaI [µL] | 2 | - | - | - |
| PstI [µL] | 2 | 1 | 1 | 1 |
| H2O [µL] | 40.4 | 10.1 | 12.5 | 10.8 |
| Total [µL] | 60 | 20 | 20 | 20 |

The DNA was restricted for 1 h for the Anderson promoters and 1 hour and 15 minutes for Taz at 37 °C. Enzymes were heat-inactivated for 20 minutes at 65 °C.
The restrictions of the three Anderson promotors were purified using the GeneJET PCR Purification Kit. DNA was eluted with 9 µL.
A second restriction was set up:

| Part | BBa\_K081005 | BBa\_K081006 | BBa\_K081007 |
| --- | --- | --- | --- |
| Volume [µL] | 17 | 17 | 17 |
| FastDigest Buffer [µL] | 2 | 2 | 2 |
| BcuI [µL] | 1 | 1 | 1 |
| Total: [µL] | 20 | 20 | 20 |

The DNA was restricted at 37 °C for 30 minutes. The DNA was purified using the GeneJET PCR Purification Kit and eluted with 10 µL. The concentration was measured at the Nanodrop.

| Part | BBa\_K081005 | BBa\_K081006 | BBa\_K081007 |
| --- | --- | --- | --- |
| Concentration [ng/µL] | 10.53 | 7.67 | 7.34 |

## Gel electrophoresis of BBa\_C0082 (restricted with XbaI and PstI)

A 2% gel was run for 45 minutes at 130 V.
![](/img/project/notebook/entry-160913-2-gel.png)
The lower band at 1.5 kb was cut out and the DNA was extracted from the gel with the GeneJET Gel Extraction Kit. DNA was eluted with 32 µL.
The DNA-concentration of Taz was 181 ng/µL.

## Ligation of BBa\_R0082/BBa\_I732018 with Terminator BBa\_B0015

| Components | Volume [µL] |
| --- | --- |
| BBa\_R0082/BBa\_I732018 (Insert) | 15 |
| BBa\_B0015 | 0.5 |
| T4 Ligase | 1 |
| T4 DNA Ligase Buffer | 2 |
| water | 1 |
| Total: | 20 |

## Ligation of Anderson Promoters with Taz

| BBa\_K081005 | BBa\_K6081006 | BBa\_K6081007 |
| --- | --- | --- |
| Vector (Anderson) [µL] | 9 | 9 | 9 |
| Insert (BBa\_C0082) [µL] | 2 | 2 | 2 |
| T4 Ligase [µL] | 1 | 1 | 1 |
| T4 DNA Ligase Buffer [µL] | 2 | 2 | 2 |
| H2O [µL] | 6 | 6 | 6 |
| Total: [µL] | 20 | 20 | 20 |

Incubation for 15 minutes at RT and heat-inactivation for 10 minutes at 65 °C.

## Fabrication of DD01-Chamber-Mask01

## Cultivation of Frozen Capsules

* Light microscopy of the capsule rest/remains and the lysed capsules showed high viability
* Encapsulation with glycerol-alginate mix does not work, capsules get fragile and start releasing bacteria
* New encapsulation of bacteria in three different approaches:
  1) Regular encapsulation; frozen on pure glycerol
  2) Regular encapsulation; frozen on glycerol / LB
  3) Encapsulation with alginate-glycerol mix; frozen on 0.9 % NaCl

## Production of 50 mL 1 M Tris-HCL Buffer

* 6.057 g TRIS
* Adjust to pH 8 with HCl
* Fill up to 50 µL with water

## Production of 0.5 L TE Buffer

* 5 mL 1M TRIS-HCl at pH 8
* 2 mL 0.5 M EDTA(Na2) also at pH 8
* Add 494 mL water

## PCR of mTaz and NOD1/EnvZ 3’/5’ OEPCR fragments

| mTaz | NOD1/EnvZ 5’ OEPCR | NOD1/EnvZ 3’ OEPCR |
| --- | --- | --- |
| Q5 2x Mastermix (Anderson) [µL] | 25 |
| Template [µL] | 2.5 | 1.5 | 1.5 |
| T4 Ligase [µL] | 1 | 1 | 1 |
| 2.5 µL fw primer (10 mM) | GCAAAAAGAATTCGCGG | GCAAAAAGAATTCGCGG | GGCAGAATCGATCAATAAAGA |
| 2.5 µL rev primer (10 mM) | GCTTTTCTGCAGCGG | ATATCTTTATTGATCGATTCTGCC | GCTTTTCTGCAGCGG |
| H2O [µL] | Add to 50 µL |

The following PCR setup was used:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1. | 98° C | 0:30 || 2. | 98° C | 0:10 |
| 3. | 47° C | 0:20 |
| 4. | 72° C | 1:00 |
| 5. | GOTO 2 repeat 28x |
| 6. | 72 °C | 2:00 |
| 7. | HOLD at 4° C |

## Gel electrophoresis of the recombinant PCR of NOD1/EnvZ 3'5' and PCR of mTaz

10 µL of PCR product was mixed with 2 µL of 6x loading dye and run on an 1% agarose gel at 130 V for 40 minutes.

![](/img/project/notebook/entry-160915-4-gel.png)
>

Bands confirmed presence of RPCR 5’ and 3’ fragment as well as mTaz. However, a second band in mTaz (~600 bp) could be observed.
The PCR reaction solutions were purified using the PCR Purification Kit. Elution in 35 µL.

* mTaz concentration: 73.64 ng/µL
* 5’ RPCR concentration: 69.92 ng/µL
* 3’ RPCR concentration: 77.95 ng/µL

All PCR products were stored at -20 °C.

## Cryostock of the overnight culture

* DH5α, Anderson 05
* DH5α, Anderson 06
* DH5α, Anderson 07

The cryostocks were stored at -80 °C.

## Miniprep of the overnight culture

GeneJET Miniprep Kit Protocol was used to the letter, and elution volume was 35 µL.

| Construct | Concentration [ng/µL] |
| --- | --- |
| DH5α, Anderson 05 | 28.01 |
| DH5α, Anderson 05 | 30.27 |
| DH5α, Anderson 05 | 42.99 |
| DH5α, Anderson 06 | 67.88 |
| DH5α, Anderson 06 | 41.91 |
| DH5α, Anderson 06 | 38.04 |
| DH5α, Anderson 07 | 41.34 |
| DH5α, Anderson 07 | 72.14 |
| DH5α, Anderson 07 | 30.26 |
| BBa\_I732018/BBa\_R0082/BBa\_B0015 | 29.99 |
| BBa\_I732018/BBa\_R0082/BBa\_B0015 | 55.97  | |
| BBa\_I732018/BBa\_R0082/BBa\_B0015 | 31.69 |

## Sequential Restriction of OmpR/GFP and OmpR/YFP

First the DNA concentrations of the plasmids were measured.

| Construct | DNA Concentration [ng/µL] |
| --- | --- |
| BBa\_R0082/BBa\_E0240 | 90.63 |
| BBa\_R0082/BBa\_E0430 | 143.74 |

First restriction with EcoRI

|  | BBa\_R0082/BBa\_E0240 | BBa\_R0082/BBa\_E0430 |
| --- | --- | --- |
| Plasmid (1 µg) [µL] | 11 7 | | |
| EcoRI [µL] | 1 | 1 |
| 10x CutSmart [µL] | 2 | 2 |
| H2O [µL] | 2 | 2 |

The restriction preparations were incubated at 37 °C for 15 min and then heat-inactivated at 65 °C for 20 min.
The restriction preparations were purified using the GeneJET PCR Purification Kit. DNA was eluted with 20 µL Elution Buffer. Afterwards DNA concentration was measured at the NanoDrop.

