iGEM 2018 — Team Edinburgh_UG

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# Team:Edinburgh UG/Notebook

Edinburgh iGEM 2018

* [Home (current)](https://2018.igem.org/Team%3AEdinburgh_UG)
* Team

  [Team Members](https://2018.igem.org/Team%3AEdinburgh_UG/Team)
  [Collaborations](https://2018.igem.org/Team%3AEdinburgh_UG/Collaborations)
* Project

  [Description](https://2018.igem.org/Team%3AEdinburgh_UG/Description)
  [Design](https://2018.igem.org/Team%3AEdinburgh_UG/Design)
  [Experiments](https://2018.igem.org/Team%3AEdinburgh_UG/Experiments)
  [Notebook](https://2018.igem.org/Team%3AEdinburgh_UG/Notebook)
  [InterLab](https://2018.igem.org/Team%3AEdinburgh_UG/InterLab)
  [Model](https://2018.igem.org/Team%3AEdinburgh_UG/Model)
  [Results](https://2018.igem.org/Team%3AEdinburgh_UG/Results)
  [Demonstrate](https://2018.igem.org/Team%3AEdinburgh_UG/Demonstrate)
  [Improve](https://2018.igem.org/Team%3AEdinburgh_UG/Improve)
  [Attributions](https://2018.igem.org/Team%3AEdinburgh_UG/Attributions)
* Parts

  [Parts Overview](https://2018.igem.org/Team%3AEdinburgh_UG/Parts)
  [Basic Parts](https://2018.igem.org/Team%3AEdinburgh_UG/Basic_Part)
  [Composite Parts](https://2018.igem.org/Team%3AEdinburgh_UG/Composite_Part)
  [Part Collection](https://2018.igem.org/Team%3AEdinburgh_UG/Part_Collection)
* [Safety (current)](https://2018.igem.org/Team%3AEdinburgh_UG/Safety)
* [Human Practices (current)](https://2018.igem.org/Team%3AEdinburgh_UG/Human_Practices)
* Awards

  [Model](https://2018.igem.org/Team%3AEdinburgh_UG/Model)
* [Judging Form (current)](https://igem.org/2018_Judging_Form?team=Edinburgh_UG)
* [Medal Criteria(current)](https://2018.igem.org/Team%3AEdinburgh_UG/Medal_Criteria)

# Notebook

We have uploaded our Lab book as a PDF (below) for you to read.

## Contact EdiGEM18

Feel free to leave us a comment on social media!

* [Twitter](https://twitter.com/EdiGEM2018)
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[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Edinburgh UG/Experiments

Edinburgh iGEM 2018

* [Home (current)](https://2018.igem.org/Team%3AEdinburgh_UG)
* Team

  [Team Members](https://2018.igem.org/Team%3AEdinburgh_UG/Team)
  [Collaborations](https://2018.igem.org/Team%3AEdinburgh_UG/Collaborations)
* Project

  [Description](https://2018.igem.org/Team%3AEdinburgh_UG/Description)
  [Design](https://2018.igem.org/Team%3AEdinburgh_UG/Design)
  [Experiments](https://2018.igem.org/Team%3AEdinburgh_UG/Experiments)
  [Notebook](https://2018.igem.org/Team%3AEdinburgh_UG/Notebook)
  [InterLab](https://2018.igem.org/Team%3AEdinburgh_UG/InterLab)
  [Model](https://2018.igem.org/Team%3AEdinburgh_UG/Model)
  [Results](https://2018.igem.org/Team%3AEdinburgh_UG/Results)
  [Demonstrate](https://2018.igem.org/Team%3AEdinburgh_UG/Demonstrate)
  [Improve](https://2018.igem.org/Team%3AEdinburgh_UG/Improve)
  [Attributions](https://2018.igem.org/Team%3AEdinburgh_UG/Attributions)
* Parts

  [Parts Overview](https://2018.igem.org/Team%3AEdinburgh_UG/Parts)
  [Basic Parts](https://2018.igem.org/Team%3AEdinburgh_UG/Basic_Part)
  [Composite Parts](https://2018.igem.org/Team%3AEdinburgh_UG/Composite_Part)
  [Part Collection](https://2018.igem.org/Team%3AEdinburgh_UG/Part_Collection)
* [Safety (current)](https://2018.igem.org/Team%3AEdinburgh_UG/Safety)
* [Human Practices (current)](https://2018.igem.org/Team%3AEdinburgh_UG/Human_Practices)
* Awards

  [Model](https://2018.igem.org/Team%3AEdinburgh_UG/Model)
* [Judging Form (current)](https://igem.org/2018_Judging_Form?team=Edinburgh_UG)
* [Medal Criteria(current)](https://2018.igem.org/Team%3AEdinburgh_UG/Medal_Criteria)

# Experiments

# General Protocols

## Agarose Gel

1. Weigh 1 g of agarose
2. Add 100 mL of TAE 1X
3. Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is
   completely dissolved
4. While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight
5. When homogeneous, add 10 µL of SYBR SAFE DNA Gel Stain to the solution and mix well
6. Pour the solution into the bed and clear all its bubbles with a pipette tip. Add in the comb and make sure it is secure
7. Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 5 µL of DNA ladder into the first well

## LB Medium (1 litre liquid)

* 10 g tryptone
* 10 g NaCl
* 5 g yeast extract
* Water
* For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)

## LB Medium (1 litre solid / 50 plates)

* 15 g agar agar
* 10 g tryptone
* 10 g NaCl
* 5 g yeast extract
* Water
* For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)

## M9 Medium (1 litre liquid)

* 1% casamino acids
* 0.2% glucose
* 2 mL 1M MgSO4
* 0.1 µg/ml thiamine (B1)
* 0.1 ml 1M CaCl2
* 60 g Na2HPO4
* 30 g KH2PO4
* 5 g NaCl
* 10 g NH4Cl
* Bring to final volume of 1l with distilled water. Autoclave.

