iGEM 2018 — Team UCopenhagen

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# Team:UCopenhagen/Notebook

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[Introduction](https://2018.igem.org/Team%3AUCopenhagen/Human_Practices)
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[Choosing a Protein](https://2018.igem.org/Team%3AUCopenhagen/Choice_of_Protein)
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[Blog on Medium](https://medium.com/igem-copenhagen)

# Lab notebook

The notebook is a chronological overview of what has happened in the lab. It is organized after each experiment.

jQuery UI Example Page

* [Cloning](#tabs-1)
* [Liposome charaterization](#tabs-3)
* [Protoplast experiments with plants](#tabs-4)
* [Leakiness characterization](#tabs-5)
* [Supported lipid bilayers](#tabs-6)
* [InterLab](#tabs-7)
* [Egg membrane experiment](#tabs-8)

# Cloning and Plasmid Construction

## Week 25

### Competent cell preparation 1 – 19/06/18

Overnight culture of MACH1 and DH5α E. coli
Inoculate MACH1 and DH5α E. coli from glycerol stock into 5 ml LB broth. Incubate at 37 ◦C with 150 rpm shaking overnight.

Prepare CCMB80 competent cell buffer (100ml)

| Components | Amount | Final concentration |
| --- | --- | --- |
| 1M pH 7.0 CH3COOK stock solution | 1 mL | 10 mM |
| CaCl2.2H2O | 1.18 g | 80 mM |
| MnCl2.4H2O | 0.4 g | 20 mM |
| MgCl2.6H2O | 0.2 g | 10 mM |
| Glycerol | 10 mL | 10 % v/v |
| MiliQ H2O | added to 100 ml | Final concentration |

Adjust pH to 6.4 by 0.01M HCl. Sterile by filtration through 0.22 μm filter. Keep in 4 ◦C

#### Nat

## Competent cell preparation 2 – 20/06/18

Protocol adjusted from iGEM [Competent Cell Production Protocol](http://parts.igem.org/Help%3AProtocols/Competent_Cells).

Inoculate 1 ml of MACH1 and DH5α E. coli O/N culture in 100 ml of fresh LB broth. Incubate at room temperature, with 50 rpm shaking until OD600 reach 0.3 - 0.5 (3.30 hours).

1. Chill the culture on ice for 30 min.- Centrifuge at 3000 g for 10 min. Discard supernatant.- Resuspend cell pellet in 50 ml ice cold sterile H20.- Centrifuge at 3000 g for 10 min. Discard supernatant.- Resuspend cell pellet gently in 20 ml ice cold CCMB80 buffer.- Centrifuge at 3000 g for 10 min. Discard supernatant.- Resuspend cell pellet gently in 8 ml ice cold CCMB80 buffer.- Aliquot 50 𝜇L to cold 2 ml Eppendorf tube. Snap freeze in liquid N2.- Store in -80 ◦C

#### Nat

#### Test for competency – 21/06/18

Test for competency according to iGEM [Competent Cell Test Kit protocol](http://parts.igem.org/Help%3A2017_Competent_Cell_Test_Kit).

Result: DH5α 1.5 x 107 CFU/μg

MACH1 5 x 106 CFU/μg

#### Nat

# Week 28

## Culture SIEC and SIECΔp1 *E. coli* – 11/07/18

SIEC and SIECΔp1 *E. coli* strain arrived from Spain.
Streak SIEC and SIECΔp1 *E. coli* from agar stab stock on LB agar plate.

#### Nat

## Culture SIEC and SIECΔp1 *E. coli* –12/07/18

Pick a single colony from SIEC and SIECΔp1 into 5 ml LB broth. Incubate at 37 ◦C with 150 rpm shaking overnight.

#### Nat

## Prepare SIEC and SIECΔp1 competent cell stocks – 13/07/18

Prepare competent cell stocks from SIEC and SIECΔp1 using the same protocol mentioned in date 19/06/18 and 20/06/18.

#### Nat

# Week 29

## pBAD and pTac Transformation – 16/07/18

Transform pBAD (BBa\_K228005) and pTac (BBa\_731500) into MACH1 E. coli according to the [iGEM transformation protocol](http://parts.igem.org/Help%3AProtocols/Transformation).

Primers arrived today! Dilute primer into 100 μM stock and 10 μM working stock.

#### Nat

## PCR reporter genes and T3SS signal – 17/07/18

PCR amplify reporter genes sfGFP (BBa\_I746916), NanoLuc (BBa\_K1159001), mCherry (BBa\_J06504 ), and beta-lactamase (BBa\_K1189008) from Biobrick distribution kit.

PCR assemble T3SS signal from 5 overlapping oligonucleotides. This signal encode 20 amino acid from N-terminal Map genes.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Template | Primer 1 | Primer 2 | Product | Size (bp) |
| 1. NanoLuc BBa\_K1159001 | Nluc-F | Nluc-R | NanoLuc | 555 |
| 2. sfGFP BBa\_I746916 | GFP-F | GFP-R | sfGFP | 748 |
| 3. mCherry BBa\_J06504 | mCH-F | mCh-R | mCherry | 740 |
| 4. Beta-lactamase BBa\_K1189008 | Blac-F | Blac-R | Beta-lactamase | 822 |
| 5. Map (no template) | Map20-1, Map20-2, Map20-3, Map20-F2, Map20-R2 (55) | | Map20 | 136 |
| 6. SIEC E. coli genomic DNA | EspD-UF | EspD-UR | EspD | 1180 |

**Pfu-X7 PCR reaction components**

|  |  |  |
| --- | --- | --- |
| Components | 50 µl reaction | Final conc. |
| 10X home-made X7 buffer | 5 µl | 1X |
| dNTP mixture (2.5 mM) | 4 µl | 200 µM |
| Pfu-X7 polymerase (2U/µl) | 0.5 µl | 1 Unit/50 µl |
| Forward primer (10 µM) | 2.5 µl | 0.5 µM |
| Reverse primer (10 µM) | 2.5 µl | 0.5 µM |
| Template | 1 µl | <250ng DNA |
| MiliQ H2O | To 50 µl | - |

**Pfu-X7 PCR temperature profile**

|  |  |  |  |
| --- | --- | --- | --- |
| Step | Temperature | time | Cycle |
| Initial denaturation | 98 ◦C | 30 sec | - |
| Denaturation | 98 ◦C | 10 sec | 35 Cycles |
| Annealing | 55 ◦C | 15 sec |
| Extension | 72 ◦C | 45 sec |
| Final Extension | 72 ◦C | 5 min | - |
| Hold | 16 ◦C | ∞ | - |

**Gel result**

![alt_text](https://static.igem.org/mediawiki/2018/thumb/1/1b/T--UCopenhagen--cloning1.jpeg/703px-T--UCopenhagen--cloning1.jpeg "image_tooltip")

1. NanoLuc Fail
2. sfGFP Fail
3. mCherry Fail
4. Beta-lactamase Fail
5. Map20 (T3SS signal) Success

6. EspD Success

The failed PCR may result from incompatible PCR polymerase enzyme or buffer. Next time change PCR enzyme to Q5 DNA polymerase.

Nat, Selma

## PCR reporter genes – 18/07/18

PCR amplify reporter genes sfGFP (BBa\_I746916), NanoLuc (BBa\_K1159001), mCherry (BBa\_J06504 ), and beta-lactamase (BBa\_K1189008) again using Q5 DNA polymerase.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Template | Primer 1 (Tm) | Primer 2 (Tm) | Product | Size(bp) |
| 1. sfGFP BBa\_I746916 | GFP-F (61) | GFP-R (55) | sfGFP | 748 |
| 2. NanoLuc BBa\_K1159001 | Nluc-F (68) | Nluc-R (60) | NanoLuc | 555 |
| 3. mCherry BBa\_J06504 | mCH-F (63) | mCh-R (58) | mCherry | 740 |
| 4. Beta-lactamase BBa\_K1189008 | Blac-F (61) | Blac-R (58) | Beta-lactamase | 822 |

Second PCR using PCR product from the first PCR as a template.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Template | Primer 1 | Primer 2 | Product | Size(bp) |
| 1. sfGFP | GFP-F | GS-His-R | sfGFP-His | 772 |
| 2. NanoLuc | Nluc-F | GS-His-R | NanoLuc-His | 579 |
| 3. mCherry | mCH-F | GS-His-R | mCherry-His | 762 |
| 4. Beta-lactamase | Blac-F | GS-His-R | Beta-lactamase-His | 846 |

**Q5 PCR reaction components**

|  |  |  |
| --- | --- | --- |
| Components | 50 µl reaction | Final concentration |
| Q5 High-Fidelity 2X Master Mix | 25 µl | 1X |
| Forward primer (10 µM) | 2.5 µl | 0.5 µM |
| Reverse primer (10 µM) | 2.5 µl | 0.5 µM |
| Template | 1 µl | <250ng DNA |
| MiliQ H2O | To 50 µl | - |

**Q5 PCR temperature profile**

|  |  |  |  |
| --- | --- | --- | --- |
| Step | Temperature | time | Cycle |
| Initial denaturation | 98 ◦C | 30 sec | - |
| Denaturation | 98 ◦C | 15 sec | 35 Cycles |
| Annealing | 55 ◦C | 25 sec |
| Extension | 72 ◦C | 45 sec |
| Final Extension | 72 ◦C | 5 min | - |
| Hold | 16 ◦C | ∞ | - |

**Gel result**

![alt_text](https://static.igem.org/mediawiki/2018/a/a4/T--UCopenhagen--clone3.jpeg "image_tooltip")

1. sfGFP-His Success

2. NanoLuc-His Success
3. mCherry-His Success
4. Beta-lactamase-His Success

Purify sfGFP-His, NanoLuc-His, mCherry-His, Beta-lactamase-His, EspD, and Map20 PCR product using E.Z.N.A.® Cycle Pure Kit.

#### Nat, Eric

## Restriction enzyme digestion – 19/07/18

**Digestion**

|  |  |  |
| --- | --- | --- |
| DNA | Restriction enzyme 1 | Restriction enzyme 2 |
| pBAD | SpeI | PstI |
| Map20 | XbaI | AgeI |
| mCherry | XmaI | PstI |
| sfGFP | XmaI | PstI |
| Beta-lactamase | XmaI | PstI |
| NanoLuc | XmaI | PstI |

**25 μl restriction enzyme digestion reaction components**

10X Cutsmart™ buffer 2.5 μl

DNA 250 ng

20U/μl Restriction enzyme 0.5 μl

H20 to 25 μl

**Temperature profile**

37 ◦C 30 min

80 ◦C 20 min

Digestion products are purified by E.Z.N.A.® Cycle Pure Kit.

#### Selma, Eric

## Ligation and transformation – 20/07/18

The digested plasmids and genes were ligated together in the four reactions below

**10 μl Ligation reaction components**

Vector DNA 20-50 ng
Insert DNA 2X - 4X molar ratio compared to vector
10U/μl T4 Ligase 0.5 μl

T4 ligase buffer 1 μl
H2O to 10 μl

|  |  |  |  |
| --- | --- | --- | --- |
|  | Vector | Insert 1 | Insert 2 |
| Ligation I | pBAD | Map20 | mCherry |
| Ligation II | pBAD | Map20 | sfGFP |
| Ligation III | pBAD | Map20 | ꞵ-lactamase |
| Ligation IV | pBAD | Map20 | NanoLuc |

Ligation reactions are performed at 16 ◦C for 1 hour.

1 μl of Ligation products were transformed into Mach1 E. coli competent cell according to [iGEM transformation protocol](http://parts.igem.org/Help%3AProtocols/Transformation).

#### Selma, Eric

## Colony PCR – 24/07/18

Ligation I, II, III grow only a few colonies while ligation IV give no colony. Colony PCR was performed to confirm reporter protein insertion.