|  | DNA concentration [ng/µL] |
| --- | --- |
| Purified BBa\_R0082/BBa\_E0240 | 90.39 |
| Purified BBa\_R0082/BBa\_E0430 | 81.81 |

Second restriction with Bcu I

|  | Purified OmpR/GFP | Purified OmpR/YFP |
| --- | --- | --- |
| Plasmid (1 µg) [µL] | 12 | 13 |
| BcuI [µL] | 1 | 1 |
| FD Buffer [µL] | 2 | 2 |
| ddH2O[µL] | 5 | 4 |

The restriction preparations were incubated at 37 °C for 10 min.
The restriction preparations were purified using the GeneJET PCR Purification Kit. DNA was eluted with 20 µL Elution Buffer. Afterwards DNA concentration was measured at the NanoDrop.

|  | DNA concentration [ng/µL] | 260/230 | 280/260 |
| --- | --- | --- | --- |
| Purified BBa\_R0082/BBa\_E0240 | 90.39 | 0.76 | 1.77 |
| Purified BBa\_R0082/BBa\_E0430 | 34.25 | 1.96 | 1.79 |

## Ligation of BBa\_R0082/BBa\_E0430 and BBa\_R0082/BBa\_E0240 with BBa\_B0015 (Terminator)

A 3:1 (insert:vector) ligation was performed:

|  | OmpR/GFP | OmpR/YFP |
| --- | --- | --- |
| Vector: restricted BBa\_B0015 ( 160909DK05) [µL] | 1 | 1 |
| Insert (71 ng) [µL] | 3 | 2 |
| T4 Ligase [µL] | 1 | 1 |
| T4 Ligase Buffer [µL] | 2 | 2 |
| ddH2O[µL] | 13 | 14 |

The ligation preparations were incubated at RT for 50 min and then heat-inactivated at 65 °C for 10 min.

## Measurement of cultivated frozen capsules

* OD600 measurements were performed on all three defrosted setups from 13th September
  1. OD600 = 2.346
  2. OD600 = 2.406
  3. OD600 = 2.458

## Chloramphenicol Stock Solution

A Chloramphenicol (CAmp) stock solution was prepared following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#xchloramphenicol-stock).

## LB-Agar Plates

LB-Agar plates were prepared following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#LB-Agar-plates).

## Transformation of JW 3367-3 with Fluorescence Reporters and B0015 - Quantification

Average amount of colonies for C2987/BBa\_E0240/B0015

|  |  |  |
| --- | --- | --- |
| 1 μL DNA, 30 μL Trafovolume | 1 μL DNA, 60 μL Trafovolume | 1 μL DNA, 90 μL Trafovolume |
| ~100 colonies | ~200 colonies | ~250 colonies |

Average amount of colonies for JW3367-3 BBa\_E0430/B0015

|  |  |  |  |
| --- | --- | --- | --- |
|  | 1 μL DNA | 2 μL DNA | 5 μL DNA |
| 50 μL Trafovolume | 20 colonies | not done | 33 colonies |
| 65 μL Trafovolume | not done | 21 colonies | not done |
| 90 μL Trafovolume | 25 colonies | ~35 colonies | ~50 colonies |

## LB-Agar Plates

LB-Agar plates were prepared following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#LB-Agar-plates).

## Vitality examination of encapsulated frozen bacteria via light microscopy

* Bacteria from the setups from the 13th September were examined via light microscopy
  1) E. coli vital
  2) E. coli vital
  3) alive but slightly lower activity

## Validation of LacZα Reporter System by β-Galactosidase Activity

20 μL of JW3367-3 cell transformed with pSB1C3/BBa\_R0082/BBa\_I732018 were plated onto a LB-Agar/CAmp plate containing 10 μL 10 mg/mL X-Gal and incubated one night at 37°C.

## Transformation of JW 3367-3 with pSB1C3/BBa\_I732020 (lacZα/Terminator)

150 μL competent JW 3367-3 cells were split into 3 x 50 μL and inoculated with DNA:

* (+) postive control: 1 μL pSB1C3/BBa\_R0082/BBa\_I732018
* (-) negative control: no addition of DNA
* 1 μL pSB1C3/BBa\_I732020, resuspended from iGEM Distribution Kit Plate 3, 2F

Transformation was carried out using the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#transformation).

## PCR purification of the linearized plasmids pSB1C3, pSB1A3, pSB1T3

A PCR was performed following the GeneJet PCR Purification Kit Protocoll.
The following concentrations were measured with Nanodrop

| Plasmid |  | Concentration [ng/µL] |
| --- | --- | --- |
| pSB1C3 | a | 158.77 |
|  | b | 184.20 |
|  | c | 132.83 |
| pSB1A3 |  | 189.96 |
| pSB1T3 |  | 145.48 |

## PCR amplification of EnvZ Fusionprotein\_Tai/RFC25, EnvZ\_First\_Transmembrane and NOD\_1LRRRFC25

|  | Volume [µL] |
| --- | --- |
| Q5 2x Mastermix | 25 |
| EnvZ 5' fw | 2.5 |
| Biobrick 3' | 2.5 |
| Template | 1 |
| H2O | 19 |

The PCR preparations were mixed according to scheme above. As a template the EnvZ Fusionsprotein\_TailRFC25 was used in preparation A.
In preparation B the EnvZ\_First\_Transmembrane and in C the NOD\_LRR\_RFC25 were used.

The following PCR setup was used:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1. | 98° C | 0:30 || 2. | 98° C | 0:10 |
| 3. | 47° C | 0:20 |
| 4. | 72° C | 1:00 |
| 5. | GOTO 2 repeat 30x |
| 6. | 72 °C | 3:00 |
| 7. | HOLD at 4° C |

## Analysis of the PCR via gel electrophoresis (plasmid pSB1C3, pSB1A3, pSB1T3)

An agarosegel (1%) was used to analyze the PCR reaction of the plasmids pSB1C3, pSB1A3 and pSB1T3.
The first lane contained pSB1C3 a, the second pSB1C3 b,the third pSB1C3 c, the fourth pSB1A3 and the fifth pSB1T3.
The gel was incubated with ethidiumbromide for 30 minutes before running it at 150 V for 30 minutes.
All preparations contained DNA fragments contained DNA fragments with the expected length.

## Analysis of the PCR via gel electrophoresis (EnvZ Fusionprotein\_Tail RFC25, EnvZ\_First Transmembrane and NOD1\_LRR RFC25)

A Agarosegel (1%) was used to analyze the PCR reaction.
The first lane contained the EnvZ Fusionprotein\_TailRFC25, the second EnvZ First Transmembrane and the third lane NOD1\_LRR RFC25.
The gel was incubated with ethidiumbromide for 30 minutes before running it at 150 V for 30 minutes.
All preparations contained DNA fragments contained DNA fragments with the expected length.