## Chemically Competent Cells: CaCl2 Method

1. Inoculate a single colony of appropriate cells into 10ml LB. Add antibiotic if needed and culture o/n at 37°C, 220rpm
2. Inoculate 100ml LB with 1 ml o/n culture
3. Incubate at 37 °C, 220rpm until OD600 = 0.3-0.6 (approx. 2 hrs)
4. Transfer to 2 x 50 ml Falcon and leave on ice for 30 mins
5. Centrifuge at 400 x g, 5 mins, 4 °C
6. Resuspend pellet GENTLY in 25 ml ice cold 0.1 M MgCl2
7. Incubate on ice for 30 min
8. Centrifuge at 4000 xg, 5 min, 4 °C
9. Resuspend pellet GENTLY in 25 ml ice cold 0.1 M CaCl2
10. Incubate on ice for 30 min
11. Centrifuge at 4000 xg, 5 min, 4 °C
12. Resuspend pellet GENTLY in 1.25 ml ice cold CaCl2/Glycerol solution (1.7 ml 0.1 M CaCl2, 0.3 ml 100 % glycerol)
13. Aliquot 100ul and flash freeze on dry ice. Store at – 80 °C

## Chemical Transformation by Heat Shock

1. Place a small glass bottle with sterile SOC (10 ml) in the 37 °C incubator to prewarm. Also prewarm at this point the plates for spreading out the transformation
2. Thaw on ice one eppi (200 µl) of self-made competent Top10 cells per ligation to be transformed (Keep the competent cells as much as possible on ice!)
3. Add the entire ligation (20 µl) to the cells (or 0.1 to 1 µl for plasmids to be re-transformed) and mix by tapping gently. Do not mix cells by pipetting, their membrane is fragile
   and shear force can kill cells at this point!
4. Incubate on ice for 30 minutes (if possible, mix once or twice by gently tapping again)
5. Switch on the water bath immediately after adding the DNA to the cells and set to 42 °C (check temperature with a thermometer, sometimes the setting needs to be adjusted to achieve
   measured 42 °C. In our lab, set to 39.5 °C to achieve 42 °C!)
6. Heat shock the cells by incubating the eppis for 60 seconds in the 42 °C water bath. Do not mix or shake!
7. Remove the eppis from the 42 °C bath and quickly place on ice, incubate for 2 min
8. Add 500 μL of pre-warmed SOC medium (SOC is a rich medium; use proper sterile technique to avoid contamination)
9. Secure the eppis in a small microcentrifuge rack with tape and place in the 37°C shaking incubator for 60- 90 min (the rack should stick down with its short side so that the eppis
   lie parallel to the platform for maximum aeration). Warning: if you label only the lids, the tape will remove the labelling when you strip it off after incubation. Label the side of the Eppendorf and secure
   the label with masking tape!
10. Plate 50- 100 µl of the cells on one selective prewarmed plate. Spin down the remainder of the cells at 3800 rpm for 3 min at RT and pour off the SN until about 50 µl of liquid
    remain in the tube. Resuspend the pellet in this remaining liquid by pipetting up and down with a 200 µl pipette and plate the entire resuspension onto a second plate
11. Wrap the plates in parafilm and incubate upside down at 37°C o.n.

## Overnight Cell Incubation

1. Add 5 ml LB into 50 ml falcon tube
2. Inoculate specific antibiotic
3. Scrape/pick glycerol stock surface/colony and transfer into falcon tube
4. Incubate at 37 °C overnight

## Glycerol Stock Solution

1. Cultures plated on LB Agar + antibiotic and grown at 37°C overnight
2. A 5ml LB culture in LB + antibiotic inoculated from a single, freshly growing colony
3. Cultivate for 16h at 37 °C, 220 rpm
4. 0.5ml of culture inoculated into sterile Eppendorf
5. Add 0.5ml of 80% glycerol
6. Vortex
7. Spin down
8. Freeze at -80 °C

## Plasmid Extraction

1. Plasmid extraction was carried out following QIAprep Spin Miniprep Kit protocol, found here: https://www.qiagen.com/us/resources/download.aspx?id=56b0162c-23b0-473c-9229-
   12e8b5c8d590&lang=en

# Maxicell Protocols

## UV Induced production

For all strains that are not *uvrA-*, all steps during and after UV exposure must be done in the dark. Store cells in a light proof container (i.e. brown flask). Photoreactivation peaks at 384 nm.

Day 1:

1. Start overnight culture in 5 ml culture medium + antibiotic. Incubate at 37 °C, 220 rpm

Day 2:

1. Dilute o.n. culture approximately 1/60 in 60 ml of medium plus appropriate concentration of antibiotic in a 250mL flask. This will yield a 60 mL culture with an OD550 ~0.05
2. Incubate 37 °C with agitation. Take OD550 readings every hour or less and plot a growth curve for future reference.
3. At OD550 = 0.4, transfer 10 mL of the culture into a glass petri dish and irradiate with UV light on a shaking platform. Shaking is essential for even exposure. Time of exposure dependent on strain – a good starting point is to expose strains for 20 seconds, 40 seconds, 60 seconds and 80 seconds. Negative control = 0 min exposure, positive control = 4 min exposure
4. Transfer to a 50 mL dark falcon tube, incubate 37 °C, 220 rpm for 45 minutes
5. Add 200 µg/ml cycloserine (1/500 dilution of stock) and continue gentle shaking 37° incubation overnight. Don’t reuse cycloserine stocks too many times, as it becomes unstable.

Day 3:

1. Centrifuge maxicells 6 mins at 6000x g (7,000 rpm in an SS34)
2. Resuspend the pellet in 5 ml of media + appropriate concentration of antibiotic
3. Centrifuge maxicells 6 mins at 6000x g
4. Resuspend the pellet in 1 ml of media + appropriate concentration of antibiotic
5. Cells can then be frozen in glycerol for future use

## I-SceI Induced production

Using the strain BW27784, with DL name DL2524, acquired from the Leach Lab (MG1655 recA::CmR araB::PBAD-I-SceI lacZ::I-SceIcs)

Day 1:

1. 5 ml overnight culture in LB + 25 μg/ml chloramphenicol + 0.5% glucose, in 50 ml falcon tube, vigorously shaking at 37 °C

Day 2:

1. Measure the OD600 in the morning
2. Dilute the culture approximately 1/60 in LB + 0.2% glucose + 25 μg/ml chloramphenicol (CAM) to OD600 0.025
3. Grow for 80 minutes at 37 °C
4. Measure the OD - should be 0.1. Dilute part of the culture 1/10 in LB + 0.2% glucose + CAM (negative control) and another part in LB + 0.5% arabinose + CAM
5. Incubate 37 °C, 220 rpm O/N

Day 3:

1. Centrifuge maxicells 6 mins at 6000 x g (7,000 rpm in an SS34)
2. Resuspend the pellet in 5 ml of media + appropriate concentration of antibiotic
3. Centrifuge maxicells 6 mins at 6000 x g
4. Resuspend the pellet in 1 ml of media + appropriate concentration of antibiotic
5. Cells can then be frozen in glycerol for future use

## sbcDC Induced Production

Using the strain BW27784, with DL name DL3355, acquired from the Leach Lab (MG1655 lacIq lacZχ- ParaBAD-sbcDC lacZ::pal460 recA::Cm). Follow protocol for I-SceI induced production (above).