**10 μl Colony PCR reaction**

|  |  |  |
| --- | --- | --- |
| Components | 10 µl reaction | Final concentration |
| Q5 High-Fidelity 2X Master Mix | 5 µl | 1X |
| Forward primer (10 µM) | 0.5 µl | 0.5 µM |
| Reverse primer (10 µM) | 0.5 µl | 0.5 µM |
| Template | E. coli cells | <250ng DNA |
| MiliQ H2O | 4 µl | - |

**Colony PCR temperature profile**

|  |  |  |  |
| --- | --- | --- | --- |
| Step | Temperature | time | Cycle |
| Initial denaturation | 98 ◦C | 30 sec | - |
| Denaturation | 98 ◦C | 15 sec | 35 Cycles |
| Annealing | 55 ◦C | 25 sec |
| Extension | 72 ◦C | 45 sec |
| Final Extension | 72 ◦C | 5 min | - |
| Hold | 16 ◦C | ∞ | - |

Colony PCR result

![drawing](https://static.igem.org/mediawiki/2018/a/ab/T--UCopenhagen--Clone3.png)

Note: Gel was overheated and image quality is poor. However, it is interpretable.

Positive results are achieved from colony I5, II1, and II2. No positive colonies from ligation III (pBAD-Map20-ꞵ-lactamase).

The positive colonies I5, II1, and II2 were picked and cultured in LB broth+chloramphenicol.

Repeat transformation of ligation III and IV into Mach1 E. coli. The repeated transformants are called IIIB and IVB.

#### Nat

## Plasmid extraction – 25/07/18

Plasmid extraction from cells with plasmid I5, II1, and II2 was done using E.Z.N.A.® plasmid mini kit. DNA Concentrations were measured using Nanodrop. Purified I5, II1, and II2 plasmids were sent to sequencing with Map20-F2 and GS-His-R primers.

Transformant IIIB and IVB grow few colonies. Perform colony PCR on 4 colonies from IIIB and 2 colonies from IIB.

Colony PCR result

![drawing](https://static.igem.org/mediawiki/2018/9/9d/T--UCopenhagen--Clone4.png)

Positive results are achieved from colony IIIB2, IIIB3, and IVB1. All positive colonies were picked and culture overnight in LB broth+chloramphenicol.

#### Eirikur

## USER cloning of EspD and chaperone cassette– 26/07/18

Plasmid extraction from overnight cultured IIIB2, IIIB3, and IVB1 was done using E.Z.N.A.® plasmid mini kit. DNA Concentrations were measured using Nanodrop. The purified plasmids were sent to sequencing with Map20-F2 and GS-His-R primers.

pBAD, pTac, synthetic CesF-CesT chaperone cassette, and EspD are PCR with USER primers.

PCR reactions are performed with X7 polymerase which is not inhibited by Uracil-containing primers.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Template | Primer 1 | Primer 2 | Product | Size(bp) |
| 1.pBAD (BBa\_K228005) | AraC-UF | AraC-UR | Linearized pBAD | 3351 |
| 2. pTac (BBa\_K731500) | LacO-UF | LacO-UR | Linearized pTac | 3342 |
| 3. SyntheticCesF-CesT chaperone cassette | CesF-UF | CesT-UR | Chaperone cassette | 965 |
| 4. EspD | EspD-UF | EspD-UR | EspD | 1180 |

Gel result

![drawing](https://static.igem.org/mediawiki/2018/4/45/T--UCopenhagen--Clone5.png)

![drawing](https://static.igem.org/mediawiki/2018/d/da/T--UCopenhagen--Clone6.png)

Purify the PCR products with EZNA Cycle Pure Kit.

#### Nat

## USER cloning and transformations of pBAD-Chaperone and pTac-EspD – 27/07/18

USER cloning of pBAD + Chaperone cassette to generate pBAD-Chaperone plasmid Mach1 E. coli.

USER cloning of pTac + EspD to generate pTac-EspD plasmid and transform into DH5α E. coli.

**USER reaction components**

Vector DNA 1 μl

Insert DNA 1 μl

10X Cutsmart buffer 0.5 μl

DpnI 0.3 μl

10X USER enzyme mix 0.5 μl

H20 0.7 μl

**USER reaction temperature profile**

37 ℃ 20 min

25 ℃ 30 min

16 ℃ hold

#### Nat

## Colony PCR pTac-EspD and pBAD-Chaperone – 28/07/18

Colony PCR pTac-EspD and pBAD-Chaperone and pick the correct clone into LB broth with chloramphenicol.

#### Nat

## pTac-EspD and pBAD-Chaperone plasmid extraction and – 29/07/18

Plasmid extraction from an overnight culture of pTac-EspD and pBAD-Chaperone was done using E.Z.N.A.® plasmid mini kit. DNA Concentrations were measured using Nanodrop. The purified plasmids were sent to sequencing with a set of sequencing primers.

#### Nat

## Construction of reporter plasmids with chaperone cassette –31/07/18

Sequencing result of pBAD-chaperone confirms a correct sequence. Reporter genes and the Map20 signal was inserted into the pBAD-chaperone vector, using the same method as described previously in date 19/07/18 - 24/07/18.

#### Nat

## Colony PCR Reporter plasmid with chaperone cassette –01/08/18

Correct constructs of the reporter plasmids with chaperone cassette are identified by colony PCR as described previously. The correct colonies are picked into LB + chloramphenicol and incubate O/N in 37 C.

#### Nat

## Plasmid extraction and sequencing –02/08/18

Reporter plasmids with chaperone are extracted using E.Z.N.A.® plasmid mini kit. DNA Concentrations were measured using Nanodrop. The purified plasmids were sent to sequencing with Map20-F2 and GS-His-R primers.

#### Nat

## Design a new primer set to clone reporter genes without signal sequence –25/08/18

Reporter plasmid without signal is needed. Forward primers for mCherry, sfGFP, ꞵ-lactamase, and Nanoluc are designed to contain RBS and XbaI restriction site to facilitate cloning.

This diagram demonstrates a plan to insert a reporter gene without signal sequence into a pBAD vector without chaperone cassette.

![alt_text](https://static.igem.org/mediawiki/2018/a/a2/T--UCopenhagen--Clone7.png "image_tooltip")

#### Nat

## Construction of reporter plasmids without signal sequence –27/08/18

mCherry, sfGFP, and Beta-lactamase are PCR from previous reporter plasmid and inserted into pBAD and pBAD-Chaperone vector using restriction-ligation as designed.

#### Nat

## Colony PCR Reporter plasmid without signal –29/08/18

Correct constructs of the reporter plasmids are identified by colony PCR as described previously. The correct colonies are picked into LB + chloramphenicol and incubate O/N in 37 C.

#### Nat

## Plasmid extraction and sequencing –30/08/18

Reporter plasmids with chaperone are extracted using E.Z.N.A.® plasmid mini kit. DNA Concentrations were measured using Nanodrop. The purified plasmids were sent to sequencing.

# Liposome injection experiments.

# Week 40

## Overnight of strains for liposome experiment 0,1 – 4/10/18

GFP I+S+C+, I-S-C-
mCherry I+S+C+, I-S-C-

Beta-lactamase I+S+C+, I-S-C-

Nanoluc I+S+C+, I-S-C-

#### Attila

## Liposome experiment 0,1 – 5/10/18

#### Liposome experiment 0,1

#### Attila

## Overnight for liposome experiment 1 – 7/10/18

GFP, all combinations of strains

#### Attila.

# Week 41

## Liposome experiment 1 – 8/10/18

liposome experiment 1.

#### Attila & nuttawat

## Overnight cultures for liposome experiment 2 – 10/10/18

mCherry, all combinations of strains.

#### Attila

## Liposome experiment 2 – 11/10/18

Liposome experiment 2

Attila

Western blot for experiment 1 & 2 part 1/2.

#### Attila & Nuttawat

## Liposome experiment 2 – 12/10/18

Western blot 2/2.

#### Attila & Nuttawat

# Week 42

## Overnight for liposome experiment 3 – 15/10/18

mCherry, all combinations of strains.

GFP, all combinations of strains

#### Attila

## Liposome experiment 3 – 16/10/18

Liposome experiment 3

#### Attila

Fluorescence Confocal microscopy

Lasse

Western blot part 1/2

Attila, Nuttawat, Lasse

## Liposome experiment 3 – 17/10/18

Western blot part 2/2

#### Lasse & Nuttawat

# Protoplast experiment with plants

## Week 36

Preparation of pilot experiment – 06/09/18

* Picked colonies from plates with the relevant strains and started an overnight liquid culture. *Note:* The colonies picked were from chose three SIEC strains, one containing GFP, another mCherry and then Beta-Lactamase to serve as a negative control.* Looked at the protocol which the 2011 Hokkaido iGEM team used for their onion experiment and adjusted it to our strains.

*Stephanie and Eiríkur*

### Further preparation for pilot experiment – 07/09/18

1. Started a new overnight culture of cells (from the 6th) in induction media containing IPTG and Arabinose.- Prepared 1L of MgM-MES buffer pH5.0 using the following recipie:

***MgM-MES buffer pH5.0:***

*170mM MES*

*5mM KCl*

*75mM (NH4)2SO4*

*0.5mM K2SO4*

*8uM MgCl2*

*38mM Glycerol*

*0.1% Casaminoacids*

Fill up to 1L of water and autoclave to sterilize.

*Stephanie and Eiríkur*

## Week 37

### Preparation of pilot experiment – 10/09/18

Due to a delay because of missing mannitol, two new overnight cultures were started with the strains mentioned above. The two cultures were made differently to see if there is a difference in time of the Induction of Injectisome production.

**Culture 1**(mCherry,GFP, beta-lactamase strains): Prepared using LB media with Chloramphinicol and 1%arabinose(to induce the production of all the reporter proteins).

**Culture 2:**(mCherry,GFP, beta-lactamase strains): Prepared using LB media with Chloramphinicol ,1%arabinose(to induce the production of all the reporter proteins) and 0.1mM IPTG(To induce the production of the injectisome)

*Stephanie and Eiríkur*

### Pilot experiment – 11/09/18

Decision was made to use D-Sorbitol instead of Manitole

* The overnight cultured cells were spun down at 3000rpm, for 10 min at 35C. Supernatant removed.* 5mL MgM-MES (pH5.0), Chloramphenicol, L-arabinose and IPTG were added to the bacterial pellet and resuspended by vortexing.cells were culteured at 37C, shaking at 200rpm for 4 hours.* While cultures were incubating, onion sheets were prepared using this [protocol](https://static.igem.org/mediawiki/2018/8/82/T--UCopenhagen--onionProt.pdf)* When cultures had incubated we spun down the culture at 3000rpm, for 10 min at 25C, supernatant was removed* Pellet was resuspended with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol and spun down at 3000rpm, for 10 min at 25C, supernatant was removed. This was repeated 3 times.* Pellet was resuspended with 2 ml of MgM-MES (pH5.0) containing 0.4 M Sorbitol, chloramphenicol, L-arabinose and IPTG.* Light absorbance was measured at 600nm using a spectrophotometer and the concentration of cells was adjusted to ΔOD600 = 0.06 by diluting with the same medium mentioned above.* To infect the onion cells 500ul of the culture fluid was put onto the pre-treatd onion cell-sheets. The whole thing was left at room temperature. We made two samples for each culture, one was left for 2 hours the other one was left overnight

* Cells were observed under a fluorescent microscope

*Stephanie and Eiríkur*

### Onion preparation– 25/09/18

* 7 Fresh epidermal sheets of onion cells were cut out and put on microscope-slides

* The onion cell sheets were attached to the microscope slide with agarose

* A solution containing 1% Cellulase, 0.1%Pectolyase, 0.4M Sorbiltole-D and MgM-MES buffer, was put onto the onion sheets

* 1 sample was a control with no solution, the rest(6) were observed after 10min, 20min and 45min(duplicates were made for each time interval), under a microscope to see how the onion cells changed according to the incubation time of the enzymes

![Hej](https://static.igem.org/mediawiki/2018/f/f3/T--UCopenhagen--NoteOnion%281%29.jpeg)

|  |  |  |
| --- | --- | --- |
| *t=0* | *T=20* | *t=45* |

* There were no conclusive results, might be because the incubation time was too short, the buffers were old or the enzymes did not work as suspected