## Restriction of pSB1A3, mTaz, EnvZ, pSB1C3 and Reporter Systems

Following scheme was used:

| Part | pSB1C3 | pSB1A3 | mTaz | EnvZSH | EmvZFH | NOD1LRR | pSB1C3/BBa\_R0082 | pSB1C3/BBa\_I532020 | pSB1C3/BBa\_K081005 | pSB1C3/BBa\_K608006 | pSB1C3/BBa\_E0430 | pSB1C3/BBa\_E0240 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Volume [μL] | 17 | 14.1 | 13.3 | 7.9 | 7.7 | 13.8 | 5.0 | 25.8 | 18 | 18 | 2.9 | 4.7 |
| Buffer | 5 μL NEB Buffer 2.1 | 5 μL NEB Buffer 2.1 | 5 μL NEB Buffer 2.1 | 5 μL NEB Buffer 2.1 | 5 μL NEB Buffer 2.1 | 5 μL NEB Buffer 2.1 | 5 μL Thermo FD | 5 μL NEB Buffer 2.1 | 5 μL Thermo FD | 5 μL Thermo FD | 5 μL NEB Buffer 2.1 | 5 μL NEB Buffer 2.1 |
| EcoRI [μL] | 1 | 1 | 1 | 1 | 1 | 1 | - | - | - | - | - | - |
| XbaI [μL] | - | - | - | - | 1 | 1 | - | - | - | - | - | - |
| BcuI [μL] | - | - | - | - | - | - | 1 | - | 1 | 1 | - | - |
| PstI [μL] | 1 | 1 | 1 | 1 | 1 | 1 | - | 1 | - | - | 1 | 1 |
| H2O [μL] | 26 | 28.9 | 29.7 | 35.1 | 35.3 | 29.2 | 39 | 17.2 | 26 | 26 | 40.1 | 38.3 |

A second restriction was set up:

| Part | pSB1C3/BBa\_R0082 restricted with BcuI | pSB1C3/BBa\_I532020 restricted with XbaI and PstI | pSB1C3/BBa\_K081005 restricted with BcuI | pSB1C3/BBa\_K608006 restricted with BcuI |
| --- | --- | --- | --- | --- |
| Volume [μL] | 50 | 25.8 | 50 | 50 |
| NEB 2.1 Buffer [μL] | 6 | 6 | 6 | 6 |
| EcoRI [μL] | 2 | - | 1.2 | 1.2 |
| PstI [μL] | - | 1 | - | - |
| XbaI [μL] | - | 1 | - | - |
| H2O [μL] | 2.8 | 17.2 | 2.8 | 2.8 |

## Ligation of pSB1A3 and pSB1C3 with the Part Precursors and Fluorescence Reporters as well as pSB1C3 with BBa\_R0082 and BBa\_I732020

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Part 1 | 5 μL pSB1C3 | 5 μLpSB1C3 | 5 μLpSB1C3 | 5 μLpSB1C3 | 5 μLpSB1A3 | 5 μLpSB1A3 | 5 μLpSB1A3 |
| Part 2 | 10 μ mTaz | 25 μ EnvZ SH | 25 μ EnvZ FH | 25 μ NOD1-LRR | 20 μ BBa\_E0430 | 20 μ BBa\_E0240 | 30 μ BBa\_R0082 |
| Part 3 | - | - | - | - | - | - | BBa\_I732020 |
| T4 Ligase Buffer [μL] | 5 | 5 | 5 | 5 | 5 | 5 | 25 |
| T4 Ligase[μL] | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 10 |
| H2O [μL] | 27.5 | 12.5 | 12.5 | 12.5 | 17.5 | 17.5 | 25 |

## Transformation of JW3367-3 with the Ligation Preparations

Competent JW 3367-3 cells were thawed on ice at 4°C and inoculated with the ligation preparations. For each ligation preparation 150 μL competent cells were used, for the positive control (transformed with pcDNA3) and negative control 75 μL were used.
Transformation was carried out using the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#transformation).

## Fabrication of Diagnostic Device DD01 (SU-8) Master02 continued

* Continuation after application of base / adhesion layer from the 27th of September
* Structural layer of SU-8 (GM1075)

  -Spin coating:
  1) 0 - 1000 rpm (100 rpm/second acceleration) in 10 s
  2) 1000 rpm for 100 seconds
  3) 1000 - 1400 rpm (400 rpm/second acceleration) in 1 s
  4) 1400 - 0 rpm (-100 rpm/second acceleration) in 14 seconds
  -30 minutes relaxation time on hotplate at 40 °C:
  The wafer was covered with a crystallization dish, but allowed for breathing due to evaporating SU-8 solvent (PGMEA), otherwise condensation occurs on covering dish and curing during prebake would be insufficient due to excessive vapour pressure
  -Soft bake:
  1) 40 - 120 °°C in about 10 minutes (avg +8 °C/minute)
  2) 120 °C for 12 minutes
  3) 120 °C - 40 °C in about 20 minutes (avg -4 °C/minute)
  -i-line exposure 3) 120 °C - 40 °C in about 20 °minutes (avg -4 °C/minute)

  -1 hour delay time at RT in wafer box before PEB

  -PEB:
  1) RT - 120 °C in about 11 minutes (avg 9 °C/minute); accidentally - 2 bubbles created on structure-free space
  2) 120 °C - 95 °C in about 3 minutes (avg -8 °C/minute)
  3) 95 °C for 50 minutes
  4) 95 °C - 36 °C in about 25 minutes (avg -2.4 °C/minute)
  -Develop:
  -In PGMEA (used) for about 8 minutes while constantly agitating the PGMEA and the wafer in the beaker
  -Wafer was rinsed with isopropanol to stop the development which caused white traces on wafer
  -Isopropanol-rinsed wafer was washed the with new, used PGMEA and subsequently rinsed with isopropanol which caused the previous white traces to vanish, however, lines of residue from isopropanol evaporation remained
  -Examination of the profile (using the DektakXT; Bruker) showed a thickness of approx. 110 μm; comparison between the depth of the large filter structure holes and the fine filter structure holes (as visible in the live video in the DektakXT using a ruler) showed similar depth (7 mm at 100x magnification) and can be checked in the captured images

## Bonding of Diagnostic Chip (Channel and Chamber) PDMS Components (PDMS batch from the 24th of September on DD01-Master01)

* Holes were drilled with 0.75 hole punch through the channel component
* Droplets of isopropanol were dripped on both components and gently rubbed with the fingertips to clean the PDMS components bonding surface; PDMS was subsequently dried using a N2-gun and leaving it at ambient air
* Effects of cleaning were examined under a light microscope:
  1) 40 μm square-shaped filter structures (of about 100 μm height) sticked onto each other with the side walls; therefore the filter structure’s columns were bent which resulted in increased filter size due to locally increased width of the filter channels
  2) Touching the filter structures with a sharp metal syringe tip causes unsticking of the filter structures from one another
  3) Wetting with isopropanol caused resticking and resticking persisted upon drying; after completed drying and touching unsticking recurred
* Both components bonding surfaces were O2-plasma treated simultaneously using the parameters: 100 seconds; about 25 W; 0,4 mbar O2
* Surfaces were examined using a light microscope; filter structures still remained largely intact and in the same state as before the O2-plasma
* Wetting of complete bonding surface of chamber component with H2O (demineralised) droplets(5 - 10 droplets)
* Surfaces were once again examined using a light microscope: filter structures still intact
* PDMS component’s bonding surfaces were brought together with remaining intermediate H2O layer
* Channel and chamber components were aligned under a light microscope using tweezers to align; used too much water - too slippery, next time use 1 - 2 droplets (formed by a syringe tip with diameter of about 0.6 mm); alignment markers / triangles do not lock into one another because the PDMS is too rigid and does not bent enough to compensate the 100 μm height difference (10:1 oligomer:initiator ratio used)
* The chips were put into an oven overnight at 40 °C to cure and bond without further touching or pressing

## Preparation of LB-Agar

1. 1 x 40 g LB-agar were put in 1 L H2O.
2. The suspension was autoclaved.

## Preparation of LB-Agar/Tetracycline-Plates

1. 1 mL of tetracycline stock solution were added into 1 L liquid LB-agar.
2. The solution was poured into petri dishes under sterile conditions.
3. Plates were stored at 4°C.