## DAPI Staining

If using UV maxicells, do this 15-18 hours post-maxicell-induction. Maxicells cannot be used after DAPI staining so do not stain whole stock.

1. Measure the OD600 - aim for 0.1 – 0.4. Add DAPI. Incubate 15 minutes at 37 °C under agitation.
2. Dissolve agarose in 25 ml distilled water in microwave 1-2 minutes. This can be kept in liquid state at 55 °C.
3. Pipette two 150 μl agarose spots onto the glass slide and immediately place a coverslip on the spots and flatten gently. Leave 10 minutes.
4. Remove coverslips - airdry for at least 5 minutes
5. Transfer 10μl sample onto the agarose spots and place coverslip on top

# SDS PAGE of Proteins in Maxicells

E. coli cells of strain DL2524 were made into maxicells using the UV maxicell protocol 48 hours, 24 hours and 0 hours (see maxicell producing protocol for details) and stored at 37 °C, 25 °C and 4°C (each timepoint had samples stored at each temperature) before being used as samples in the following protocol:

(A Bio-rad brand SDS page electrophoresis system was used for this experiment)

## Mixtures

#### Running Buffer

The following was added to a beaker to make 2x running buffer:

* 6 g of Tris base
* 28.8 g of glycine
* 2 g of SDS
* Added enough distilled water to make it to 1 liter

The buffer was then diluted 1 to 1 buffer to distilled water to make it 1x running buffer.

#### Separating Gel:

First the following was added to a beaker:

* 3.35 ml of Distilled Water
* 2.5 ml of 1.5M Tris pH 8.8
* 0.1 of 10% SDS
* 4 ml of 30% acrylamide

Immediately before transferring into the casting frames/gel mould the following was added to the beaker:

* 50 μl of 10% APS
* 5 μl of TEMED

#### Stacking Gel:

First the following was added to a beaker:

* 6.1 ml of Distilled Water
* 2.5 ml of 0.5 M Tris pH 6.8
* 100 μl of 10% SDS
* 1.3 ml of 30% acrylamide

Immediately before transferring into the casting frames/gel mould the following was added to the beaker:

* 50 μl of 10% APS
* 10 μl of TEMED

#### Sample Buffer

The sample buffer was prepared and was:

* 10% w/v SDS
* 0.2 M Tris-HCl, pH 6.8
* 20% w/v Glycerol
* 10 mM beta-mercapto-ethanol
* 0.05 % w/v Bromophenolblue

## Protocol

1. The SDS electrophoresis system was set up and the glass plates were placed in their clamps.
2. The Running buffer was poured into the buffer tank, enough to submerge the gels which will eventually be placed in the tank.
3. The separating gel was pipetted into the gap between the glass plates of the casting frame and a small amount if isopropanol was pipetted on top to level out the separating gel. The gel was left to set for 20 mins.
4. The isopropanol was discarded from the casting frame leaving only the separating gel. The stacking gel was then pipetted between the glass plates off the casting frame (on top of the separating gel) until it reached the top of the casting frame then a well forming comb was inserted into the top of the casting frame taking care not to trap any air bubbles. The stacking gel was left for 20 mins to set.
5. The O.D. 600 nm of the maxicell samples were taken and the samples were diluted till they all had the same O.D. 600 nm as the lowest sample and the total volume was 500 μl. 500 μl of sample buffer was added to the microfuge tube and then the tube was boiled at 95 – 100 °C for 7 mins.
6. After gelation, the comb was removed and the gel in the casting frame was submerged in the running buffer which was in the buffer tank. A protein ladder was loaded into the left most well and the maxicell protein samples were each loaded into separate wells taking care not to let the sample overflow the well.
7. The lid and anode and cathode were attached to the tank and the gel was left to run for approximately 1 hour at 120 V.
8. Before the gels had over run the voltage was turned off and the gels were removed from the casting frames and placed into separate containers coomassie stain (enough to just cover the gel). The containers containing the gel and coomassie stain were gently agitated for approximately 1 hour (until the protein bands were clearly visible but before the whole gel was stained too much).
9. The gel was destained in coomassie blue destain with several washes and pictures were taken of the gel

# ATP Determination Protocol

To Determine ATP levels over Time and ATP Assay was preformed. The Kit used to preform the assay was bough at Thermofisher. A lot of the protocols used are adapted from their protocols. Only THE Strain DL2524 was used to preform the ATP assay as they were the fastest method for producing Maxicells.

## Making Reagents and Solutions:

#### Preparation of Assay Solution:

Standard assay solution was prepared as as stated in the Thermofisher ATP Determination Kit, 200-1,000 assays kit (Catalogue Number: A22066) with the addition of Triton X-100. The following were used to make the solution:

1. 8.4 mL dH2O (deionised);
2. 0.5 mL 20X Reaction Buffer;
3. 0.1 mL of 0.1 M DTT (see below);
4. 0.5 mL of 10 mM D-luciferin (see below);
5. 2.5 µL of firefly luciferase;
6. 0.5 mL detergent solution (8% Triton X-100, 300 mM potassium phosphate, pH 7.2).

Gently invert to mix, do not vortex; as the firefly luciferase enzyme will denatured. Keep solution protected from light. Solution may be stored at 2–6°C protected from light for several days, assay sensitivity will diminish with time. This ATP assay solution is Mix B

#### Reagent Preparation:

1. Add 50 µL of 20X Reaction Buffer to 950 µL of deionized water (dH2O).
2. To make 1 mL of a 10 mM D-luciferin stock solution,, add adding 1 mL of 1X Reaction Buffer (prepared earlier) to one vial of D-luciferin. Protect from light until use and store ≤–20°C.
3. Prepare a 100 mM DTT stock solution by adding 1.62 mL of dH2O to the bottle containing 25 mg of DTT. Aliquot into ten 160 µL volumes and store frozen at ≤–20°C. Thawed aliquots should be kept on ice or at 4°C until ready for use.
4. Prepare low-concentration ATP standard solutions by diluting the 5 mM ATP solution in dH2O. The concentrations and volumes to make depend upon the sensitivity and design of the luminometer to be used. Range used in Experiment wa from 0.5-3mM of ATP. These dilute solutions are stable for several weeks when stored at ≤–20°C. This is then used to make standard curve and convert fluorescence into mM of ATP.