### Further preparation of onion sheets and tobacco plant observations– 27/09/18

New buffers and solutions were made:

* 1 liter MgM-MES buffer,* 500ml 0.4M Sorbitol-D solution in MgM-MES buffer,* 50ml of 1%Cellulase and 0.2% pectolyase in a 0.4M Sorbitol-D solution in MgM-MES
      + 7 Fresh epidermal sheets of onion cells were cut out and put on microscope-slides

* The onion cell sheets were attached to the microscope slide with agarose

* A solution containing 1% Cellulase, 0.1%Pectolyase, 0.4M Sorbiltol-D and MgM-MES buffer, was put onto the onion sheets

* 1 sample was a control with no solution, the rest(6) were observed after 10min, 20min and 60min(duplicates were made for each time interval), under a microscope to see how the onion cells changed according to the incubation time of the enzymes

* A single tobacco plant was obtained for the purpose of trying to observe protoplasts* The cuticule was scraped off as best as possible* A solution containing 1% Cellulase, 0.1%Pectolyase, 0.4M Sorbiltol-D and MgM-MES buffer, was put into a petri dish, the piece of leaf without it´s cuticle was put on top of the solution and left there for an hour* The treatment was done on a big leaf from the plant, a smaller leaf, and a tiny leaf, to see of there was any visible difference in amount of protoplasts* The leafs and its solution were observed under through a microscope which revealed burst cells and very few protoplast

### Osmolarity test on onion sheets and tobacco plant – 02/10/18

We had been using 0.4M solution of Sorbitol through the entire experiment so we decided to test different concentrations, to see if it made any difference. We decided furthermore to switch from Sorbitol to Mannitol since a new batch of mannitol just arrived

* A 12 well culture plate was obtained. Each well was filled with a solution containing MgM-MES buffer, 0.1% pectolyase, 1%cellulase and Mannitol in 9 different concentrations ranging from 0.4M-0.8M.* The cuticle of the tobacco leaves were scraped of and but downside-down into each of the wells* The first 9 wells contained 0.4;0.45;0.5;0.55;0.6;0.65;0.7;0.75 and 0.8 M Mannitol,respectively, plus the MgM-MES and enzymes. The last three contained onion cell sheets in 0.6,0.7 and 0.8 M Mannitol with the solution mentioned above.* Samples were taken after 3 hours and looked at under a fluorescent microscope.* We saw tobacco protoplasts in most samples but the sample with the highest concentration protoplast appeared to be the one containing 0.6-0.65 M of Mannitol* From looking at the onion cells, they appeared to be best treated with a 0.8M of Mannitol* We decided to move forward with 0.6M Mannitol for the tobacco leaves and 0.8M for the Onion cells

# Onion and tobacco assay – 05/10/18

*8/9/18 - Stephanie & Eirikur*

Onion assay pilot experiment:

Delayed due to missing Mannitol

1. Spin down the cultured cells at 3000rpm, for 10 min at 35C. Remove supernatant.- Add 5mL MgM-MES (pH5.0), Chloramphenicol, L-arabinose and IPTG to the bacterial pellet. Resuspend it using vortex mixer. Culture cells at 37C, shaking at 200rpm for 4 hours. This step is required because genes encoding salmonella T3SS are expressed in acidic environment.- While cultures are incubating, prepare onion sheets:- Cut an onion into four pieces.- Take out a fresh sheets of epidermal cells (second or third layer from the surface of onion).- Cut the layer into rectangular shape (about 1.5cm x 3cm)- Tear off a cell sheet from the inner surface of the layer.- Put the sheet onto a slide glass at back face up because the upper face deflects enzyme solution, and fix the edge of the sheet onto slide glass with 1% agarose gel.- Fill a petri dish with 1% cellulase and 0.1% pectolyase in 0.4 M Mannitol solution (pH7.0), then submerge the cell sheet fixed on slide glass into the enzyme solution. Incubate for 8 min at RT.- Prepare 3 petri dishes filled with MgM-MES pH5.0 containing 0.4 M Mannitol. After processing, wash cells stepwise soaking in these petri dishes to remove enzyme solution.- Remove excess moisture, and transfer the sample to another petri dish for infection.- Spin down the culture fluid at 3000rpm, for 10 min at 25C. Remove supernatant.- Resuspend the pellet with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol, then spin down the culture fluid at 3000rpm, for 10 min at 25C. Remove supernatant. Repeat this step 3 times. (This is done to remove the toxic substances that are produced by *E. coli* and adjust the osmotic pressure to that in onion cells)- Resuspend the pellet with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol, Tetracycline, chloramphenicol and L-arabinose.- Measure the light absorbance at 600 nm using a spectrophotometer, then adjust the concentration of cells to ΔOD600 = 0.06 by diluting with the same medium mentioned above.- To infect onion cells with *E. coli* add 500 ul of the culture fluid onto pre-treated onion cell-sheets. Leave at RT in petri dish (Prevent drying)- Remove the bacterial culture fluid carefully by micropipette so that the cell-sheets are not to be torn. Observe the cells under a fluorescencent microscope.

## Week 41

### Remaking protoplast and preparing bacteria– 10/10/18

We needed to remake protoplast in 5,0 pH and induce older bacteria samples to mix and check for secretion.

*Victoria*

Collecting protoplasts made 09/10/18

1. Identify which well contains protoplasts for onion and tobacco samples with a microscope- Add 2 ml sample to an eppendorf tube- Spin down samples at low tempo for 3 min- Remove supernatant- Flick tubes to mix- Check new samples for protoplast using a microscope- Steps 1-6 were repeated 2x, with batch number 1 containing the most protoplasts

#### Inducing bacteria

Samples

mCherry: I-S-C-, I+S+C+

GFP: I-S-C-, I+S+C+

EspD:

1. bacteria (older sample from the fridge, since no new overnight culture was started beforehand) was spun down and pellet resuspended in 5 ml fresh LB+CaM media- 400 microliter resuspended culture added to 10 ml LB+CaM + 500 microliter 20 % Arabinose + 2mM IPTG to induce bacteria- Incubation
       1. 1st batch start: 12:53- 2nd batch start: 13:20

#### Remaking onion and tobacco protoplasts at pH 5,0

Prepared 500 ml of MgM-MES buffer pH5.0 using the following recipe:

***MgM-MES buffer pH5.0:***

*170mM MES: 19,66 g*

*5mM KCl: 0,19 g*

*75mM (NH4)2SO4: 4,96 g*

*0.5mM K2SO4: 0,044 g*

*8uM MgCl2: 0,068 g*

*38mM Glycerol: 1,4 ml*

*0.1% Casaminoacids: 0,5 g*

1. pH was adjusted with 5,0 M KOH- Dilute buffer to 500 ml- 0,8 M and 0,6 M Mannitole buffer was prepared (250 ml 0,6 M Mannitole = 27,5, 250 ml 0,6 M Mannitole = 36,5 g )- Peel inner epidermis from onion and tobacco- Leave in Mannitole buffer for min 4. hours- Mix bacteria and protoplasts- Incubate overnight

## Week 41

### Washing protoplasts to remove bacteria – 11/10/18

We needed to separate bacteria from protoplasts to use a fluorescent microscope to check for secretion into protoplasts. It turned out washing by spinning down protoplasts, removing supernatant containing bacteria and resuspending pellet was an inefficient way to do this.

*Victoria*

Note: Only the onion protoplast samples were used

1. Mix samples gently by flicking- Centrifuge for 3 min at 150 RCF (or until pellet appear)- Remove supernatant- Resuspend pellet in 200 microliter 0,8 M mannitole buffer
         1. Check samples in microrscope to ensure protoplast are stable and kept in sample- All samples were washed 6 times by following step 1-4
              1. Note: This wasn't sufficient to remove bacteria entirely- Note: Protoplast seemed to shrink during the time of the experiment- Check samples in fluorescent microscope

### Using Lyzozyme to remove bacteria – 11/10/18

We wanted to see if digesting the bacteria would be more efficient than washing the samples. This experiment showed that separating protoplast from bacteria was innificient.

Note: This experiment used the tobacco samples left over from the day before

*Victoria*

1. Spin tobacco 3 min 150 RCF- Remove supernatant- Add 200 microliter of lyzozyme solution to samples
       1. Lyzozyme solution: 10 g Lysozyme from chicken egg white,1 ml Mannitole buffer- Check samples every 30 min in microscope
            1. Note: Bacteria did not get removed

# Leakiness characterization

# Week 36

## Overnight of strains (1) – 06/09/18

An overnight of the following strains was made:

1: I+, S+, C+ mCherry

2: I+, S+, C- mCherry

3: I-, S+, C+ mCherry

4: I-, S-, C- mCherry

5: I-, S+, C- mCherry

6: I+, S-, C+ mCherry

7: I+, S+, C+ beta-lactamase (TEM1) (negative control for mCherry fluorescent measurement).

#### Nat

## Induction of strains + sample taking over time (1) – 07/09/18

The first experiment in relation to the examination of the injectisome leakiness was conducted with the strains from the overnight. We followed this [protocol](https://static.igem.org/mediawiki/2018/1/14/T--UCopenhagen--LeakProt.jpeg) (protocol 1).

The bacteria were induced with IPTG and arabinose and a sample was taking at T=0 for each strain. Thereafter a sample was taken every 2 hours. The samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

#### Attila, Nat and Lasse

## Induction of strains + sample taking over time (1) – 08/09/18

The last sample of this experiment, T=overnight, was taken for each strain. The samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

#### Attila, Nat and Lasse

# Week 37

## Overnight of strains (2) – 13/09/18

An overnight of the following strains was made:

1: I+, S+, C+ mCherry

2: I+, S+, C- mCherry

3: I-, S+, C+ mCherry

4: I-, S-, C- mCherry

5: I-, S+, C- mCherry

6: I+, S-, C+ mCherry

7: I+, S+, C+ beta-lactamase (TEM1) (negative control for mCherry fluorescent measurement).

#### Nat

## Induction of strains + sample taking over time (2) – 14/09/18

The second experiment in relation to the examination of the injectisome leakiness was conducted with the strains from the overnight. We followed this [protocol](https://static.igem.org/mediawiki/2018/1/14/T--UCopenhagen--LeakProt.jpeg) (protocol 2).

The bacteria were induced with IPTG and arabinose and a sample was taking at T=day0 for each strain. Thereafter the bacteria were placed at 16 oC. The T=day0 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

#### Attila, Nat and Lasse

## Induction of strains + sample taking over time (2) – 15/09/18

A sample, T=day1, was taken from the tubes incubated at 16 CO for each strain. The T=day1 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

#### Attila, Nat and Lasse

## Induction of strains + sample taking over time (2) – 16/09/18

A sample, T=day2, was taken from the tubes incubated at 16 CO for each strain. The T=day2 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

#### Attila, Nat and Lasse

# Week 38

## Induction of strains + sample taking over time (2) – 17/09/18

A sample, T=day3, was taken from the tubes incubated at 16 CO for each strain. The T=day3 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

#### Attila, Nat and Lasse

## Induction of strains + sample taking over time (2) – 18/09/18

A sample, T=day4, was taken from the tubes incubated at 16 CO for each strain. The T=day4 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

#### Attila, Nat and Lasse

## Induction of strains + sample taking over time (2) – 19/09/18

A sample, T=day5, was taken from the tubes incubated at 16 CO for each strain. The T=day5 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

#### Attila, Nat and Lasse

## Induction of strains + sample taking over time (2) – 20/09/18

A sample, T=day6, was taken from the tubes incubated at 16 CO for each strain. The T=day6 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

#### Attila, Nat and Lasse

## Measurement of fluorescence of supernatant and pellet (2) – 21/09/18

The fluorescence was measured of the samples of the supernatant and the pellet, for the experiment where samples were taken once a day for six days. The supernatant was measured directly whereas the pellet was first resuspended and diluted 10-fold using LB+CAM media.

#### Attila and Lasse

# Week 39

## Western blot of the samples (2) - 26/09/18

Western blot was conducted for the samples following this [protocol](https://static.igem.org/mediawiki/2018/c/cd/T--UCopenhagen--WestAndSDS.pdf).