## Preparation of Tetracycline Stock Solution

1. 3 x 10 mg tetracycline were solved in 1 mL H2O.
2. Steril filtered with a 0.22 μm filter.
3. Stored at -20°C.

## Preparation of LB-Medium

1. 2 x 25 g LB-medium were put in 1 L H2O.
2. The suspension was autoclaved.
3. Stored at 4°C.

## PCR of EnvZ RFC25

Following scheme was used for the different preparations.

|  |  |
| --- | --- |
| Template | 1 μL |
| Primer fw | 2.5 μL |
| Primer rv | 2.5 μL |
| 10 x Dream Taq buffer | 5 μL |
| dNTPs (10 mM) | 1 μL |
| Taq polymerase | 0.1 μL |
| H2O | 39.5 μL |
|  | 50 μL |

There were three preparations:

| Template | Primer fw | Primer rv |
| --- | --- | --- |
| EnvZ RFC25 TM1 | RFC23 TMFirst fw | RFC23 TMFirst rv |
| Nod1/EnvZ RFC25 Tail | RFC23 EnvZ Tail fw | RFC23 EnvZ Tail rv |
| Nod1/LRR RFC25 | RFC23 NOD1-LRR fw | RFC23 NOD1-LRR rv |

## Preparations of Competent Cells DH5α

1. Overnight cultures of the DH5α were splitted into two 1000 mL Erlenmeyer flask in 500 mL LB-medium and grown at 37°C, 250 rpm.
2. The OD was checked till it reached 0.4

   | Time after incubation start [min] | OD (1.=flask 1; 2.=flask 2 ) |
   | --- | --- |
   | 20 | 1. 0.044; 2. 0.026 |
   | 50 | 1. 0.053; 2. 0.030 |
   | 80 | 1. 0.055; 2. 0.026 |
   | 120 | 1. 0.278; 2. 0.144 |
   |  | 2. 0.352 |
   |  | 2. 0.420 -> done |
3. Cells were chilled on ice for at least 5 min.
4. Cells were centrifuged at 5000 rpm for 10 min at 4°C.
5. Flow-through was discarded.
6. Cells were resuspended in 40 mL cold Ca/glycerol buffer on ice.
7. Cells were chilled on ice for at least 30 min.
8. Cells were centrifuged at 4000 rpm for 10 min at 4°C.
9. Flow-through was discarded.
10. Cells were resuspended in 40 mL cold Ca/glycerol buffer and chilled on ice for 90 min.
11. Cells were collected by centrifugation (5000 rpm, 15 min, 4°C)
12. Cells were resuspended in 6 mL cold Ca/glycerol buffer on ice.
13. Aliquots of 150 μL each were made.
14. Aliquoted competent cells were frozen in liquid N2 and stored at -80°C.

## PCR of EnvZ RFC25

Following scheme was used for the different preparations.

|  |  |
| --- | --- |
| Template | 1 μL |
| Primer fw | 2.5 μL |
| Primer rv | 2.5 μL |
| 10 x Dream Taq buffer | 5 μL |
| dNTPs (10 mM) | 1 μL |
| Taq polymerase | 0.1 μL |
| H2O | 39.5 μL |
|  | 50 μL |

There were three preparations:

| Template | Primer fw | Primer rv |
| --- | --- | --- |
| EnvZ RFC25 TM1 | RFC23 TMFirst fw | RFC23 TMFirst rv |
| Nod1/EnvZ RFC25 Tail | RFC23 EnvZ Tail fw | RFC23 EnvZ Tail rv |
| Nod1/LRR RFC25 | RFC23 NOD1-LRR fw | RFC23 NOD1-LRR rv |

## PCR Purification of ENvZ/LRR

The PCR products were purified using the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-PCR-Purification-Kit).
DNA concentrations were measured:

| DNA | DNA concentration [ng/μL] |
| --- | --- |
| EnvZ RFC25 TM1 | 36.36 |
| Nod1/EnvZ RFC25 Tail | 97.89 |
| Nod1/LRR RFC25 | 33.72 |

## PCR of mTaz

Following scheme was used:

|  |  |
| --- | --- |
| Template | 1 μL |
| Primer EnvZ 5' fw | 2.5 μL |
| Primer EnvZ 3' rv | 2.5 μL |
| Q5 Master Mix 2x | 25 μL |
| H2O | 19 μL |
|  | 50 μL |

Following programme was used for the PCR:

|  |  |
| --- | --- |
| 1. 98 °C | 30 sec |
| 2. 98 °C | 10 sec |
| 3. 47 °C | 20 sec |
| 3. 72 °C | 1.5 min |
| 4. GOTO 2. | Repeat 34x |
| 5. 72 °C | 3 min |
| 4 °C | ∞ |

## PCR Purification of mTaz

The PCR product was purified using the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-PCR-Purification-Kit).
DNA concentration was measured:

| DNA | DNA concentration [ng/μL] |
| --- | --- |
| mTaz | 104.36 |

## PCR of mTaz

Following scheme was used:

|  |  |
| --- | --- |
| Template | 1 μL |
| Primer EnvZ 5' fw | 2.5 μL |
| Primer EnvZ 3' rv | 2.5 μL |
| Q5 Master Mix 2x | 25 μL |
| H2O | 19 μL |
|  | 50 μL |

Following programme was used for the PCR:

|  |  |
| --- | --- |
| 1. 98 °C | 30 sec |
| 2. 98 °C | 10 sec |
| 3. 47 °C | 20 sec |
| 3. 72 °C | 1.5 min |
| 4. GOTO 2. | Repeat 34x |
| 5. 72 °C | 3 min |
| 4 °C | ∞ |

The PCR products were loaded onto a 1%-gel. There was one band at the expected size 1.500 bp along with four other bands, that weren't the expected size.

## PCR of mTaz

Following scheme was used:

|  |  |
| --- | --- |
| Template | 1 μL |
| Primer EnvZ 5' fw | 2.5 μL |
| Primer EnvZ 3' rv | 2.5 μL |
| 10 *Pfu* buffer | 5 μL |
| dNTPs (10 mM each) | 1.5 μL |
| *Pfu* polymerase | 1 μL |
| H2O | 36.5 μL |
|  | 50 μL |

Following programme was used for the PCR:

|  |  |
| --- | --- |
| 1. 98 °C | 30 sec |
| 2. 98 °C | 10 sec |
| 3. 47 °C | 20 sec |
| 3. 72 °C | 4 min |
| 4. GOTO 2. | Repeat 34x |
| 5. 72 °C | 3 min |
| 4 °C | ∞ |

## Restriction of pSB1C3, pSB1A3, mTaz and Taz

| Part | EnvZ TM first | NOD1-LRR | EnvZ Tail |
| --- | --- | --- | --- |
| Volume [μL] | 3.8 | 8.4 | 2.8 |
| NEB Buffer 2.1 [μL] | 2 | 2 | 2 |
| EcoRI [μL] | 0.5 | 0.5 | 0.5 |
| PstI [μL] | 1 | 1 | 1 | 1 | 1 | 1 |
| H2O [μL] | 3 | 3 | 9.5 | 31.8 | 13 | 20.1 |
| Total [μL] | 50 | 50 | 50 | 50 | 50 | 50 |

The DNA was restricted for 1 h at 37 °C. Enzymes were heat-inactivated for 20 minutes at 80°C.