## Assay Protocol:

1. E. coli cells (DL2524 strain) were grown to the stationary phase in Luria-Bertani (LB) medium containing 2.8% glucose at 37° C for 24 h.
2. After 24h, cells were extracted via centrifugation (6 minutes at 10,000 rpm). Then Induction (or no indcution for control - similar to WT) needs to be made (i.e. cells need to be converted to Maxicells). Therefore, cells were either re suspended in LB + 0.2% Arabinose OR LB + 2.8 % Glucose (Depending if you are testing Maxicells or Non-Maxicells respectively). Cells are then allowed to incubate for an hour.
3. 100µg/mL of Cycloserine is then added to kill live cell in Maxicell samples only. Cell allowed to incubate for another hour.
4. An hour after the cycloserine is added, the time of induction is zero (see below). Measurements were taken at every 2 hour intervals (at times labs were open). Two inductions were made one at the beginning of the day and one at the end of the day to get allow all possible timepoints to be measured (again, see below).
5. 50µL of sample placed in 2mL eppendorf and centrifuged (3 minutes at 4,000rpm), sample then re-suspended in 100 mM Tris-HCl buffer (pH 7.4). Cells were then subsequently washed washed with the same buffer, again.
6. Cell suspension then mixed with an equal volume of pre-treatment solution (40% [w/v] glucose, 0.8% [v/v] Triton X-100) and allowed to sit for 20 minutes at room temperature. This is Mix A.
7. To begin Reaction to measure fluorescence of sample: Add 10 µL of Mix A (sample) to 90 µL of Mix B (Assay solution). Invert eppendorf gently to mix and read Luminescence. Make sure to subtract background luminescence (i.e. luminescence of Mix B - assay solution).

![](https://static.igem.org/mediawiki/2018/6/62/T--Edinburgh_UG--ATP_TIMELINE.png)

Figure 1. Timeline of ATP Assay. Yellow highlight shows time points when samples were taken(see  [Results page](https://2018.igem.org/Team%3AEdinburgh_UG/Results)  for the results of experiment)

NOTE: To make standard curve mix 10 µL of Standard ATP Stock solution (ranging from 0.5-3mM of ATP) with Mix B (assay solution) and read luminescence. Make sure to subtract background luminescence. Plot this to create standard curve of [ATP] (mM) vs Luminescence.

## Protocol for Freezing Maxicells:

DL2524 was induced with arabinose and stored in a freezer (<-20°C) to test the effect of freezing Maxicells after induction. This was done to see how ell these cells can be transported (after induction). The Protocol for Freezing Maxicells is as follows:

1. Prepare an overnight Culture of DL2524 with LB + 2.8% Glucose
2. Next day Spin cells down in centrifuge (6 minutes at 10,000rpm)
3. Cells then Re-suspended in either LB + 0.2% Arabinose for Maxicells OR LB + 2.8% glucose for Non-Maxicells. Cell allowed to incubate for 1 hour.
4. After an hour add 100μg/mL of cycloserine, to Maxicell sample only, to kill any living cells. Leave cells to incubate for an hour.
5. Add equal volume of 50% [v/v] glyerol stock solution to the samples (both Maxicells and Non-Maxicells)
6. Samples then placed in <-20°C freezer.
7. To get range of hours; sample was taken out of freezer at 2 timepoitns during testing which occured 2 weeks after freezing (once at beginning of day (11am) and second at end of the day (5pm) this is to mirror the other ATP assays).
8. ATP assay protocol was followed from step 5 onwards to measure ATP conc. (Sample was stored at room temp in between tests after initial de-thaw).

# Triclosan Protocols

## Minimal Inhibitory Concentration (MIC) Assay for *Escherichia coli* (adapted from [Bio-protocol.org](https://bio-protocol.org/e1308) )

1. Dissolve a single colony of Escherichia coli in 5 ml LB broth and incubate overnight at 37 °C, 220 rpm
2. Make up serial dilution of triclosan of desired concentration range
3. Check OD600 (1 OD600 = 109 CFU/ml) with a spectrophotometer
4. Dilute the bacterial solution with LB broth to get 0.1 OD600 suspension and incubate at 37 °C, 220 rpm till mid-log phase (~2 h)
5. Put 1 ml mid-log phase bacterial solution in 1.5 ml Eppendorf tube, centrifuge at 6,000 rpm for five min, and wash with 1 ml PBS. Repeat the washing procedure twice. Dissolve the
   bacterial pellet with 1 ml LB
6. Get 100 µl the above bacterial solution and mix it with 900 µl PBS, then check OD600 with a spectrophotometer. The bacterial concentration can be deduced from the
   measured value x 10
7. Adjust the bacterial concentration to 1 x 107 CFU/ml with LB (1 OD600 ~109 CFU/ml)
8. In a well of a 96-well plate, mix 10 µl adjust E. coli bacterial solution (1x107 CFU/ml), 170 µl LB and 20 µl of serial diluted triclosan. Repeat for each well for range of
   triclosan concentrations
9. Use 180 µl LB and 20 µl triclosan dilution series for OD600 measurement as a negative control
10. On last row of plate, add 200 µl LB to be used as a blank
11. Incubate at 37 °C, 220rpm in a plate reader, taking OD600 measurements over a period of 20-24 h

## Triclosan Preparation

* Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) was ordered in powdered form from Alfa Aesar
* 58 mg powdered triclosan was dissolved in 500 µl dimethyl sulfoxide (DMSO) to make 400 mM stock solution
* 400 mM stock solution was diluted 1/1000 in DMSO to make 400 µM stock
* Add 125 µl of 400 µM stock to LB to a final volume of 5 ml for overnight culture at 10 µM triclosan
* For 1 µM triclosan agar plates, add 125 µl of 400 µM stock to a 50 ml falcon tub. Pour melted agar into falcon tube, mix and pour into 2 empty
  plates

## Contact EdiGEM18

Feel free to leave us a comment on social media!