#### Attila and Nat

# Week 42

## Western blot of the samples (1) - 16/10/2018

Western blot was conducted for the samples following this [protocol](https://static.igem.org/mediawiki/2018/c/cd/T--UCopenhagen--WestAndSDS.pdf).

#### Attila, Nat and Lasse

## Western blot of the samples (1) - 17/10/2018

Western blot was conducted for the samples following this [protocol](https://static.igem.org/mediawiki/2018/c/cd/T--UCopenhagen--WestAndSDS.pdf).

#### Attila, Nat and Lasse

# Experiments with supported lipid bilayers

## Week 28

### Learning to make liposomes – 12/07/18

We were introduced to the process of making liposomes by the nice people in Nikos Hatzakis lab, Søren Schmidt-Rasmussen Nielsen and Mette Galsgaard Malle who gave us the [Liposome preparation protocol](https://static.igem.org/mediawiki/2018/6/6d/T--UCopenhagen--Protocol_Liposome_preparation_step_by_step_NH_lab.pdf). We made some tryout liposomes, that were flashed freezed and stored in -20 **°**C.

*Selma, Sofia and Attila*

## Week 34

### Transforming SIEC strains with GFP plasmids – 24/08/18

To visualize our bacteria in the microscope together with a membrane we needed it transformed with a GFP plasmid. We used plasmids from the distribution kit that functioned as Test Device 1 (BBa\_J36400) and positive control (BBa\_I20270) in the InterLab study.

Transformation as described in [Transformation protocol](https://static.igem.org/mediawiki/parts/6/67/IGEM_Registry_-_Transformation_Protocol.pdf) until step 14, the following combinations of strain and plasmid was made:

* SIEC x BBa\_I20270* SIECΔp1 x BBa\_I20270

Plates were made from agar and LB with chloramphenicol (34 μg/mL) (CAM). Plates with bacteria spent 17 hours in the 37 **°**C incubator before being kept in fridge.

*Selma*

## Week 35

### Testing transformed bacteria for fluorescence – 27/08/18

To test if the transformation was successful fluorescence were measured on a plate reader.

Colonies from transformation 24/08/18 was picked into 50 mL falcon tubes with 5 mL LB media (that also contained 1% arabinose). Incubated at 37 **°**C at 130 rpm for 6 hours. Triplicates of each 100 μL of culture was pipetted into wells of a 96 well-plate. Fluorescence and absorbance was measured. LB media was used as blank.

Glycerol stocks were made from the cultures.

*Selma*

### Making lipid film stocks 1 – 29/08/18

In order to make the liposomes for later experiments quickly and to ensure that the liposomes have the same composition, we made lipid film stocks from a lipid mastermix.

First, we weighed about 25 mg of powdered lipids.

|  |  |
| --- | --- |
| **Lipid** | **Mass (mg)** |
| DOPC | 24,8 |
| DOPS | 25,2 |
| SM | 25\* |
| Cholesterol | 25\* |

\*lipids were obtained as powder in this amount from the manufacturer

Lipids were dissolved in 1 mL chloroform.

To properly store the lipids, we used nitrogen flow to exchange air in the glass vials with nitrogen. Because some of the solvent evaporated during preparation due to the nitrogen flow, the lipid film stocks had to be redone 31/08/18.

Second, we preparred biotin stock 2. The biotin stock concentration was 0.5 μg/μL (stock 1). 150 μL of biotin stock 1 was added to 600 μL chloroform to make biotin stock 2 (0.1 μg/μL).

*Selma and Lasse*

### Microscope tryout – 30/08/18

We got an introduction to the microscope and saw our bacteria in the microscope for the first time. We used the confocal microscope in Associate Professor Nikos Hatzakis' laboratory. Introduction to the microscope was kindly performed by Søren Schmidt-Rasmussen.

#### Bacteria incubation

We added 20 μL of glycerol stock from the 27/08/18 to 5 mL LB media with CAM in 50 mL falcon tubes (SIEC and SIECΔp1 with GFP, BBa\_I20270).

Put in incubator at 37**°**C at 130 rpm for 3 hours.

#### Preparation of supported lipid bilayer

Supported lipid bilayer was made as described in the [Moran-Mirabel protocol](https://www.chemistry.mcmaster.ca/moran-mirabal/resources/SUPPORTED-LIPID-BILAYER-PREPARATION-SOP-9-2015.pdf), step 8 to 12. For this first tryout, we used the liposomes made 12/07/18 (not right membrane composition). Microscopy slides were washed with Tris buffer. Membrane contained the dye Atto655.

#### Microscopy

Microscope slides were washed with the same media as used for cultivation of the bacteria. 100 μL bacteria culture was added to a slide well. We used lasers of 488 nm and 635 for excitation. FRAP was used to see if the membrane was intact, which was not the case.

*Selma, Attila, Lasse and Nat*

### Making lipid film stocks 2 – 31/08/18

We wanted to make a mixture containing sphingomyelin (SM), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and cholesterol (Chl) was used for the supported lipid bilayer and liposomes in a ratio of 44:24:12:20 (SM:DOPC:DOPS:Chl).

Chloroform in stocks from 29/08/18 was evaporated with nitrogen flow. Masses of lipids left were weighed. 1 mL solvent was added to each lipid vial to make stock 1.

|  |  |
| --- | --- |
| **Lipid** | **Mass (mg)** |
| DOPC | 25,6\* |
| DOPS | 21 |
| SM | 24,4 |
| Cholesterol | 24,9 |

\*This number is higher than 29/08/18. This means that the concentration for DOPC is not exact.

Dilutions of each lipid stock ( called "Stock 2") were made:

|  |  |  |  |
| --- | --- | --- | --- |
| **Lipid** | **Volume of Stock 1** | **Methanol** | **Chloroform** |
| DOPC | 39 μL\*\* | 0 μL | 960 μL |
| DOPS | 48 μL | 476 μL | 476 μL |
| SM | 41 μL | 480 μL | 480μL |
| Cholesterol | 40 μL | 0 μL | 960 μL |
| Atto655 (0.5 μg/μL) | 20 μL | 0 μL | 980 μL |

\*\*If the mass had corresponded to what was measured 29/08/18 this would have been 40 μL instead of 39 μL.

Three mixtures were made from the stock 2.

|  |  |  |  |
| --- | --- | --- | --- |
| **Lipid, stock 2** | **Mix 1** | **Mix 2** | **Mix 3** |
| DOPC (1μg/μL) | 75 μL | 75 μL | 75 μL |
| DOPS (1 μg/μL) | 39 μL | 39 μL | 39 μL |
| SM (1 μg/μL) | 133 μL | 134 μL | 134 μL |
| Cholesterol (1 μg/μL) | 31 μL | 31 μL | 31 μL |
| Atto655 (0.01 μg/μL) | 27 μL | 27 μL | 0 μL |
| Biotin (0.1 μg/μL) | 60 μL | 0 μL | 0 μL |

Mixtures were aliquoted into glass vials to contain 0.2 μmol lipids:

|  |  |  |  |
| --- | --- | --- | --- |
|  | **Mix 1** | **Mix 2** | **Mix 3** |
| μL mastermix to reach 0,2 μmol | 37 μL/vial | 31 μL/vial | 28 μL/vial |

Vials were kept in -20**°**C.

*Selma*

## Week 36

### Making lipid film stocks 3 – 05/09/18

To finish the lipid film stocks, solvent was evaporated with nitrogen flow for about 5 minutes. Then vials were centrifuged on a ScanVac for 15 minutes under vacuum. Vials containing lipid films were stored at -20 **°**C.

#### Selma

### Streaking bacteria – 05/09/18

Bacteria from glycerol stock 27/08/18 (transformations 24/08/18) were streaked on LB agar plates with CAM (34 μg/mL). Incubated overnight and kept in fridge.

*Selma*

## Week 37

### Full protocol tryout 1 – 12/09/18

We inoculated colonies from plates that were made the 05/09/18 in LB media with arabinose (1%), IPTG (0.1 mM) and CAM (34 μg/mL). The cultures were incubated for 2.5 hours at 37 **°**C 160 rpm. Meanwhile lipid films were rehydrated, extruded through a 200 nm filter and underwent 10 cycles of freezing in nitrogen and heating in a water bath, see [Liposome preparation protocol](https://static.igem.org/mediawiki/2018/6/6d/T--UCopenhagen--Protocol_Liposome_preparation_step_by_step_NH_lab.pdf). Liposomes were used to make a supported lipid bilayer on a microscope slide. The rest of the liposomes were kept in -80 **°**C freezer.

After 2.5 hours of incubation, we measured OD of the bacterial culture (results below) and pipetted 100 uL of bacteria culture onto each slide with bilayer. This was incubated at 37 **°**C in 3 hours.

After incubation, we looked at each slide in the microscope, taking pictures before and after washing with 100 uL media 3 times.

See the full protocol [here](https://static.igem.org/mediawiki/2018/4/4e/T--UCopenhagen--Protocol_SLB_Experiments_iGEM_2018.pdf)

*Sofia and Selma*

# InterLab

Throughout the interlab experiments, we followed the protocol provided by the measurement committee. The protocol can be seen [here](https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf).

In the following notebook we have focused on what we have done and then linked to the interlab page on this wiki. We have chosen not to incorporate the data not used as final result however, we states the number of times we have done the experiments. If you want to read more about the different experiments you can read more here (link to wiki interlab page).

## Week 26

### Calibration 1: LUDOX – 27/06/18

OD600 was measured for the four replicates, and this was performed three times for the LUDOX and one time for ddH2O. The data from one of these replicants were used to the submission as a final result. Description and final results of the experiment can be found here (2.1 LUDOX).

*Selma, Attila, Eirikur and Lasse*

#### Calibration 2: Silica beads (1) – 27/06/18

OD600 was measured for the dilutions of silica beads. The results from this measurement was not used. Description and final results of the experiment can be found here (2.2 Silica beads).

*Selma, Attila, Eirikur and Lasse*

#### Transformation of E. coli DH5 α with the six test devices (1) – 27/06/18

Competent E. coli DH5 α were transformed with six test devices obtained from the distribution kit. The protocol used for the transformation were the one produced by iGEM, see here http://parts.igem.org/Help:Protocols/Transformation. Changes from the protocol were:
- The use of LB media instead of soc in step 10.
These transformants were not used.

*Selma, Attila, Eirikur and Lasse*

#### Transformation of E. coli DH5 α with the six test devices (2) – 28/06/18

Competent E. coli DH5 α were transformed with six test devices obtained from the distribution kit. The protocol used for the transformation were the one produced by iGEM, see here http://parts.igem.org/Help:Protocols/Transformation. Changes from the protocol were:
- The use of LB media instead of soc in step 10.
These transformants were used for the rest of the interlab experiments.

 *Selma, Attila, Eirikur and Lasse*

#### Calibration 3: Fluorescein (1)– 01/07/18

The pH of PBS buffer from common stock was adjusted to pH=7,45.

The fluorescence was measured for the dilutions of fluorescein. The results from this measurement was not used. Description and final results of the experiment can be found here (2.3 Fluorescein).

*Selma, Attila and Eirikur*

#### Calibration 2: Silica beads (2) – 01/07/18

OD600 was measured for the dilutions of silica beads. The results from this measurement was used for submission. Description and final results of the experiment can be found here (2.2 Silica beads).

*Selma, Attila and Eirikur*

#### Cell measurement: Overnight of transformants (1) – 01/07/18

An overnight of the transformants were set over in 5 mL LB + Chloramphenicol (CAM) (1:1000).

*Selma, Attila and Eirikur*

### Week 27

#### Cell measurement: Cell growth, sampling, and assay (1) – 02/07/18

The overnight were diluted, abs600 measured and diluted further to an abs600 og 0,02 in a final volume of 12 mL. The samples were not measured and the experiment was therefore performed again. Description and final results of the experiment can be found here (3 Absorbance and fluorescence of transformed cells).