## Ligation

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Part 1 | pSB1C3 | pSB1C3 | pSB1A3 | pSB1A3 | pSB1A3 | pSB1A3 | pSB1A3 | pSB1A3 |
| Part 2 | mTaz (IDT) | mTaz aliquot 2 | BBa\_K081005 | BBa\_K608006 | BBa\_K608007 | BBa\_K081005 | BBa\_K608006 | BBa\_K608007 |
| Part 3 | - | - | mTaz | mTaz | mTaz | Taz | Taz | Taz |
| T4 Ligase Buffer [μL] | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 |
| T4 [μL] | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 |
| H2O [μL] | 2.1 | 38.3 | 7.6 | 7.6 | 7.6 | 7.6 | 7.6 | 7.6 |

## Restriction of pSB1C3, pSB1A3, mTaz and Taz

| Part | pSB1C3 | pSB1A3 | mTaz aliquot 1 | mTaz aliquot 2 | mTaz (IDT) | mTaz |
| --- | --- | --- | --- | --- | --- | --- |
| Volume [μL] | 40 | 40 | 33.5 | 11.2 | 30 | 22.9 |
| NEB Buffer 2.1 [μL] | 5 | 5 | 5 | 5 | 5 | 5 |
| EcoRI [μL] | 1 | 1 | - | 1 | 1 | - |
| XbaI [μL] | - | - | 1 | - | - | 1 |
| PstI [μL] | 1 | 1 | 1 | 1 | 1 | 1 |
| H2O [μL] | 3 | 3 | 9.5 | 31.8 | 13 | 20.1 |
| Total [μL] | 50 | 50 | 50 | 50 | 50 | 50 |

The DNA was restricted for 1 h at 37 °C. Enzymes were heat-inactivated for 20 minutes at 80°C.

## Transformation of JW3367-3 and NEB competent E. coli with ligations

Following plasmids were transformed into following cells:

| cells | Plasmids |
| --- | --- |
| NEB cc | pSB1C3/mTaz(IDT) |
| NEB cc | pSB1A3/BBa\_K081005/mTaz |
| NEB cc | pSB1A3/BBa\_K608006/mTaz |
| NEB cc | pSB1A3/BBa\_K608007/mTaz |
| JW3367-3 | pSB1C3/mTaz |
| JW3367-3 | pSB1A3/BBa\_K081005/mTaz |
| JW3367-3 | pSB1A3/BBa\_K608006/mTaz |
| JW3367-3 | pSB1A3/BBa\_K608007/mTaz |
| JW3367-3 | pSB1A3/BBa\_K081005/BBa\_C0082 |
| JW3367-3 | pSB1A3/BBa\_K608006/BBa\_C0082 |
| JW3367-3 | pSB1A3/BBa\_K608007/BBa\_C0082 |
| JW3367-3 | (+) positive control: pcDNA (1:20) |
| JW3367-3 | (-) negative control: - (plated onto CAmp plate) |
| JW3367-3 | (-) negative control: - (plated onto Amp plate) |
| DH5α | competent cell control: pcDNA (1:20) |

Transformation was carried out using the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#transformation). At the end cells were spun down, resuspended in 50 μL LB-medium and plated onto LB-agar plates with the suitable antibiotic.

## Colony PCRs of cells transformed with pSB1C3/mTaz

Following mastermix for 30 PCR reactions was set up:

|  |  |
| --- | --- |
| VF2 Primer fw | 15 μL |
| VR Primer rv | 15 μL |
| 10 x Dream Taq buffer | 30 μL |
| dNTPs (10 mM) | 6 μL |
| Dream Taq polymerase | 7.5 μL |
| H2O | 226.5 μL |
| total | 300 μL |

28 x 10 μL of the mastermix was pipetted into PCR tubes, which were inoculated with:

| Colony numbering | Content |
| --- | --- |
| 1 - 2 | old mTaz from 27.09. |
| 3 - 28 | different colonies from the transformations from 03.10. |

Positive results (bands with expected sizes) for 4 - 6, 10, 13, 15, 20, 24 and 26.

## Colony PCRs of cells transformed with pSB1C3/mTaz

Following mastermix for 90 PCR reactions was set up:

|  |  |
| --- | --- |
| VF2 Primer fw | 45 μL |
| VR Primer rv | 45 μL |
| 10 x Dream Taq buffer | 90 μL |
| dNTPs (10 mM) | 18 μL |
| Dream Taq polymerase | 22.5 μL |
| H2O | 679.5 μL |
| total | 900 μL |

80 x 10 μL of the mastermix was pipetted into PCR tubes, which were inoculated with:

| Colony numbering | Content |
| --- | --- |
| 29 | NEB cc pSB1C3/mTaz (IDT) |
| 30 - 31 | NEB cc pSB1A3/BBa\_K608007/mTaz 2 |
| 32 - 36 | NEB cc pSB1A3/BBa\_K081005/mTaz 2 |
| 37 - 44 | NEB cc pSB1A3/BBa\_K608007/mTaz 1 |
| 45 - 52 | NEB cc pSB1A3/BBa\_K608006/mTaz 1 |
| 53 - 60 | NEB cc pSB1A3/BBa\_K608007/mTaz 1 |
| 61 - 68 | JW3367-3 pSB1A3/BBa\_K081005/mTaz |
| 69-76 | JW3367-3 pSB1A3/BBa\_K608006/mTaz |
| 77 - 84 | JW3367-3 pSB1A3/BBa\_K608007/mTaz |
| 85 - 92 | JW3367-3 pSB1A3/BBa\_K081005/BBa\_C0082 |
| 93-100 | JW3367-3 pSB1A3/BBa\_K608006/BBa\_C0082 |
| 101 - 108 | JW3367-3 pSB1A3/BBa\_K608007/BBa\_C0082 |

Positive results (bands with expected sizes) for 29, 31, 37, 55, 68.

## Minipreparation of Overnight Cultures

The minipreparation was carried out following the instructions of the [GeneJET Plasmid Miniprep Kit](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-Plasmid-Miniprep-Kit).

| Plasmid | DNA concentration |
| --- | --- |
| pSB1C3/mTaz colony 4 | 158.29 ng/μL |
| pSB1C3/mTaz colony 5 | 143.37 ng/μL |
| pSB1C3/mTaz colony 6 | 141.55 ng/μL |
| pSB1C3/mTaz colony 10 | 98.89 ng/μL |
| pSB1C3/mTaz colony 13 | 134.68 ng/μL |
| pSB1C3/mTaz colony 15 | 188.91 ng/μL |
| pSB1C3/mTaz colony 20 | 176.11 ng/μL |
| pSB1C3/mTaz colony 24 | 165.42 ng/μL |

## Restriction of EnvZ TM first, NOD1-LRR and EnvZ Tail for RFC23 Assembly

| Part | EnvZ TM first | NOD1-LRR | EnvZ Tail |
| --- | --- | --- | --- |
| Volume [μL] | 3.8 | 8.4 | 2.8 |
| NEB Buffer 2.1 [μL] | 2 | 2 | 2 |
| EcoRI [μL] | 0.5 | 0.5 | 0.5 |
| PstI [μL] | 0.5 | 0.5 | 0.5 |
| H2O [μL] | 13.2 | 8.6 | 14.2 |
| Total [μL] | 20 | 20 | 20 |

The DNA was restricted for 20  at 37°C. Enzymes were heat-inactivated for 30 minutes at 80°C.