* [Twitter](https://twitter.com/EdiGEM2018)
* [Instagram](https://www.instagram.com/igemedinburgh/)
* [Facebook](https://www.facebook.com/EdiGEM2018/?ref=br_rs)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Edinburgh UG/Results

Edinburgh iGEM 2018

* [Home (current)](https://2018.igem.org/Team%3AEdinburgh_UG)
* Team

  [Team Members](https://2018.igem.org/Team%3AEdinburgh_UG/Team)
  [Collaborations](https://2018.igem.org/Team%3AEdinburgh_UG/Collaborations)
* Project

  [Description](https://2018.igem.org/Team%3AEdinburgh_UG/Description)
  [Design](https://2018.igem.org/Team%3AEdinburgh_UG/Design)
  [Experiments](https://2018.igem.org/Team%3AEdinburgh_UG/Experiments)
  [Notebook](https://2018.igem.org/Team%3AEdinburgh_UG/Notebook)
  [InterLab](https://2018.igem.org/Team%3AEdinburgh_UG/InterLab)
  [Model](https://2018.igem.org/Team%3AEdinburgh_UG/Model)
  [Results](https://2018.igem.org/Team%3AEdinburgh_UG/Results)
  [Demonstrate](https://2018.igem.org/Team%3AEdinburgh_UG/Demonstrate)
  [Improve](https://2018.igem.org/Team%3AEdinburgh_UG/Improve)
  [Attributions](https://2018.igem.org/Team%3AEdinburgh_UG/Attributions)
* Parts

  [Parts Overview](https://2018.igem.org/Team%3AEdinburgh_UG/Parts)
  [Basic Parts](https://2018.igem.org/Team%3AEdinburgh_UG/Basic_Part)
  [Composite Parts](https://2018.igem.org/Team%3AEdinburgh_UG/Composite_Part)
  [Part Collection](https://2018.igem.org/Team%3AEdinburgh_UG/Part_Collection)
* [Safety (current)](https://2018.igem.org/Team%3AEdinburgh_UG/Safety)
* [Human Practices (current)](https://2018.igem.org/Team%3AEdinburgh_UG/Human_Practices)
* Awards

  [Model](https://2018.igem.org/Team%3AEdinburgh_UG/Model)
* [Judging Form (current)](https://igem.org/2018_Judging_Form?team=Edinburgh_UG)
* [Medal Criteria(current)](https://2018.igem.org/Team%3AEdinburgh_UG/Medal_Criteria)

# Results

## Achievements

* We have characterised various methods of maxicell production, to optimise their manufacture for synthetic biology
* The active time frame of maxicells was quantified, to determine their potential “lifespan”
* We have created parts which facilitate maxicell production in any E. coli strain of your choosing
* We have introduced a functional biosensor into the maxicell chassis, which has shown that maxicells have the potential for gene expression for at least 18 hours after their
  production
* Various methods of preventing horizontal gene transfer were explored, to increase the security of maxicells as a chassis designed for environmental release
* Triclosan resistance was introduced into a standard backbone as an alternative to antibiotic selection, which would prevent the unwanted release of antibiotic resistance genes

# Evaluating and Optimising Maxicell Protocols

Three methods of maxicell production were evaluated to identify the most efficient process. Each method causes a double-strand break in the chromosome, leading to its degradation by native exonucleases (the rec system, in recA knockout cells). After maxicell induction, all of these methods involved treatment by cycloserine to kill the non-maxicells. Cycloserine is an antibiotic that targets cell wall synthesis, thereby killing any actively dividing cells. The level of maxicell production was determined by fluorescence microscopy of samples stained with DAPI; a fluorescent stain that binds to double stranded DNA.

## UV Production Method

We first started by looking at the *Escherichia coli* strains DH5-alpha (*recA-*, *uvrA+*) and CSR603 (*recA-*, *uvrA-*). It was found that when attempting maxicell formation in strains with functional uvrA, such as DH5-aplha, all stages post-UV exposure until the end of a 15-18 hour incubation must be carried out in complete darkness, to prevent photoreactivation by the UVR system. We also found that while CSR603 is a more efficient maxicell producer than DH5-alpha, it grows slower and the cells are significantly shorter in length and diameter than DH5-a. We surmised that this was because the cells are, in essence, sicker due to the mutations in DNA repair systems. When cultured in LB and exposed to 40 seconds of UV and then incubated for a further 18 hours, 70.0% CSR603 showed no DAPI fluorescence. When grown under the same conditions but exposed to no UV, 2.9% CSR603 showed no DAPI fluorescence.

Next, we used the *E. coli* strain MC4100 to produce a *recA*, *uvrA* knockdown, as this strain was recommended in literature for maxicell production. Through utilisation of RNA interference by MicC sRNAs, we created a *recA*, *uvrA* knockdown construct. The objective was two-fold: to develop a method of maxicell production which could be theoretically applied to any strain of E. coli; and to create a healthier maxicell producing strain - after it was observed that CSR603 has impaired growth due to its sickness. Both of these objectives were successful. For the knockdown strain (MC4100), we found that the colony forming units per mL culture 18 hours post UV-exposure was vastly lower than that of MG1655 (fully functioning *recA* and *uvrA*), and only slightly higher than CSR603 (fully non-functioning *recA* and *uvrA*) (Figure 1.). Furthermore, MC4100 growth rate in LB media and M9 glucose media is vastly higher than that of CSR603. When cells are cultured in M9 glucose media, there is no significant difference in CFU/mL 18 hours post-UV exposure when cells are exposed to light or kept in complete darkness. When cells are cultured in LB media, CFU/mL is significantly higher if the post-UV culture is exposed to light (Figure 2.).

![](https://static.igem.org/mediawiki/2018/a/a2/T--Edinburgh_UG--strains.jpeg)

Figure 1. Colony forming units/mL after 18 hours of UV treatment, when cells are exposed to light afterwards.

![](https://static.igem.org/mediawiki/2018/b/bc/T--Edinburgh_UG--4100lightdark.jpeg)

Figure 2. Colony forming units/mL of MC4100 starin after 18 hours of UV treatment, when cells are exposed or not to light afterwards.