*Selma, Attila, Eirikur and Lasse*

#### Cell measurement: Overnight of transformants (2) – 02/07/18

An overnight was made from the overnight from the same day (Cell measurement: Overnight of transformants (1) – 01/07/18).

*Selma, Attila, Eirikur and Lasse*

#### Cell measurement: Cell growth, sampling, and assay (2) – 03/07/18

The overnight were diluted, abs600 measured and diluted further to an abs600 og 0,02 in a final volume of 12 mL. Abs600 and fluorescence were measured. The results from this measurement was used for submission. Description and final results of the experiment can be found here (3 Absorbance and fluorescence of transformed cells).

*Selma, Attila, Eirikur and Lasse*

#### CFU per 0,1 OD600: Starting sample preparation and dilution series (1) – 03/07/18

Abs600 were measured on samples from the overnight culture (Overnight of transformants (2)). Triplicates were made after measurement of OD600=0,1. After the dilution series the samples were plated. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD600 E. coli cultures).

*Selma, Attila, Eirikur and Lasse*

#### CFU per 0,1 OD600: CFU/mL/OD calculation (1) – 03/07/18

The plates were counted, but as we figured that the triplicate was not correct, we did not use the result. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD600 E. coli cultures).

*Selma, Attila, Eirikur and Lasse*

#### CFU per 0,1 OD600: Overnight culture (2) – 03/07/18

An overnight culture was made from the transformants.

*Selma, Attila, Eirikur and Lasse*

#### CFU per 0,1 OD600: Starting sample preparation and dilution series (2) – 04/07/18

Abs600 were measured on samples from the overnight culture. Triplicates were made and each of the triplicates were diluted to an OD600=0,1. After the dilution series the samples were plated. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD600 E. coli cultures).

*Attila and Lasse*

#### CFU per 0,1 OD600: CFU/mL/OD calculation (2) – 05/07/18

The plates were counted, but as we were not satisfied with the result, we did not use the result and decided to do it once more. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD600 E. coli cultures).

*Attila and Lasse*

### Week 29

#### CFU per 0,1 OD600: Overnight culture (3) – 16/07/18

An overnight culture was made from the transformants.

*Attila and Lasse*

#### CFU per 0,1 OD600: Starting sample preparation and dilution series (3) – 17/07/18

Abs600 were measured on samples from the overnight culture. Triplicates were made and each of the triplicates were diluted to an OD600=0,1. After the dilution series the samples were plated. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD600 E. coli cultures).

*Attila and Lasse*

#### CFU per 0,1 OD600: CFU/mL/OD calculation (3) – 18/07/18

The plates were counted, but as we were not satisfied with the result, we did not use the result and decided to do it once more. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD600 E. coli cultures).

*Attila and Lasse*

#### CFU per 0,1 OD600: Overnight culture (4) – 18/07/18

An overnight culture was made from the transformants.

*Attila and Lasse*

#### CFU per 0,1 OD600: Starting sample preparation and dilution series (4) – 19/07/18

Abs600 were measured on samples from the overnight culture. Triplicates were made and each of the triplicates were diluted to an OD600=0,1. The calculation for the OD600=0,1 were made based on an average of the measurements of each of the singletons from the triplicate. After the dilution series the samples were plated. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD600 E. coli cultures).

*Attila and Lasse*

#### CFU per 0,1 OD600: CFU/mL/OD calculation (4) – 20/07/18

The plates were counted and The results from this measurement/calculation was used for submission. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD600 E. coli cultures).

*Attila and Lasse*

### Week 30

#### Calibration 3: Fluorescein (2)– 24/07/18

As we figured out that a wrong setting on the plate reader had been used for Calibration 3: Fluorescein (1) we performed the experiment again.

The pH of PBS buffer from common stock was adjusted to pH=7,45.

The fluorescence was measured for the dilutions of fluorescein. The results from this measurement was used for submission. Description and final results of the experiment can be found here (2.3 Fluorescein).

*Attila and Lasse*

# Egg membrane experiment

## Up to week 35

### Egg yolk membrane extractions

* Various attempts to cut the egg yolk and obtain the intact membrane.* Experimenting with "fixation" of the membrane on various surfaces, integrity of membrane, etc...

Note: fresh eggs have stronger membrane

* Learning the "technique" and manufacturing appropriate tools (bend wire) for handling delicate membrane

*Eric & Natt*

## Week 34

#### Manufacturing the chamber and testing the setup – 24-26/08/18

The polystyrene plastic cup (PS) was found to be the most appropriate material for our setup.

* Small holes were made at the bottom of the upper cup with heated nail* Membrane was placed across the holes, left to settle and fixed with wax

The next step was testing the leakiness of the setup. Cups were filled with water up to 2 cm and left for 24h. Upon any sign of leakiness the membrane was discarded.

*Eric*

## Week 35

#### Manufacturing the chamber and testing the setup2 – 1-2/09/18

Upon further testing the simple "gluing" the membrane with universal glue was found to be superior in comparison to "waxing".

* Same testing for leakines was performed with similar amount of water and therefore hydrostatic pressure.* It was found out that letting the membres dry even for 1h causes leakiness upon further testing for leakiness. All leaky membranes were discarded.* 4 membranes that passed all the tests were stored in a plastic box with wet tissue to preserve the humidity and prevent drying of the membranes.

## Week 36

#### Preparing the bacteria – 6/09/18

For preliminary experiments the following strains of SIEC bacteria were used:

(All had chaperones and mCherry as a reporter protein)

|  |  |  |
| --- | --- | --- |
| numbering | Genetic material | Description |
| 1. | I+, S+, C+ | Fully functional strain |
| 2. | I-,S+,C+ | Strain with disabled injectisome |
| 3. | I+,S-,C+ | Strain with injectosome but without secretion signal |

Legend:

* I = injectosome, (+) means functional injectosome, while (-) means the plasmid is lacking promoter in front DNA that in encoding injectisome.* S = secretion signal, a sequence of 20 amino acids which is believed to provide selectivity when it comes to secretion via injectosome.* C = chaperone. In this experiment all strains had chaperone. It is believed that they facilitate unfolding of the protein prior to injecting, however we tried to validate this in some other experiments.

Strains were picked from the araginoze plates and and overnight culture was grown in 10mL of LB media + Chloramphenicol.

* Incubated overnight at 28°C since the other incubator was broken (for 37°C)

#### Incubation– 7/09/18

After measuring OD600 of overnight cultures the calculated amount of arabinose and IPTG was added to get the final concentration of 1% (w/w%) for arabinose and 1mM for IPTG. After incubating for 2h at 28°C (the 37°C incubator was still out of function) the bacteria solution was poured over membrane (without centrifugation) so that the upper chamber was filled approximately to the half (5mL of culture).

In lower chamber LB was poured (approx 3mL) for easier comparison of fluorescence.

#### Measurements

The samples were taken after 20 and 47 hours and compared with LB media serving as a blank.

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# Team:UCopenhagen/Experiments

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# Experiments

This is the page where we describe our different experiments.

* [Cloning](#tabs-1)
* [Experiments with biomimetic membranes](#tabs-3)
* [Protoplast experiments with plants](#tabs-4)
* [Leakiness characterization](#tabs-5)
* [Egg membrane experiment](#tabs-6)

# Cloning of expression constructs

# 1. Introduction

Our experiments were based on SIEC and SIECΔp1 bacteria transformed with plasmids encoding reporter proteins, Map20 and Chaperones (read more about the different components [here](https://2018.igem.org/Team%3AUCopenhagen/Gene_Construct_Design)). We used both USER cloning and restriction-ligation to assemble the expression plasmids. Protocols used for molecular cloning can be found [here](https://2018.igem.org/Team%3AUCopenhagen/Protocols).

# 2. Cloning strategies

The primers, that we used throughout the cloning process can be viewed in the bottom of this page.

The reporter genes were PCR amplified in two rounds. First to add a C-terminal 6x-His tag and secondly to add restriction sites. XmaI and PstI restriction sites were added to the reporters that were to be assembled with both Map20 the vector and XbaI and PstI restriction sites were added reporter genes that were inserted into the vector without Map20. The reporter genes were then inserted into a BBa\_K228005 backbone with or without chaperones.

The Map20 part sequence was constructed using five different primers and assembled together in a PCR reaction. Restrictions sites for XbaI and AgeI were also added by PCR. This method gave a successful result in first try.

A vector backbone with chaperones genes was also constructed. We used USER cloning to insert the CesT and CesF chaperone genes into the *pBAD* vector as can be seen **figure 1.**

![](https://static.igem.org/mediawiki/2018/d/dc/T--UCopenhagen--vectormap5.jpeg)

**Figure 1.** Assembly of the *pBAD* vector with chaperones. Both the chaperone (CesF and CesT) genes and vector backbone (BBa\_K228005) are first PCR amplified with uracil primers to allow for USER cloning. The figure is made using SnapGene.

The strategy used to assemble expression plasmids with the T3SS signal sequence Map20 can be seen on **figure 2**. The same approach was used for insertion into the vector with and without chaperone genes. We used 3A assembly to insert the reporter genes with Map20 signal downstream of the pBAD promoter in the vectors. The reporter genes are digested with XmaI leaving overhang complementary that of the Map20 gene digested with AgeI. This way Map20 is inserted upstream of the reporter. Additionally, the reporter is cut with PstI and Map20 with XbaI allowing them to be assembled with the vector backbone which is digested with SpeI and PstI.

The same strategy was used to assemble the expression plasmids with reporter without Map20 signal sequence. This time both the vector backbone and the reporter protein sequence were digested with XbaI and PstI.

![](https://static.igem.org/mediawiki/2018/e/e1/T--UCopenhagen--vectormap6.jpeg)

**A**

![](https://static.igem.org/mediawiki/2018/8/8e/T--UCopenhagen--vectormap4.jpeg)

**B**

**Figure 2.** Outline of the 3A cloning strategy used to assemble the expression plasmid with reporter and T3SS signal sequence. Plasmid backbone is either without (**A**) or with chaperones (**B**). The figure is made using SnapGene.

The expression plasmid with the EspD translocon protein was assembled with USER cloning as well. USER overhangs were added to the EspD genes and the BBA\_K731500 vector by PCR. EspD genes and vector backbone were mixed and treated with USER™ enzyme to create single stranded overhangs on both the gene and vector. The overhangs allow for hybridization to form a circular construct. The assembly method of the EspD expression plasmid can be seen in **figure 3**.

![](https://static.igem.org/mediawiki/2018/b/b7/T--UCopenhagen--vectormap2.jpeg)

**Figure 3.** Outline of USER cloning strategy used to assemble the expression plasmid with EspD translocon. The figure was made using SnapGene.

# 3. Primers

The following primers were used for cloning.