## Ligation of pSB1C3 with the EnvZ Parts

Three ligations were set up using restricted, dephosphorylated linear pSB1C3 and the restricted EnvZ parts.

|  |  |  |  |
| --- | --- | --- | --- |
| Part 1 | 10.5 μL pSB1C3 | 4.6 μL pSB1C3 | 4.9 μL pSB1C3 |
| Part 2 | 10 μL EnvZ TM first | 10 μL NOD1-LRR | 10 μL EnvZ Tail |
| T4 Ligase Buffer [μL] | 2.5 | 2 | 2 |
| T4 Ligase [μL] | 0.5 | 0.5 | 0.5 |
| H2O [μL] | 1.5 | 2.9 | 2.6 |

Ligations were incubated at 22°C for 1 h. Ligase was heat inactivated at 80°C for 20 min.

## Transformation of JW3367-3 with pSB1C3/EnvZ-parts

8 x 150 μL competent JW 3367-3 cells were thawed on ice at 4°C and inoculated with DNA as following:

| cell amount [μL] | DNA | DNA amount [ng] |
| --- | --- | --- |
| 150 | pSB1C3/EnvZ TM first ligation | 200 |
| 150 | pSB1C3/NOD1-LRR ligation | 200 |
| 150 | pSB1C3/EnvZ Tail ligation | 200 |
| 50 | pSB1C3/BBa\_B0015 | 50 ng |
| 50 | - (negative control) | - |

Transformation was carried out using the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#transformation). At the end cells were spun down, resuspended in 50 μL LB-medium and plated onto LB-agar/CAmp-plates.

## Colony PCRs

Following mastermix for 40 PCR reactions was set up:

|  |  |
| --- | --- |
| VF2 Primer fw | 20 μL |
| VR Primer rv | 20 μL |
| 10 x Dream Taq buffer | 40 μL |
| dNTPs (10 mM) | 8 μL |
| Dream Taq polymerase | 2 μL |
| H2O | 310 μL |
| total | 400 μL |

33 x 10 μL of the mastermix was pipetted into PCR tubes, which were inoculated with:

| Colony numbering | Content |
| --- | --- |
| 1 - 3 | NEB cc pSB1A3/BBa\_K081005/mTaz 2 |
| 4 - 6 | NEB cc pSB1A3/BBa\_K081005/mTaz 1 |
| 7 - 9 | NEB cc pSB1A3/BBa\_K608006/mTaz |
| 10 - 12 | NEB cc pSB1A3/BBa\_K608007/mTaz 1 |
| 13 - 15 | JW3367-3 pSB1A3/BBa\_K081005/mTaz |
| 16 - 18 | JW3367-3 pSB1A3/BBa\_K608006/mTaz |
| 19 - 21 | JW3367-3 pSB1A3/BBa\_K608007/mTaz |
| 22 - 24 | JW3367-3 pSB1A3/BBa\_K081005/BBa\_C0082 |
| 25 - 27 | JW3367-3 pSB1A3/BBa\_K608006/BBa\_C0082 |
| 28 - 30 | JW3367-3 pSB1A3/BBa\_K608007/BBa\_C0082 |
| 31 | NEB cc pSB1A3/BBa\_K608007/mTaz 2 |
| 32 | NEB cc pSB1A3/BBa\_K608007/mTaz |
| 33 | JW3367-3 pSB1C3/BBa\_R0082/BBa\_I7302020 |

Positive results (bands with expected sizes) for 12, 22, 25, 26, 31 and 32.

## LB-Agar Plates with antibodies

1. **CAmp/Amp**
   700&nbp;mL LB-agar were mixed with 700&nbp;μL CAmp and 700&nbp;μAmp. The solution was distributed onto petri dishes.
2. **Amp**
   700&nbp;mL LB-agar were mixed with 700&nbp;μAmp. The solution was distributed onto petri dishes.
3. **CAmp**
   600&nbp;mL LB-agar were mixed with 600&nbp;μL CAmp The solution was distributed onto petri dishes.

## Transformation of JW3367-3 with the (m)Taz ligations and pSB1C3/OmpR/GFP

8 x 150 μL competent JW 3367-3 cells were thawed on ice at 4°C and inoculated with DNA as following:

| cell amount [μL] | DNA | DNA amount [μL/100 ng] |
| --- | --- | --- |
| 150 | pSB1A3/BBa\_K081005/mTaz + pSB1C3/BBa\_R0082/BBa\_E0240 | 1.16 + 1.16 |
| 150 | pSB1A3/BBa\_K081005/BBa\_C0082 + pSB1C3/BBa\_R0082/BBa\_E0240 | 1.39 + 1.16 |
| 150 | pSB1A3/BBa\_K608006/BBa\_C0082 + pSB1C3/BBa\_R0082/BBa\_E0240 | 0.77 + 1.16 |
| 150 | pSB1A3/BBa\_K608007/BBa\_C0082 + pSB1C3/BBa\_R0082/BBa\_E0240 | 1.58 + 1.16 |
| negative control: 150 | - | - |
| negative control: 150 | pSB1A3/BBa\_K608006/BBa\_C0082 | 1.54/200 ng |
| negative control: 150 | pSB1C3/BBa\_R0082/BBa\_E0240 | 2.32/200 ng |
| positive control: 150 | pSB1A3/BBa\_K608006/BBa\_C0082 (plated onto LB-agar/Amp plates) | 1.54/200 ng |
| positive control: 150 | pSB1C3/BBa\_R0082/BBa\_E0240 (plated onto LB-agar/CAmp plates) | 2.32/200 ng |

Transformation was carried out using the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#transformation). At the end cells were spun down, resuspended in 50 μL LB-medium and plated onto LB-agar/CAmp/Amp plates (if not stated otherwise).

## Colony PCRs of cells transformed with pSB1C3/mTaz

Following mastermix for 20 PCR reactions was set up:

|  |  |
| --- | --- |
| VF2 Primer fw | 10 μL |
| VR Primer rv | 10 μL |
| 10 x Dream Taq buffer | 20 μL |
| dNTPs (10 mM) | 4 μL |
| Dream Taq polymerase | 1 μL |
| H2O | 155 μL |
| total | 200 μL |

18 x 10 μL of the mastermix was pipetted into PCR tubes, which were inoculated with:

| Colony numbering | Content |
| --- | --- |
| 1 - 6 | JW3367-3/pSB1C3/NOD1-LRR |
| 7 - 12 | JW3367-3/pSB1C3/EnzZ Tail |
| 13 - 18 | JW3367-3/pSB1C3/EnvZ TM first |

Positive results (bands with expected sizes) for 6, 7, 10, 14 and 15.

## Minipreparation of Overnight Cultures from the PCRs from 05.10. and 06.10

The minipreparation was carried out following the instructions of the [GeneJET Plasmid Miniprep Kit](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-Plasmid-Miniprep-Kit).

| Plasmid | DNA concentration |
| --- | --- |
| pSB1A3/BBa\_K608006/BBa\_C0082 | 93.04 ng/μL |
| pSB1A3/BBa\_K608006/BBa\_C0082 | 129.88 ng/μL |
| pSB1A3/BBa\_K608007/mTaz | 98.40 ng/μL |
| pSB1A3/BBa\_K608007/mTaz | 63.21 ng/μL |
| pSB1A3/BBa\_K081005/mTaz | 86.03 ng/μL |
| pSB1A3/BBa\_K081005/mTaz | 90.59 ng/μL |
| pSB1A3/BBa\_K081005/BBa\_C0082 | 71.80 ng/μL |

## Preparation of Aspartate Solution

2.5 g in 100 mL H2O

## Measurement of Aspartate sensor (BBa\_C0082)

16 samples JW 3367-3 cells with pSB1A3/BBa\_K608006/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240 were inoculated in 5 mL LB-medium with CAmp/Amp resistance and grown until OD600=0.178.