The MicC constructs we produced were as follows:

* [BBa\_K2725006](http://parts.igem.org/Part%3ABBa_K2725006): micC construct for recA knock down
* [BBa\_K2725007](http://parts.igem.org/Part%3ABBa_K2725007): micC construct for uvrA knock down
* [BBa\_K2725008](http://parts.igem.org/Part%3ABBa_K2725008): micC construct for uvrA and recA knock down
* [BBa\_K2725009](http://parts.igem.org/Part%3ABBa_K2725009): micC construct for the mCherry gene that produces increased mCherry fluorescence

## Palindromic Cleavage Method

Another method of maxicell induction that was investigated involved using a strain of *E. coli*, DL3355 ( lacIq lacZχ- ParaBAD-sbcDC lacZ::pal460 recA::Cm), which contains a palindromic site in the chromosome that forms a hairpin loop. This hairpin loop can be cut by an sbcCD exonuclease that was introduced to the chromosome under control of an arabinose-sensitive promoter. This causes a double strand break in the chromosome. leading to digestion by native exonucleases. However, this method was found to be largely unsuccessful: a spot test of *E. coli* DL3355 with the *sbcCD* exonuclease gene under an arabinose inducible promoter inserted into the chromosome showed that there was no decrease in colony forming units after *sbcCD* induction. It was surmised that this was due to a high rate of mutation in this strain, as the palindromic sequence is a burden on the cell there is an inherent rate of loss of the palindrome site of 1/10000 per genome per generation. The rate of strain reversion is therefore very high, so this method of maxicell production is inefficient and not recommended.

## Homing Endonuclease Method

The last method we looked at centres around *E. coli* DL2524 (MG1655 recA::CmR araB::PBAD-I-SceI lacZ::I-SceIcs fnr-267), a strain that contains in its chromosome an 18 base pair recognition site for the homing endonuclease I-SceI, and the *I-SceI* gene under control of an arabinose-sensitive promoter. Maxicell production by this strain was initiated by culturing in arabinose, which activates *I-SceI* expression, leading to a double-strand break in the chromosome at the I-SceI recognition site. This method was the most successful at maxicell production: the lowest percentage of cells seen to be fluorescing with DAPI were in DL2524 arabinose induced samples (compared to all UV induced maximal methods and DL3355).

![](https://static.igem.org/mediawiki/2018/e/e1/T--Edinburgh_UG--DL2524_images.png)

Figure 3. Microscopy images of DL2524 cells incubated in glucose (top - no induction of homing endonuclease expression) and in arabinose (bottom - induction of homing endonuclease expression).

# Quantifying Maxicell Timeframe

## ATP Quantification

All ATP assays was conducted in the DL2524 *E. coli* strain (Endonuclease Method of producing maxicells). This strain and method were used as it was the easiest way of forming Maxicells as it was controlled by an inducible promoter. The ATP levels in Maxicells were compared at 3 Different temperature (37 °C; 21 °C; 4 °C, Figure 4) as we wanted to test the effect of varying temperature on the functionality of our chassis.

#### ATP Decline in Maxicells when Stored in the Incubator (37 °C):

Variation of ATP levels seem normal over 18 hours after induction between maxicells and non-maxicells (Fig 3a). As expected non-maxicells showed an increase of ATP after 18 hours as they used the nutrients in the medium to proliferate and produce more ATP. However, after 18 hours we start the decline in ATP in our maxicell strain. At 20 hours the concentration of ATP in maxicells is 0.54 mM; while the concentrations of ATP in non-maxicells is 0.62 mM. This divergence in ATP levels continues until ~26 hours in maxicells where it appears to have plateaued at ~0.48 mM while non-maxicells are at 0.61 mM and continue to increase over the rest of the time frame. It appears maxicells have ~24-hour time frame before the ATP inside the cell is depleted to baseline and the cell stops to function when stored at 37 °C. However, this still needs to be confirmed via protein synthesis assays.

#### ATP Decline in Maxicells when Stored at Room Temperature (21 °C):

ATP concentration for non-maxicells show a similar trajectory to non-maxicells at 37 °C, however with a lower overall concentration (Fig 3b). This is because *E. coli* proliferate better at 37 °C compared to 21 °C, therefore the non-maxicells at 37 °C have a higher ATP concentration on average. We can also see that maxicells at 21 °C show a similar trajectory as maxicells at 37 °C. However, seem to plateau earlier than the maxicells at 37 °C. They plateau at ~16 hours to 0.51mM. This is unusual as our initial thought were the cells at the 37 °C will metabolise ATP faster as enzymes are at their optimal temperature, however, this data suggests that this may not be the case and that the main factor driving ATP concentration decline is natural degradation/hydrolysis of ATP over time. This could be confirmed as there may just be discrepancy recording or other errors.

#### ATP Decline in Maxicells when Stored in the Fridge (4 °C):

Again, non-maxicells increase in ATP concentration albeit at a lower rate the non-maxicells at 21 °C or 37 °C (Fig 3c). The maxicells show plateauing at 24 hours to 0.49 mM of ATP. Again, very similar to the other two temperatures. However, this data supports the theory that the main factor driving ATP degradation in our maxicells is not an enzymatic activity but rather natural degradation/hydrolysis of ATP over time.

![](https://static.igem.org/mediawiki/2018/b/b1/T--Edinburgh_UG--ATP_Graph_37C.png)

![](https://static.igem.org/mediawiki/2018/0/08/T--Edinburgh_UG--ATP_Graph_21C.png)

![](https://static.igem.org/mediawiki/2018/2/2b/T--Edinburgh_UG--ATP_Graph_4C.png)

Figure 4. Concentration of ATP (mM) over Time (hours) in DL2524 either induced - Maxicells (grown in Arabinose) OR not induced - Non-Maxicells (grown in glucose). a: shows Maxicell and Non-Maxicell ATP conc. when stored at 37 °C. b: shows Maxicell and Non-Maxicell ATP conc. when stored at 21 °C. c: shows Maxicell and Non-Maxicell ATP conc. when stored at 4 °C

#### Conclusions:

The general trend seems to follow a pattern between all the data sets and temperatures that is: Plateau of ATP concentration at ~24 hours to ~0.5mM. This was similar for all temperatures (37 °C; 21 °C and 4 °C) which suggests may suggest the main factor that causes ATP degradation in our maxicells is naturally degradation of the molecule; as if the main factor was metabolism we would expect to see a larger difference in the time it takes to plateau for each temperature. This is because enzymes within the cell are most optimal at 37 °C and least at 4 °C. However, they plateau at the same time. Therefore this raises the question of the cause of ATP decline over time.
It is also important to not that the natural concentration of ATP within *E. coli* is 1.54 ± 1.22 mM. Which our system does fall into as 1 hour after induction we see both maxicells and non-maxicells are ~0.62 mM of ATP, which fall within the normal range. Albeit the DL2524 seems to fall at the lower normal range that wild-type, however, this may be due to the fact our cells seem to be sicker than wild-type and therefore may not be able to produce ATP as efficiently as wild type cells.
The potential active time frame of ~24 hours in the environment may not be limited to this maximum number as we know we can make our cells healthier by not using *recA* KO mutant and instead using *recA* knockdown, as seen in the MicC constructs. As the cell is healthier it will be able to proliferate quicker and able to store more ATP. It may also be possible to increase intracellular ATP before induction to boost potential active timeframe via other methods. These are all experiments future iGEM teams can conduct to further characterise our chassis and boost potential usage.