**Map20-signal synthetic primers**

Map20-1 5' CTAAAGAGGAGAAATACTAGATGTTTAGTCCAATGACAATGGTAGGTC 3'
Map20-2 5' AGTCCAATGACAATGGTAGGTCGTTCGTTAGCTCAGGCGGCTACACA 3'
Map20-3 5' ACCTGAGCCAGAACCACCAAGAGTTTGTGTAGCCGCCTGAGCTAAC 3'
Map20-F2 5' GGCCGCTTCTAGAGAATCTAAAGAGGAGAAATACTAGATGTTTAG 3'
Map20-R2 5' AATCACTTAACCGGTACCAGAACCTGAGCCAGAACCACCAAG 3'

**Primers for pBAD Vector**

araC-UF 5' AGATCACTACUAGAGCCAGGCATCAAATAAA 3'
araC-UR 5' ACTTTCCTGUGTGACTCTAGTTATTAAGCTACTAAAGCGTAGT 3'
Primers for CesT/CesF chaperone
CesF-UF 5' ACAGGAAAGUACTAGATGAATGAACAA 3'
CesT-UR 5' AGTAGTGATCUACACTAGCACTATTTATTAATTG 3'

**Sequencing primers for pBAD (BBA\_K228005)**

AraC-SF 5' GACGATCAACTCTATTTCTCGCGAG 3'
rrnB-SR 5' AGTGTGACTCTAGTAGAGAGCGTTC 3'
CmR-SF 5' CATGATGAACCTGAATCGCCAG 3'
Ori-SF 5' 5' CGCTTTCTCATAGCTCACGCTGTA 3'
Ori-SR 5' GTTGGACTCAAGACGATAGTTACCG 3'
BBP-SF 5' CTGGAATTCGCGGCCGCTTCTAGA 3'
BBS-SR 5' CGGACTGCAGCGGCCGCTACTAGTA 3'

**Primers for Reporter genes**

NLuc-F 5' AGTAGACCCGGGATGGCCGGCGTGTTCACCCTCG 3'
NLuc-R 5' GTGATGGTGGCTACCGCTACCGGTAGCAAGGATTCTCTCG 3'
mCh-F 5' AGTAGACCCGGGATGGTGAGCAAGGGCGAGGAG 3'
mCh-R 5' GTGATGGTGGCTACCGCTACCCTTGTACAGCTCGTCCATGC 3'
GFP-F2 5' AGTAGACCCGGGATGCGTAAAGGCGAAGAGCTGT 3'
GFP-R 5' GTGATGGTGGCTACCGCTACCTTTGTACAGTTCATCCATACCATG 3'
Blac-F 5' AGTAGACCCGGGCACCCAGAAACGCTGGTGAAAG 3'
Blac-R 5' GTGATGGTGGCTACCGCTACCCCAATGCTTAATCAGTGAGGCAC 3'
GS-His-R 5' ACTGACCTGCAGCTAGTGATGATGGTGATGGTGGCTACCGCTACC 3'

**Primers for EspD translocon expression vectors**

EspD-UF 5' AGAGGAGAAA**U**ACTAGATGCTTAATGTAAATAACGATATCCA 3'
EspD-UR 5' ATGGTGATGG**U**GAGATCCTGAGCCAACTCGACCGCTGACAATACG 3'
LacO-UF 5' ACCATCACCA**U**CATCACTAATAGTACTAGTAGCGGCCGCTGCAGTC 3'
LacO-UR 5' ATTTCTCCTC**U**TTCTCTAGTATTTCATGAGGGAATTGTTATCCG 3'

**Primers to identify SIEC and SIEC∆P1 strain**

pTac-F 5' TTGACAATTAATCATCGGCTCGTATAATG 3'
Ler-F 5' AACTCATCGAAAGGTGTTTACTACCG 3'
CesAB-R 5' TTCTTGTTTGGCTCACAATACTCATCC 3'

**Primers for reporter genes without signal**

mCherry 5’ GCT TCT AGA GAA TCT AAA GAG GAG AAA TAC TAG ATG GTG AGC AAG GGC GA 3’
GFP 5’ GCT TCT AGA GAA TCT AAA GAG GAG AAA TAC TAG ATG CGT AAA GGC GAA GA 3’
Beta-lactamase 5’ GCT TCT AGA GAA TCT AAA GAG GAG AAA TAC TAG ATG GCC GGC GTG TTC AC 3’
NanoLuc 5’ GCT TCT AGA GAA TCT AAA GAG GAG AAA TAC TAG ATG CAC CCA GAA ACG CTG 3’

# 5. Conclusion

To verify successful cloning we did either colony PCR or sequencing, revealing that we managed to make 14 out of the 16 possible constructs seen in the table below.

|  |  |  |  |
| --- | --- | --- | --- |
| Plasmid constructed | Reporter | N-terminal Map20 | CesT and CesF |
| + | sfGFP | + | + |
| + | sfGFP | + | - |
| + | sfGFP | - | + |
| + | sfGFP | - | - |
| + | mCherry | + | + |
| + | mCherry | + | - |
| + | mCherry | - | + |
| + | mCherry | - | - |
| + | nanoluc | + | + |
| + | nanoluc | + | - |
| - | nanoluc | - | - |
| - | nanoluc | - | + |
| + | β-lactamase | + | + |
| + | β-lactamase | + | - |
| + | β-lactamase | - | - |
| + | β-lactamase | - | + |

# Experiments with biomimetic membranes

# 1. Introduction

The membrane is an important component of our protein production system as it separates the bacterial culture from the protein collection chamber. In the final protein production chamber the membrane would be an artificial biomimetic membrane.

In this experiment, we investigated the use of lipid membranes using supported lipid bilayers and liposomes.

# 2. Aim

The first aim of this part of our project was to investigate if the SIEC strain (i.e. the *E. coli* strain expressing the injectisome) would bind to a biomimetic membrane and secrete
proteins through it.

The second aim was to investigate if the secretion signal Map20 (BBa\_K2871000) would increase the amount of protein secreted into liposomes.

# 3. Membrane composition

A mixture containing sphingomyelin (SM), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and cholesterol (Chl) was used for the supported lipid bilayer and liposomes in a ratio of 44:24:12:20 (SM:DOPC:DOPS:Chl). The ratio of the membrane components was inspired from Chatterjee and colleagues [1]who showed that EspD, which is required for formation of pores in the host membrane, spontaneously bound to lipid vesicles with this lipid composition. This lipid composition is also similar to the composition of the outer leaflet of a mammalian cell plasma membrane [2].

The dye Atto655 was also included in some of the lipid mixtures to enable detection of the lipid bilayers and liposome by fluorescence microscopy.

# 4. Binding to supported lipid bilayers

In this experiment, binding of SIEC and SIECΔp1 to supported lipid bilayers were investigated using fluorescence microscopy. The protocols used can be found [here](https://2018.igem.org/Team%3AUCopenhagen/Protocols).The bacterial strains expressed GFP (BBa\_I20270) for visualization in the microscope. We expected that the SIEC strain would bind to the membrane due to expression of the injectisome whereas SIECΔp1 would not bind to the membrane. Microscopy slides covered with supported lipid bilayers with the dye Atto655 were prepared and incubated with bacterial culture. After incubation, the microscopy slides were washed in LB media to remove unbound bacteria. An initial test where SIECΔp1 bacteria expressing GFP were incubated with supported lipid bilayers showed GFP signal after washing the microscopy slide 15 times. The integrity of the supported lipid bilayer was not investigated here, but it could be investigated using fluorescence recovery after photobleaching (FRAP). Due to time limitations, this experiment was not pursued further.

# 5. Injection into liposome experiment

## Outline

The purpose of this experiment is to see if the bacteria with the right genetic constructs is able to inject a specific protein into liposomes that mimic the [injectisomes](https://2018.igem.org/Team%3AUCopenhagen/Background) natural membrane-targets. By varying the presence of the injectisome, specific [chaperones](https://2018.igem.org/Team%3AUCopenhagen/Background) and the [signal peptide](https://2018.igem.org/Team%3AUCopenhagen/Background), the experiment is set up to give information about the effect of these genetic products concerning the secretion into liposomes. Any differences of injection that is observed when varying these components provide information about if the parts are needed for secretion or if the components increase secretion rates.

Bacterial strain abbreviations are **"I"**, **"S"** and **"C"** which corresponds to **"injectisome",** **"signal"** and **"Chaperones (CesT, CesF)"** respectively. For example strain **I+S+C-** have all components present, except the chaperones.

The strains that are used are: **I+S+C+, I+S+C-, I+S-C+, I+S-C-, I-S-C-, I-S+C-, I-S-C+, I-S+C+.** All constructs are available with either mCherry or GFP functioning as a reporter protein.

Separating bacteria, liposomes and possible media soluble reporter proteins from each other in an intact form proved troublesome. Method is still not fully developed. Centrifugation with spin columns containing a 450 µm pore size filter was used to separate bacteria and liposomes. Proteinase K was used as a method to sever the injectisome needle complex, in case of strong attachment to the liposomes, that could prevent separation. Moreover proteinase k would remove residual media soluble reporter proteins, so.

## Experimental overview

Protocols can be found [here.](https://static.igem.org/mediawiki/2018/f/f2/T--UCopenhagen--Protocol_Injection_into_Liposome.pdf)

**For liposome injection experiment 1**

1. Overnight of strains.- Rehydration of previously made and 'dried' lipids to form liposomes.- Recovery of strain cultures in fresh media.- Induction of injectisome and reporter protein.- Incubation of bacteria and liposomes together.- Separation of bacteria and liposomes.- Measure the absorption and fluorescence of the liposomes.- Do western blot of liposomes.

**For liposome injection experiment 2 & 3**

1. Overnight of strains with induction.- Rehydration of previously made and 'dried' lipids to form liposomes.- Incubation of bacteria and liposomes together.- Separation of bacteria and liposomes.- Measure the absorption and fluorescence of the liposomes.- Do western blot of liposomes.

**[1]** Chatterjee, A., Caballero-Franco, C., Bakker, D., Totten, S., Jardim, A. (2015) Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD. J Biol Chem. 290 (42) pp. 25579-25594.

**[2]** Zachowski, A. (1993) Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. Biochem. J. 294, pp. 1-14.

# Protoplast experiment with plants

# Aim

The aim of the experiment was to check, using onion and tobacco protoplasts and *E. Coli* bacteria expressing GFP, Mcherry and the injectisome, if the injectisome(T3SS) was successful in injecting proteins across a membrane.

# Background and Introduction

## The Idea

The inspiration for this project came from the 2011 Hokkaido iGEM team and their "injection assay using onion cells". We wanted to replicate the experiment to show that the Injectisome served its function, a proof of concept of sorts. First we were focused on using onion cells but in the process of the experiment some problems arose which led us to look to other options. We decided on that the other option should be tobacco cells.

## Bacteria and reporter proteins

The *E. coli* strain we wanted the proof of concept for contained the injectisome, secretion signal and a reporter protein(GFP and Mcherry). We wanted to use Mcherry for the assay on the onion and GFP for the assay on the tobacco due to ease of analysis of results in a fluorescent microscope. IPTG induces the formation of the injectisome and arabinose induces the reporter protein.

## Protoplasts

Tobacco protoplasts and onion protoplasts differ quite a bit. Onion cells are large and without any chlorophyll but tobacco cells are small and contain chlorophyll. Protoplasts are in essence plant cells without a cell wall. To make our protoplasts, the cells with an intact cell wall, have to be incubated with a solution with cell wall degrading enzymes, an optimal osmotic pressure and PH value, so the cells don't shrink and/or burst. The optimization of the production and handling of the protoplasts was one of the biggest barriers to overcome, during the experimental process.

The reason for making the protoplast in the first place is that a plant cell with an intact cell wall might pose a problem to the attachment of the injectisome to the cell and the injection of the protein into the cell.

![plant](https://static.igem.org/mediawiki/2018/thumb/3/34/T--UCopenhagen--plant1.jpeg/800px-T--UCopenhagen--plant1.jpeg)

**Figure 1** showing experimental overview

# Methods, process and results

## Intro

We used the protocol from the 2011 Hokkaido iGEM team as a start point and tried to follow their protocol as best as possible. When that didn't give useful results other than burst cells, flabs of apparent onion remnants and fluorescent bacteria floating about, we had to optimise several aspects of the protocol to our needs.

## Protoplast production and preperation

After trying effortlessly attaching onion cell sheets with agarose to a slide glass and incubating with an MgM-MES buffer(ph 5) with 0.4M Sorbitol and cell wall degrading enzymes to obtain protoplasts, we got the idea of making tobacco protoplasts instead and keep the onion to the side.

We then tried incubating a few pieces of a tobacco leafs (downside-down without their cuticle) in a culture plate with the same solution mentioned above. Looking at a sample of the solution after a few hours of incubation revealed burst protoplasts and other plant materials. the production of onion protoplast was tried again on glass slides and revealed no apparent degradation of the cell wall, it looked like the cells had shrunk.

After mostly getting burst or shrunk cells or protoplasts we decided to look at the osmolarity. It was decided to try out different concentrations of Mannitol in an MgM-MES buffer of PH 5. This time the onion cell sheets were treated the same way as the tobacco cells. both were put in a cell wall degrading enzyme solution in an MgM-MES buffer with different concentrations of Mannitol in a culture plate. We observed the solutions in a microscope after incubating the onion and tobacco cells for a couple of hours. This time we successfully produced protoplasts, both onion and tobacco. We decided from quantitative observations in the microscope to use 0.8M Mannitol for the onion cell sheets and 0.6M Mannitol for the tobacco leaves.