## Making BBa\_K081005/mTaz, BBa\_K608006/mTaz and BBa\_K608007/mTaz competent cells

1. JW 3367-3/pSB1A3/BBa\_K081005/mTaz (colony 68)
2. NEB competent cells/pSB1A3/BBa\_K608007/mTaz (colony 55)
3. NEB competent cells/pSB1A3/BBa\_K081005/mTaz (colony 37)
4. NEB competent cells/pSB1A3/BBa\_K608007/mTaz (colony 31)

This samples were incolated overnight in LB/Amp-medium. Cryostocks were produced, following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#glycerol-stocks) and the cells were incolated in 50 mL LB/Amp-medium flasks until OD600=0.27. The cultered were chilled on ice for 5 min and collected after centrifugation at 5000 rpm, 10 min, 4 °C.

## Transformation

according to [Zang Gong Competent Cell Protocol](https://2016.igem.org/Team%3AHamburg/Experiments#transformation)

|  | cell amount [μL] | DNA | DNA amount[μL;ng] |
| --- | --- | --- | --- |
| NEB competent cells/pSB1A3/BBa\_K608007/mTaz (colony 31) | 50.0 | pSB1C3/BBa\_R0082/BBa\_E0240 | 0.58;50 |
| NEB competent cells/pSB1A3/BBa\_K081005/mTaz (colony 37) | 50.0 | pSB1C3/BBa\_R0082/BBa\_E0240 | 0.58;50 |
| NEB competent cells/pSB1A3/BBa\_K608007/mTaz (colony 55) | 50.0 | pSB1C3/BBa\_R0082/BBa\_E0240 | 0.58;50 |
| JW 3367-3/pSB1A3/BBa\_K081005/mTaz (colony 68) | 100.0 | pSB1C3/BBa\_R0082/BBa\_E0240 | 2.32;200 |
| NEB competent cells/pSB1A3/BBa\_K608007/mTaz (colony 31) | 50.0 | - | - |
| NEB competent cells/pSB1A3/BBa\_K081005/mTaz (colony 37) | 50.0 | - | - |
| JW 3367-3 | 50.0 | pSB1A3/BBa\_K608007/mTaz  pSB1C3/BBa\_R0082/BBa\_E0240 | 0.79; 50  0.58; 50 |
| JW 3367-3 | 50.0 | pSB1A3/BBa\_K081005/mTaz  pSB1C3/BBa\_R0082/BBa\_E0240 | 0.59; 50  0.58; 50 |
| JW 3367-3 | 50.0 | pSB1A3/BBa\_K081005/BBa\_C0082  pSB1C3/BBa\_R0082/BBa\_E0240 | 0.70; 50  0.58; 50 |
| JW 3367-3 | 50.0 | pSB1A3/BBa\_K608006/BBa\_C0082  pSB1C3/BBa\_R0082/BBa\_E0240 | 0.39; 50  0.58; 50 |
| JW 3367-3 | 50.0 | - | - |
| JW 3367-3 (negativ) | 50.0 | pSB1A3/BBa\_K608006/BBa\_C0082 | 0.39; 50 |
| JW 3367-3 (negativ) | 50.0 | pSB1C3/BBa\_R0082/BBa\_E0240 | 0.58; 50 |
| JW 3367-3 (positiv) | 50.0 | pSB1A3/BBa\_K608006/BBa\_C0082 | 0.39; 50 |
| JW 3367-3 (positiv) | 50.0 | pSB1C3/BBa\_R0082/BBa\_E0240 | 0.58; 50 |

The CAmp/Amp plates are preparated with 20 μL (40 %) Amp, the same for the Amp-plates for positiv and negativ control.

Plates incubated at 37 °C

## Colony PCR I

PCR reaction Master Mix (20x)

|  |  |
| --- | --- |
|  | amount [μL] |
| 10x DreamTaq Buffer | 40.0 |
| dNTPs, 10 mM | 8.0 |
| VF2 Primer, 10 μM | 20.0 |
| VR Primer, 10 μM | 20.0 |
| DreamTaq | 2.0 |
| H2O | 310.0 |
|  | 400.0 |

| Colony | cell with plasmids |
| --- | --- |
| 1 | NEB Competent Cells with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 2 | NEB Competent Cells with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 3 | NEB Competent Cells with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 4 | NEB Competent Cells with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 5 | JW 3367-3 with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 6 | JW 3367-3 with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 7 | JW 3367-3 with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 8 | JW 3367-3 with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 9 | JW 3367-3 with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 10 | JW 3367-3 with pSB1A3/BBa\_K608006/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 11 | JW 3367-3 with pSB1A3/BBa\_K608006/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 12 | JW 3367-3 with pSB1A3/BBa\_K608006/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 13 | JW 3367-3 with pSB1A3/BBa\_K608006/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 14 | JW 3367-3 with pSB1A3/BBa\_K608006/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 15 | JW 3367-3 with pSB1A3/BBa\_K608006/BBa\_C0082 |

Colonies were picked and resuspended in 4 μL H2O

3 μL H2O were transformed into 5 mL LB/CAmp/Amp-medium, the rest was used as template for the PCR.

Program: TAQSCREEN

result: all samples are positiv!

## Colony PCR II

PCR reaction Master Mix (20x)

|  |  |
| --- | --- |
|  | amount [μL] |
| 10x DreamTaq Buffer | 40.0 |
| dNTPs, 10 mM | 8.0 |
| VF2 Primer, 10 μM | 20.0 |
| VR Primer, 10 μM | 20.0 |
| DreamTaq | 2.0 |
| H2O | 310.0 |
|  | 400.0 |

| Colony | cell with plasmids |
| --- | --- |
| 1 | NEB Competent Cells with pSB1A3/BBa\_K608007/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 2 | NEB Competent Cells with pSB1A3/BBa\_K608007/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 3 | NEB Competent Cells with pSB1A3/BBa\_K608007/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 4 | NEB Competent Cells with pSB1A3/BBa\_K608007/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 5 | JW 3367-3 with pSB1A3/BBa\_K608007/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 6 | JW 3367-3 with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 7 | JW 3367-3 with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 8 | JW 3367-3 with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 9 | JW 3367-3 with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 10 | JW 3367-3 with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 11 | JW 3367-3 with pSB1A3/BBa\_K081005/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 12 | JW 3367-3 with pSB1A3/BBa\_K081005/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 13 | JW 3367-3 with pSB1A3/BBa\_K081005/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 14 | JW 3367-3 with pSB1A3/BBa\_K081005/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240 |
| 15 | JW 3367-3 with pSB1A3/BBa\_K081005/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240 |

Colonies were picked and resuspended in 4 μL H2O

3 μL H2O were transformed into 5 mL LB/CAmp/Amp-medium, the rest was used as template for the PCR.

Program: TAQSCREEN

## Inoculate for FACS

1. 2x 50 μL JW 3367-3 with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240
2. 2x 50 μL JW 3367-3 with pSB1A3/BBa\_K608006/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240
3. 2x 50 μL JW 3367-3 with pSB1A3/BBa\_K608007/BBa\_C0082 and pSB1C3/BBa\_R0082/BBa\_E0240

negative controls

1. 20x 50 μL in 45 mL JW 3367-3/pSB1C3/BBa\_R0082/BBa\_E0240
2. 20x 500 ΜL in 45 mL JW 3367-3/pSB1A3/BBa\_K081005/mTaz

## Preparation of DAP solutions

DAP solution was prepared in a 1 g/ 100 mL concentration on a 40 mL volumina with autoclaved water.

## FACs

Since ONCs failed to grow (fast enough) the following cultures were prepared for FACs measurement.