#### ATP levels of Induced Maxicells when stored in the Freezer (-20°C) for 2 weeks:

To see if our maxicell could be frozen for transport we decided to test ATP concentrations of maxicells that were frozen and compared this to our other temperatures to see if they held a similar trajectory. The maxicells were frozen along with non-maxicells (see method here). They were kept in the freezer for two weeks before being thawed to simulate transport in real life. They were then assayed. Figure 5 shows that non-maxicell act as normal; as they gradually increase in ATP concentrations.
However, our maxicells seem to decline in ATP concentration much quicker than our other temperatures. They seem to reach 0.46mM of ATP at ~16 hours after being thawed. This may be due to lysis of the cells during the freezing or thawing process which results in reduced ATP within the cell, or there may be some unknown factor for the rapid decline in ATP seen in the frozen maxicells.
Due to time restraint, we could not conduct more experiments, but we would have like to repeat this experiment at different time points to see the effect of storage on the cell, i.e. test at 2 days frozen; 1 week frozen; 1 month frozen etc. We would have also liked to try other methods of storage such as freeze-drying and the effect of this on ATP levels.

![](https://static.igem.org/mediawiki/2018/0/0a/T--Edinburgh_UG--ATP_Graph_-20C.png)

Figure 5. Conc. of ATP (mM) over Time (hours) in DL2524 either induced - maxicells (grown in arabinose then frozen) OR not induced - non-maxicells (grown in glucose then frozen). Shows maxicell and non-maxicell ATP concentration over time for maxicells that have been induced at then frozen.

## Protein Retention

Next, we decided to investigate the level of protein degradation in our maxicells, to find out whether or not all the cellular machinery remains intact over long periods of time. To do so, we took samples of maxicells stored 4°C for 0 h and 24 h, and at 25°C & 37°C for 0 h, 24 h and 48 h. We then ran these samples through an SDS-PAGE gel, the results of which can be seen in Figure 6. below. These results show that there is no major decrease in protein concentration within maxicells over a period of 48 h, not even at 37°C, suggesting that the level of protein degradation is minimal. Although we cannot determine absolute protein concentrations between the samples due to potential differences in sample preparation, we can see that they are not orders of magnitude different. It can, therefore, be inferred that protein degradation within maxicells is not a significant factor for their functionality over long periods of time.

![](https://static.igem.org/mediawiki/2018/1/15/T--Edinburgh_UG--sdspage.png)

Figure 6. SDS Page of All Maxicell Proteins Over Time when Stored at Different Tempratures. (see  [experiments page](https://2018.igem.org/Team%3AEdinburgh_UG/Experiments)  for details)

## Functional Gene Expression

Finally, CSR603 UV-induced maxicells were used to investigate the ability of maxicells to transcribe and translate a chosen gene, in this case mCherry under an inducible arabinose promoter ([BBa\_K2725010](http://parts.igem.org/Part%3ABBa_K2725010)). These maxicells were induced by arabinose to produce mCherry 18-25 hours after UV exposure. Fluorescence by mCherry was observed after arabinose treatment 24 hours after each sample was induced. The proportion of cells fluorescing that were induced 18 hours after UV exposure was 57.9%, which significantly decreases to 4.7% in samples induced 20 hours after UV exposure, and a maximum of 6% of cells fluorescing was seen in samples induced 25 hours post-UV exposure (figure 7.). In contrast, it was found that cells with intact chromosomes fluoresce only faintly after arabinose treatment, and mCherry is not visible to the naked eye in colonies cultured on LB+arabinose plates. It can be surmised, therefore, that maxicells can be induced to produce a gene product after their production, and can act as a biosensor: for arabinose in this case.

Unfortunately, due to difficulties in transformation of CSR603 we were unable to transform our ars-mCherry construct ([BBa\_K2725011](http://parts.igem.org/Part%3ABBa_K2725011)) into this strain. This made it impossible to test the ability of maxicells to act as a biosensor for arsenic.

![](https://static.igem.org/mediawiki/parts/1/1d/T--Edinburgh_UG--mCherry_fluorescence.png)

Figure 7. Graph of mCherry fluorescence 24 h after arabinose induction against "age" of maxicells (time in hours after maxicell production).

# The Triple Lock System

## Colicin Kill Switch

The results for the modelling section of our colicin kill switch can be seen [here](https://2018.igem.org/Team%3AEdinburgh_UG/Degradation_Switch). We [collaborated with Team Vilnius to develop a protocol for applying this model.](https://2018.igem.org/Team%3AEdinburgh_UG/Collaborations)

## Semantic Containment

Our aim was to recode the kanamycin resistance gene with differing numbers of serine codons replaced with amber codons (1, 2, 5 and 10). E. coli TOP 10 was transformed with each of these 4 plasmids and their growth measured on 8 concentrations of kanamycin - concentrations decreasing 2 fold from 400 ug/mL to 3.125 ug/mL. The outcome of this can be seen in Figure 8, which shows that replacing a single serine codon with an amber STOP codon does not fully inhibit kanamycin resistance in this strain. The fact that 1\* showed growth on kanamycin is due to an inherent level of amber suppression. This phenomenon has been investigated by [*in silico* modelling](https://2018.igem.org/Team%3AEdinburgh_UG/Semantic_Containment_Modelling) to determine the rates of read-through for the different numbers of mutations.

![](https://static.igem.org/mediawiki/2018/a/ab/T--Edinburgh_UG--400C.png)
![](https://static.igem.org/mediawiki/2018/a/a6/T--Edinburgh_UG--semcom100c.png)
![](https://static.igem.org/mediawiki/2018/9/99/T--Edinburgh_UG--semcom25c.png)
![](https://static.igem.org/mediawiki/2018/a/ac/T--Edinburgh_UG--semcom3125c.png)

Figure 8. Growth curves of Top10 *E.coli* transformed with P1003, P1003\* (1\*), P1003\*\* (2\*), P1003 5\* (5\*) and P1003 10\* (10\*) grown in the given concentration of kanamycin. These growth curves show the extent of kanamycin resistance conferred by each part.