![plant](https://static.igem.org/mediawiki/2018/0/06/T--UCopenhagen--onion.jpeg)

**Figure 2**  showing experimental setup of culture plates with plant samples and enzyme buffer

## Bacteria preperation

We mostly followed the Hokkaido 2011 team's protocol in preparation of our bacteria. We incubated them overnight and induced them with IPTG and arabinose. the next day the bacteria were washed and resuspended, in the solution mentioned above(The solution differs in Osmolarity between onion cells and tobacco cells). To check that our bacteria actually emit fluorescence we looked at them in a fluorescence microscope. In the microscope we saw clearly that our strains expressing Mcherry and GFP without a signal sequence gave of clear and bright fluorescence but our strains which contained the signal sequence showed much weaker fluorescence.

# Leakiness characterization

## 1. Aim

The aim of this experiment was to examine if injectisome (T3SS)-bearing bacteria secrete proteins into the surrounding media without interaction with any target membrane. In other words we wanted to test if the *E. coli*  expressing the injectisome was leaky and whether the leakiness was affected by presence of the reporter signal and/or co-expression of chaperones. In order to do this we needed to ensure that the signal tagged reporter protein was expressed by bacteria.

## 2. Introduction

The principle of the experiment is to measure fluorescence of the media in which injectisome expressing *E. coli* have been grown for different time periods (0, 3 and 6 days) at 16 ℃. The experiment was conducted to investigate whether the injectisome leaks and quantify the degree of leakiness

## 3. Method

We used the plate reader SpectraMax M5 to measure the flourescence of the supernatant after different time periods. We also did Western Blots in order to detect presence of unfolded protein (that wouldn't be detected by the flourescence).

## Experimental overview

1) Grow bacteria

2) Induce the bacteria with IPTG and arabinose to get expression of the injectisome and reporter protein

4) Take samples at different time intervals

5) Measure fluorescence of the media (supernatant) and pellet

6) Perform a western blot to detect non-folded proteins

Strains

For the experiment a variety of different strains were used. We have use "I", "S", "C" to describe the different strains.

**'I+/-'** corresponds to presence or absence **injectisome** (control strain vs. strain that is induced to express the injectisome).

**'C+/-'** corresponds to whether the **chaperone** (the CesF-CesT chaperone cassette) is present or not

**'S+/-'** corresponds to presence or absence of T3SS protein **signal** tag ( in our experiment the N-terminal Map20).

For example strain **I+S+C-** have all components present, except the chaperones.

**IPTG** is used to induce the expression of the injectisome.

**Arabinose** is used toinduce the expression of reporter proteins (e.g. mCherry or GFP)

In our experiment we examined the following strains:

![](https://static.igem.org/mediawiki/2018/3/35/T--UCopenhagen--Stains.jpeg)

**Figure 1**  Strains used in leakiness

## Introduction: Egg membrane experiment

The final product of PharMARSy would be a device that allows for growth of bacteria in one chamber and secretion of pure protein into another. Chambers would be divided by a biomimetic membrane, which should allow for bacteria to "dock"on it and use injectosome for secretion, yet it should also be robust to prevent bacteria from crossing across to other chamber. Having bacteria residues or living bacteria in collection chamber would be very dangerous for patient (could result in sepsis) if no additional purification steps are implemented.

It is thus quite evident that membrane is probably one of the most important component of our final product.

However as we couldn't obtain satisfactory biomimetic membrane we were forced to improvise and look into what nature could offer us. One of the possibilities was to use egg yolk membrane - the biggest membrane we could obtain and robust enough for experimenting.

![Schematic of device](https://static.igem.org/mediawiki/2018/thumb/0/0c/T--UCopenhagen--EY1.jpeg/799px-T--UCopenhagen--EY1.jpeg)

**Figure 1**: Schematic of the device on which membrane was tested.

## Experiment: Hypothesis and setup

The goal was to prove that only the secreted proteins can pass the membrane and not the bacteria. We believed that bacteria would recognize the phospholipid bilayer characteristic for eukaryotic cells and secrete the protein via injectosome.

Detection of increase in fluorescence from samples taken from collection chamber would mean a successful demonstration of our idea. However the solution in collection chamber has to remain sterile, otherwise we would get false positive result. (i.e. bacteria could pass to collection chamber and start producing protein which increases the fluorescence).

The following illustration provides the somewhat simpler explanation of the setup of the experiment .

![Description of the experiment](https://static.igem.org/mediawiki/2018/thumb/1/19/T--UCopenhagen--EY2.jpeg/800px-T--UCopenhagen--EY2.jpeg)

**Figure 2**: Description of the experiment. In time intervals a sample was taken from the collection chamber. Fluorescence was measured to detect if protein was being secreted (mCherry), while plating was performed to check whether bacteria crossed the membrane. Since our strain was resistant to Chloramphenicol, we used agarose plates with added Chloramphenicol. This way only bacterial strains with resistance to this antibiotic could grow on the plates. In order to prove that the colonies on the plate were indeed the strain we were working with arabinose was added since it induces production of mCherry protein in our strain. If colonies turned red then we can safely assume that the bacteria has somehow breached the membrane and that detected increase in fluorescence in collection chamber was indeed a false positive result.

### Technical details

The following experiment has probably not yet been done. The experiment therefore required a great deal of improvisation, flexibility and imagination, not mentioning the number of trials before optimizing the protocols.

### Strains used

For preliminary experiments the following strains of SIEC (*Synthetic injector E. coli)* bacteria were used:

All had chaperone and mCherry as a reporter protein.

Legend:

* I = injectisome, (+) means functional injectisome, while (-) means the plasmid is lacking promoter in front DNA that in encoding injectisome.* S = secretion signal, a sequence of 20 amino acids which is believed to provide selectivity when it comes to secretion via injectosome.* C = chaperone. In this experiment all strains had chaperone. It is believed that they facilitate unfolding of the protein prior to injecting, however we tried to validate this in some other experiments.

|  |
| --- |
| numbering |

 Genetic material |

| 1. |

 I+, S+, C+ || 2. | I-,S+,C+ |
| 3. | I+,S-,C+ |

### Egg yolk extraction

In contrary to popular belief - it is actually possible to extract the membrane separating yolk from the white. However, some practice is needed beforehand and a specialized tools which are not part of the standard laboratory inventory.

First step is to separate the white of the egg, which can be done by simply cracking the egg and carefully pouring the white of the egg out. Once the intact yolk is obtained it should be submerged into water to allow for easier manipulation afterwards.

Next, the membrane should be carefully cut - there are many ways that this can be done, but the best results are usually obtained with sharp scissors and a pincet. First a hole should be made into yolk carefully to serve as a starting point for cutting. The hole should be ideally on the top of the yolk to prevent excessive spilling which quickly clouds the water and makes any further operations much more difficult.

Starting from this hole a line can be cut in any direction. The easiest way seems to be by making (almost) 180° circle cut from the side and allowing for yolk to "open" and spill to the opposite side. It is worth noting that movements should be relatively slow and gentle, since we don't want to spill the yolk and cloud the water before finishing the cutting.

Once the desired cut is made the yolk will spill out. Careful exchange of water either with syringe or any other method will gradually return the visibility in the media to initial levels. The purification should be done gently, though, as membrane may very well rupture or be flushed out with exiting water. It is advisable to hold membrane gently with pincers where membrane is the strongest i.e. on the "poles" or on "chalaza", the white membrane structure on the "poles" of the egg yolk. Chalaza can be seen on the figure 3.

![A submerged egg yolk](https://static.igem.org/mediawiki/2018/thumb/1/1a/T--UCopenhagen--EY3.jpeg/450px-T--UCopenhagen--EY3.jpeg)

**Figure 3:** A submerged egg yolk with visible white membrane structure (chalaza).

After some purification relatively pure membranes can be obtained. White like sheet can be manipulated using pincers or any similarly shaped tool with no sharp edges.

The membrane should be cut to appropriate size (smaller is better).

![Purified membrane in petri dish](https://static.igem.org/mediawiki/2018/thumb/3/35/T--UCopenhagen--EY4.jpeg/450px-T--UCopenhagen--EY4.jpeg )

**Figure 4**: Purified membrane in a petri dish.

### "Fixation" of the membrane

After several attempts a glue was found to be a better alternative to wax. The membrane had to be held tightly against the bottom of the chamber covering the holes, so that no liquid can pass between the membrane and the chamber bottom. Since any mechanical pressure system i.e. with rubber bands pressing against the membrane would inevitably result in its penetration, a glue was used instead. Dropping melted wax seemed to be a better solution at first, since it allowed for a very fine manipulation, but after it hardened it tended to separate from the base of the chamber thus destroying its purpose. The following figure (figure 5) illustrates the crude design of the chamber:

![Schematics of the hardware](https://static.igem.org/mediawiki/2018/thumb/3/38/T--UCopenhagen--EY5.jpeg/800px-T--UCopenhagen--EY5.jpeg)

**Figure 5**: A schematics of the "hardware". Into plastic cups the holes were made, which were later covered with membrane.

### Tests for quality - leakiness

Each membrane was tested for leakines. Some water was placed in the chamber and a paper tissue was placed below. Upon any signs of water on the tissue the chamber was discarded.

### Final setup

In the final setup two chambers were separated by glued egg yolk membrane with different orientations. Initially the outer side of the membrane (that was originally facing the white of the egg) was facing the medium with bacteria.

The overnight culture of needed strain was incubated in 1% arabinose (w/w%) and 1mM IPTG for 2 hours prior to being poured over membrane (In next experiments this time was increased to an overnight incubation). Approximately 1 mL of overnight culture was poured into upper chamber while in collection chamber there was LB media (3-4mL) with antibiotic Chloramphenicol. Previous experiments characterizing the pBAD arabinose promoter suggested that 1% arabinose was optimal for our setup.

![Improvisation of experiment](https://static.igem.org/mediawiki/2018/thumb/0/07/T--UCopenhagen--EY6.jpeg/450px-T--UCopenhagen--EY6.jpeg)

**Figure 6**: As can be seen from the pictures improvisation was an important part of the experiment. Two plastic cups were cut and on the upper one holes were made with heated needle. After gluing the egg yolk membrane and testing for leakage, the setup was placed on the petri dish to minimize further contamination. In the presented picture the violet red colour comes from the mCherry fluorescent protein that was produced by strain having functional injectosome and chaperone but no secretion signal sequence.

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# Overview of results for the different experiments

# 1. Leakiness

### Detection of signal peptide-dependent secretion without membrane

To create an E. coli strain with a different combination of injectisome, signal, reporter, and chaperone. The reporter plasmids with and without chaperone cassette were transformed into SIEC E. coli strain which express injectisome, and SIECΔP1 strain which doesn’t have injectisome. The E. coli strains with reporter plasmid were cultured in LB media overnight at 37 C. Reporter protein and injectisome expression was induced by adding 1% L-arabinose and 0.1mM IPTG respectively. The induced cultures were incubated at 16 C with 180 rpm shaking for 6 days. We collected 1 ml of sample from the induced culture every 24 hours and separate cell and supernatant by centrifugation. Cellular fraction were lysed by sonication and separated into soluble and insoluble parts by centrifugation. The supernatants, cellular soluble parts, and cellular insoluble parts from day 0, 3, and 6 were gel electrophoresis on 12% SDS-polyacrylamide gel (SDS-PAGE). The reporter protein was detected western blot with anti-his tag antibody. The constructs tested all contained the reporter protein, being mCherry or ꞵ-lactamase.

## Result

### Map20-mCherry fusion protein is expressed and present in both soluble and insoluble part of E. coli cell.

The SDS-PAGE(Figure 1.1) and Western blot(Figure 1.2) result shows that our mCherry reporter proteins with and without Map20 are present in both cellular soluble and insoluble part which confirm its expression in SIEC and SIECΔP1 E. coli cell. However, the expression level of mCherry with Map20 signal is significantly lower than normal mCherry. This data suggest that N-terminal Map20 signal might decrease translation rate or decrease stability of its fusion protein.