1. NEB Competent Cells with pSB1A3/BBa\_K081005/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240
2. JW 3367-3 with pSB1C3/BBa\_K608007/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240
3. JW 3367-3 with pSB1C3/BBa\_K608007/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240
4. JW 3367-3 with pSB1C3/BBa\_K608006/mTaz and pSB1C3/BBa\_R0082/BBa\_E0240

Preparation included centrifugation, resuspended in new LB-medium, splitting the culture into nine samples and treating them with:

0; 0.1; 1.0; 5.0; 10.0 [mmol/L] of DAP and Aspartate.

At timepoints of 0, 30, 90 minutes 1 mL culture was centrifuged at 3000 rpm for 15 minutes to get cell pellett. The supernatant was discarted and the cells werde resuspended in 1 mL PBS.

The cells were centrifuged at 13400 rpm for 1 minuts.

The supernatant were discarded and the cells resuspended in 1 mL PBS for FACs analysis.

## Preparations of FACs Samples

1. JW 3367-3 cells
2. DH5α cells
3. JW 3367-3/pSB1C3/BBa\_R0082/BBa\_E0240
4. DH5α/pSB1C3/BBa\_R0082/BBa\_E0240

Cells were splitted in nine 5 mL cultures after centrifugation and resuspention in 45 mL LB-medium.

Cell cultures 3 and 4 were treated with CAmp.

At timepoints of 0, 30, 90 minutes 1 mL culture was centrifuged at 3000 rpm for 15 minutes to get cell pellett. The supernatant was discarted and the cells werde resuspended in 1 mL PBS.

The cells were centrifuged at 13400 rpm for 1 minuts.

The supernatant were discarded and the cells resuspended in 1 mL PBS for FACs analysis.

## Get constructs from pSB1K3 into pSB1C3

mTaz and BBa\_C0082 with Anderson Promoters [BBa\_K081005, BBa\_K608006, BBa\_K608007] (respectivly)

**Restriction**

* 5 μL pSB1C3 (50 ng/μL)
* 3.4 μL pSB1A3/BBa\_K608007/mTaz
* 2.3 μL pSB1A3/BBa\_K081005/mTaz
* 2.8 μL pSB1A3/BBa\_K081007/BBa\_C0082
* 2.1 μL pSB1A3/BBa\_K608006/BBa\_C0082

Add 0.5 μL PstI and EcoRI and 2 μL 2.1NEB Buffer for each reaction, add H2O to 20 μL

Incubate for 20 min (37 °C); heat inactivate for 20 min (80 °C)

**Dephosphorylation of Vector**

Add 0.5 μL FastAP and 2 μL AP Buffer

Incubate for 10 min (37 μC), heat inactivation 5 min (75 °C)

**Ligation**

* Vektor and Insert (1:5)
* 4.5 μL pSB1C3 (50 ng)
* 20.0 μL of each BBa\_C0082 and mTaz Restriction sample
* 1.0 μL T4 Ligase
* 3.0 μL T4 Buffer
* 1.5 μL H2O

Incubation for 1 h (22 °C), heat inactivation for 10 min (65 °C)

**Transformation**

Transformation following the [Zhang Gongs Transformation protocol](https://2016.igem.org/Team%3AHamburg/Experiments#transformation)

25.0 μL of each ligation product.

## Colony PCR and Over Night Cultures

Master Mix

|  |  |
| --- | --- |
| 10x DreamTaq Buffer | 35.0 μL |
| dNTPs, 10 mM | 7.0 μL |
| VF2 Primer, 10 μM | 5.0 μL |
| VR Primer, 10 μM | 5.0 μL |
| DreamTaq | 2.0 μL |
| H2O | 331.0 μL |
|  | to 350 μL |

Eight colonies of each construct were picked.

positiv bands

1. pSB1C3/BBa\_K081005/mTaz colonies 1, 2, 4, 6 and 7
2. pSB1C3/BBa\_K608006/BBa\_C0082 colonies 10, 11, 12, 13, 14, 15 and 16
3. pSB1C3/BBa\_K081005/BBa\_C0082 colonies 17, 18, 19, 20, 21, 22 and 24
4. pSB1C3/BBa\_K608007/mTaz colonies 25, 28, 29, 30, 31 and 32

first three of each sample there minipreped, following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#GeneJET-Plasmid-Miniprep-Kit)

of each cryostock were generated, following the [respective protocol](https://2016.igem.org/Team%3AHamburg/Experiments#glycerol-stocks)

## Fabrication of ED02 (SU-8) Master01

* 4-inch Si-wafer was dehydrated for 20 minutes at 130 °C in oven
* Base / adhesion layer of SU-8 (GM1060):

  -Spin coating:
  1) 0 - 4000 rpm (100 rpm/second acceleration) in 40 s
  2) 4000 rpm for 40 seconds
  3) 4000 - 0 rpm (-100 rpm/second acceleration) in 40 seconds
  -30 minutes relaxation time at RT

  -Soft bake:
  1) 37 °C - 65 °C in about 5 minutes (avg +6 °C/minute)
  2) 65 °C for 5 minutes
  3) 65 °C - 95 °C in about 5 minutes (avg +5 °C/minute)
  4) 95 °C for 5 minutes
  5) 95 °C - 49 °C in about 13 minutes (avg -3.5 °C/minute)
  i-line exposure (13 mW/cm2) for 30 seconds without mask (complete exposure)

  -10 minutes delay time before PEB

  -PEB:
  1) 36 °C - 65 °C in about 5 minutes (avg +6 °C/minute)
  2) 65 °C for 5 minutes
  3) 65 °C - 95 °C in about 5 minutes (avg +6 °C/minute)
  4) 95 °C for 15 minutes
  5) 95 °C - 45 °C in about 15 minutes (avg -3.3 °C/minute)
* Structural layer of SU-8 (GM1075):

  -Spin coating:
  1) 0 - 1000 rpm (100 rpm/second acceleration) in 10 s
  2) 1000 rpm for 100 seconds
  3) 1000 - 1400 rpm (10000 rpm/second acceleration) in 1 s
  4) 1400 - 0 rpm (-100 rpm/second acceleration) in 14 seconds
  -30 minutes relaxation time on hotplate at 40 °C; covered with glass dish

  -Soft bake:
  1) 40 - 120 °C in about 9 minutes (avg +9 °C/minute)
  2) 120 °C for 5 minutes
  3) 120 °C - 37 °C in about 25 minutes (avg -3.3 °C/minute)
  -i-line exposure (13 mW/cm2 at 365 nm) for 30 seconds (corresponding 390 mJ/cm2 instead of recommended 350 mJ/cm2) using the ED02-Mask01

  -1 hour delay time at RT in wafer box before PEB

  -PEB:
  1) 28 °C- 95 °C in about 7 minutes (avg 9.5 °C/minute);
  2) 95 °C for 30 minutes
  3) 95 °C - 41 °C in about 16 minutes (avg -2.4 °C/minute)
  -Develop:
  -In PGMEA (new) for about 8 minutes while constantly agitating the PGMEA and the wafer in the beaker
  -Wafer was rinsed with isopropanol to stop the development which caused white traces on wafer
  -Isopropanol-rinsed wafer was washed the with new, used PGMEA and subsequently rinsed with isopropanol which caused the previous white traces to vanish, however, lines of residue from isopropanol evaporation remained
  -Examination of the profile (using the DektakXT; Bruker) showed a thickness of approx. 110 μm; comparison between the depth of the large filter structure holes and the fine filter structure holes (as visible in the live video in the DektakXT using a ruler) showed similar depth (7 mm at 100x magnification) and can be checked in the captured images

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