![](https://static.igem.org/mediawiki/parts/7/7b/SDS-PAGE_of_cellular_insoluble_protein_UCPH.png)

**Figure 1.1:**  SDS-PAGE of cellular insoluble protein. M1: Stained ladder, M2: Unstained ladder (UV visible), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones.

![](https://static.igem.org/mediawiki/parts/e/e3/Western_blot_of_cellular_insoluble_protein.png)

**Figure 1.2:**Western blot of cellular insoluble protein. mCherry bands are present in day 3 and day 6 of all groups that use mCherry as a reporter. Bands corresponding to mCherry protein are present in day 3 and day 6 of almost all groups that use mCherry as a reporter. Note that the beta-lactamase does not get a band. M1: Stained ladder, M2: Unstained ladder (UV visible), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones.

![](https://static.igem.org/mediawiki/parts/3/35/SDS-PAGE_of_cellular_soluble_protein.png)

**Figure 1.3:**  SDS-PAGE of cellular soluble protein. In lanes 8/9 and 16/17 an mCherry bands is readily visible as a prominent band, which indicate high expression level. The other lanes show no distinguishable bands for mCherry tagged with Map20. M1: Stained ladder, M2: Unstained ladder (UV visible), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones.

![](https://static.igem.org/mediawiki/parts/4/4a/Western_blot_of_cellular_soluble_protein.png)

**Figure 1.4.a:**  Western blot of cellular soluble protein. mCherry bands are present in day 3 and day 6 of all groups that use mCherry as a reporter. Note that the beta-lactamase does not get a band. M1: Stained ladder, M2: Unstained ladder (UV visible), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones.

![](https://static.igem.org/mediawiki/parts/5/52/Western_blot_of_cellular_soluble_protein_lane1.png)

**Figure 1.4.b:** Western blot of cellular soluble protein. mCherry bands are present in day 3 and day 6 of all groups that use mCherry as a reporter. Note: The shown sample was run on another gel than the rest of the samples from Figure 1.4.a

![](https://static.igem.org/mediawiki/parts/e/ee/SDS-PAGE_of_supernatant.png)

**Figure 1.5:** SDS-PAGE of supernatant with no distinguishable lanes other than identifiable markers M1 and M2.M1: Stained ladder, M2: Unstained ladder (UV visible), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones.
The signal Map20 does not cause the Map20+mCherry to be secreted through the membrane and into the media - The signal does not cause the injectisome to "leak".

![](https://static.igem.org/mediawiki/parts/0/0c/Western_blot_with_anti-his_tag_antibody_of_supernatant.png)

**Figure 1.6:**  Western blot with anti-his tag antibody of supernatant.SDS-PAGE of cellular soluble protein. In lanes 8/9 and 16/17 an mCherry bands is visible as a prominent band, which indicate high expression level. The other lanes show no distinguishable bands for mCherry tagged with Map20. M1: Stained ladder, M2: Unstained ladder (UV visible), D0: Day 0, D3: Day 3, D6: Day 6. I+/-: With/without injectisome, S+/-: With/without signal, C+/-: With/without chaperones. The antibody binds to something unknown from possibly the media, which appears as a faint band. No other bands are visible.

![xxx](https://static.igem.org/mediawiki/2018/1/1b/T--UCopenhagen--leakyness_fluorescence_pellet.png)

**Figure 1.7:**  Graph showing the fluorescence of the bacteria for day 0, 3 and 6 in the leakiness experiment. The two strains without signal peptide (I+S-C+ mCherry and I-S-C- mCherry) shows remarkably stronger fluorescence compared to the other strains. For the other strains with mCherry as reporter protein ( I+,S+,C+ mCherry, I+S+C- mCherry, I-S+C+ mCherry, I-S+C- mCherry) a slight increasement in the fluorescence is seen. As expected, I+S+C+ beta-lactamase shows no fluorescence.

![xxx](https://static.igem.org/mediawiki/2018/9/9d/T--UCopenhagen--leakyness_absorbance_pellet.png)

**Figure 1.8:**  Graph showing the OD600 of the bacteria for day 0, 3 and 6 in the leakiness experiment. A similar growth curve is seen for all of the strains.

# 2. Liposome and biomimetic membrane

## Fluorescence confocal microscope

From the liposome experiment, where bacteria were incubated together with liposomes, we examined samples in a fluorescence confocal microscope. As seen on figure 2.1 and 2.2, we were able to visualize bacteria expressing mCherry and GFP indicating that the signal peptide not completely eliminate expressing and folding of the reporter proteins.

![](https://static.igem.org/mediawiki/parts/a/a8/MCherry_bact_%2B_liposomes.png)

**Figure 2.1:** Bacteria expressing Map20+mCherry visualized in a fluorescence confocal microscope. As seen in the lower left square, fluorescence is seen from the bacteria. The upper right square shows the bright field view with bacteria. Note, no fluorescence is seen in the lower left square (fluorescence from GFP). The results show that the Map20 signal+mCherry reporter protein is fluorescent and doesn't misfold enough to lose fluorescence because of the signal sequence.

![](https://static.igem.org/mediawiki/parts/4/4d/GFP_bact_%2B_liposomes.png)

**Figure 2.2:** Bacteria expressing Map20+GFP visualized in a fluorescence confocal microscope. As seen in the lower left square, fluorescence is seen from the bacteria. The upper right square shows the bright field view with bacteria. Note, no fluorescence is seen in the lower right square (fluorescence from mCherry). Again the results show that the Map20 signal+GFP reporter protein is fluorescent and doesn't misfold enough to lose fluorescence because of the signal sequence.

## Conclusion

Map20-tagged mCherry is expressed in lower amount compared to mCherry without tag. No protein secretion is detected in the supernate, meaning that the Map20 signal does not cause the tagged Map20+mCherry to be secreted into the media without membrane attachment. Figure 2.1 and 2.2 shows fluorescent reporter proteins tagged with the Map20 secretion signal, which indicates that the fusionprotein folds correctly.

# 3. Protoplast experiment with plants

![Protoplast](https://static.igem.org/mediawiki/2018/thumb/a/a2/T--UCopenhagen--onion6.jpeg/180px-T--UCopenhagen--onion6.jpeg)

**Figure 3.1** Onion protoplasts

After getting the production and preparation of the protoplasts and the bacteria right we incubated them together in a solution of 0.8M and 0.6M Mannitol ( for onion and tobacco respectively) in MgM-MES buffer with a PH of 5. samples from the solutions were taken after 2hours, 3 hours and the day after(approx 18hours). To evalute the samples we used a fluorescent microscope. The results seem to be negative or inconclusive.

Samples incubated with the strains that didn’t have the signal sequence showed bright fluorescence(of both GFP and Mcherry) but no apparent secretion into protoplasts, although some cell membranes appeared to have a greater fluorescence than their surroundings, it was too inconclusive for making any qualitative assumptions. Some of the protoplasts appeared to be full of bacteria in which case the protoplasts had probably burst, forming a “bag-like” structureand filled with bacteria.

![Protoplast](https://static.igem.org/mediawiki/2018/thumb/e/e6/T--UCopenhagen--proto.jpeg/800px-T--UCopenhagen--proto.jpeg)

**Figure 3.2 and 3.3** Birght field vs fluorescence filter showing tobacco proplasts forming “bag-lige” structures filled with GFP.

Samples incubated with the strains containing the signal sequence showed a very weak fluorescence(of both GFP and Mcherry) and no clear fluorescence around or inside the protoplasts. We hoped to see some proteins inside the protoplasts and a greater fluorescence around the protoplasts.

We can therefore conclude that the preliminary results from the injection assay using onion and tobacco protoplasts, were inconclusive. It could be that the strains containing the signal sequence attached to the protoplasts and injected protein into them but the fluorescence emitted was to weak to observe it using a fluorescence microscope. it could also be that the signal sequence somehow disrupts the production of the proteins, perhaps during folding. Furthermore, it could simply be due to the fact that the injectisome does not recognize and bind to the membrane of the protoplasts.

# 4. Egg membrane experiment

In the first experiment 2 samples were taken, 20h after filling the chambers and after 47h. The bacterial load of lower chamber solution was tested by plating it on agarose gel plate with added chloramphenicol and arabinose. If the colonies appeared the following day and they were red in appearance (due to mCherry) that meant that the membrane was breached and lower chamber compromised.

As a control, a setup with LB media in both chambers was used.

Unfortunately all plates showed red colonies of different sizes the following days, meaning we got false positive results. Red colour came from produced mCherry protein (after induction with arabinose), since we were working with mCherry-expressing E coli in this experiment.

![Bacteria breaching membrane](https://static.igem.org/mediawiki/2018/thumb/1/1e/T--UCopenhagen--EY7.jpeg/320px-T--UCopenhagen--EY7.jpeg)

**Figure 4.1**: Presence of red violet colour meant that bacteria had indeed breached the membrane.

### Troubleshooting

Few more tests were performed similar to the experiment described above. Increase of fluorescence was always connected with breaching of the membrane, however there were a few experiments where sterility of the collection chamber remained uncompromised, but also no increase in fluorescence was detected.

The strain used in that experiment was I+,S-,C+ meaning that it had the injectisome and chaperone but lacked the secretion signal. It was noted that the overnight culture turned red after incubation in 1% (w/w) arabinose already after 12 hours. However this strain was unable to secrete the protein through membrane - we speculated that this may be due to the lack of secretion signal.

To test this theory the only logical thing to do would have been to try the strain with secretion signal but the strain I+S+C+ didn't show enough fluorescence in overnight culture to even start the experiment. We believed that signal sequence was somehow disrupting the folding process of mCherry protein and therefore tried growing bacteria at lower temperature (28°C instead of 37°C as in the first experiments). Bacteria was allowed to grow for 5 days yet it still didn't produce enough of the fluorescent protein.

The same issue was encountered when trying to repeat the experiment with E coli producing sfGFP (superfolder green fluorescent protein). The strain without secretion signal produced enough fluorescent protein for experiments, yet the one with secretion signal produced protein below detectable levels for fluorescent plate reader. As the strains without secretin signal failed to secrete the fluorescent protein across the membrane and the strains with secretion signal failed to produce enough of it the experiments were eventually discontinued.

### Conclusions

After initial difficulties with the "hardware" i.e. leaky membranes, the manufacturing process was eventually perfected to produce a reasonable number of leak free membrane systems of required quality. The protocols were also eventually optimized at least concerning the incubation time, volumes and similar, but the problem with signal interfering with the folding of the protein remained unsolved.

Nevertheless, had the issue with folding of the protein (with secretion signal) been solved the developed setup would be the best possible option to test whether the whole idea of secreting the proteins across membrane worked. Instead of egg yolk membrane, different substitutes could be used, either using membranes from nature or biomimetic membranes.

An interesting thing to investigate however would be how long can a membrane be exposed to bacteria before being damaged.

# 5. Other

### Map20-mCherry fusion protein expressing E. coli lack visible mCherry colour characteristic

Visual observation of E. coli cell pellet on day 6 shows a difference in color between E. coli that express mCherry and Map20-mCherry fusion protein. The mCherry-expressing E. coli pellet (tube no. 4 and 6) has pink color while Map20-mCherry fusion protein-expressing E. coli pellet (tube no. 1, 2, 3 and 5) has pale white-yellowish color as shown in Figure 3. This observation suggests that Map20 signal might decrease mCherry reporter protein expression or interferes with its folding.

![](https://static.igem.org/mediawiki/parts/c/c8/Visual_observation_of_different_E._coli_strains_after_6_days_of_incubation.png
)

**Figure 1.1** Visual observation of different E. coli strains after 6 days of incubation in 16 ℃

## References

[1] Charpentier, X., Oswald, E. (2004) Identification of the Secretion and Translocation Domain of the Enteropathogenic and Enterohemorrhagic Escherichia coli Effector Cif, Using TEM-1 β-Lactamase as a New Fluorescence-Based Reporter. J Bacteriol, 186(16), pp 5486-5495.