After it was determined that sufficient kanamycin resistance could not be conferred by the 2\*, 5\* ([BBa\_K2725012](http://parts.igem.org/Part%3ABBa_K2725012)) and 10\* ([BBa\_K2725013](http://parts.igem.org/Part%3ABBa_K2725013)) genes when *supD* is functional, we then sought to test for resistance when *supD* is mutated as an amber suppressor. We also tested the extent of kanamycin resistance when the amber suppressor *supD* was expressed under different strength Anderson promoters. The results of this can be seen in Figure 9 below.

![](https://static.igem.org/mediawiki/2018/e/ec/T--Edinburgh_UG--400.png)
![](https://static.igem.org/mediawiki/2018/1/1b/T--Edinburgh_UG--semcom100.png)
![](https://static.igem.org/mediawiki/2018/6/63/T--Edinburgh_UG--semcom25.png)
![](https://static.igem.org/mediawiki/2018/3/38/T--Edinburgh_UG--semcom3125.png)

Figure 9. Growth curves of 8 Top10 *E.coli* double transformants in the given concentration of kanamycin . These growth curves show the extent of kanamycin resistance conferred by each part combination.

It is clear that when the amber suppressor *supD* is expressed under medium strength promoter J23108, P1003 with 1, 2, and 5 amber codons can be expressed at a sufficient enough level that confers kanamycin resistance. This means the rate of serine integration at amber codons is much higher than the removal of the nascent polypeptide by Release Factor 1. However, there was no kanamycin resistance conferred by the P1003 10\* gene. This means that the amber suppressor *supD* was not expressed at a high enough level to outcompete Release Factor 1 at 10 successive amber STOP codons. We attempted higher level expression of amber suppressor *supD* ([BBa\_K2725014](http://parts.igem.org/Part%3ABBa_K2725014)), however no double transformants with this part had any kanamycin resistance. We think this is due to the presence of amber STOP codons in the chromosome. If chromosomal genes that use amber STOP codons for termination of translation consistently have serine incorporated (and continued translation) instead, this would place a significant metabolic burden on the cell. This, therefore, generates significant selection pressure for a mutation that decreases the expression level of the amber suppressor *supD* or prevents it completely. Because the construct was under a constitutive promoter this mutation likely occurred before the cells were transferred to a kanamycin medium.

The Anderson Promoter-SupD constructs are as follows:

* [BBa\_K2725014](http://parts.igem.org/Part%3ABBa_K2725014): Serine amber suppressor tRNA gene under J23102 Anderson promoter
* [BBa\_K2725015](http://parts.igem.org/Part%3ABBa_K2725015): Serine amber suppressor tRNA gene under J23103 Anderson promoter
* [BBa\_K2725016](http://parts.igem.org/Part%3ABBa_K2725016): Serine amber suppressor tRNA gene under J23108 Anderson promoter

## Alternative Selection

FabI

The *fabI* gene was PCR amplified from DH5-α genomic DNA and the biobrick prefix and suffix was added. The PCR product was introduced into the biobrick site of pSB1C3. The FabI biobrick was then expressed under a high constitutive expression cassette ([BBa\_K314100](http://parts.igem.org/Part%3ABBa_K314100)) to produce [BBa\_K2725001](http://parts.igem.org/Part%3ABBa_K2725001) and growth was observed on triclosan at 1 µM (Figure 10.), while untransformed DH5-alpha showed no growth (Figure 11.).

![](https://static.igem.org/mediawiki/2018/a/aa/T--Edinburgh_UG--BBa_K2725001.jpeg)

Figure 10. 1 µM triclosan agar plate, plated with *E. coli* DH5-alpha transformed with BBa\_2725001

![](https://static.igem.org/mediawiki/2018/d/dc/T--Edinburgh_UG--DH5-alpha_triclosan.png)

Figure 11. 1 µM triclosan agar plate, plated with untransformed *E. coli* DH5-alpha

FabV

As a proof of concept for our final design, the *fabV* gene was introduced into pSB1C3, replacing chloramphenicol resistance by Gibson Assembly to produce a new plasmid backbone: [pSB1Tcs1](http://parts.igem.org/Part%3ABBa_K2725005). The *fabV* gene was therefore expressed under the chloramphenicol acetyltransferase promoter and ribosome binding site. This new plasmid conferred resistance to triclosan above 16 mM, showing similar growth profiles at all concentrations below 16 mM, while the growth of untransformed DH5-α was inhibited at 0.25 µM and was severely inhibited above 4 µM (Figure 12.). It is therefore advised that 4-16 µM triclosan be used for selection in liquid culture. However, It was found that 1 µM triclosan is sufficient for selection on agar plates, with no colonies being observed when untransformed DH5-ɑ was plated at 1 µM (Figure 11.), as lower concentrations can be used for selection when plating.

![](https://static.igem.org/mediawiki/2018/archive/7/70/20181018013010%21T--Edinburgh_UG--Triclosan.jpeg)

Figure 12. Growth curves of untransformed DH5-alpha at triclosan concentrations ranging from 0 to 64 µM (left) and DH5-alpha transformed with pSB1Tcs1 (BBa\_K2725005) at concentrations of triclosan ranging from 0 to 16 mM.

The *fabV* gene was then PCR amplified from our pSB1Tr1 plasmid to add the Biobrick prefix and suffix. This *fabV* Biobrick ([BBa\_K2725002](http://parts.igem.org/Part%3ABBa_K2725002)) was inserted after a high constitutive expression cassette ([BBa\_K314100](http://parts.igem.org/Part%3ABBa_K314100)) to produce [BBa\_K2725003](http://parts.igem.org/Part%3ABBa_K2725003) and a low constitutive expression cassette ([BBa\_K314101](http://parts.igem.org/Part%3ABBa_K314101)) to produce [BBa\_K2725004](http://parts.igem.org/Part%3ABBa_K2725004), both of which showed growth on triclosan at 1 µM (Figure 13.).

![](https://static.igem.org/mediawiki/2018/e/e7/T--Edinburgh_UG--BBa_K2725003_BBa_K2725004.png)

Figure 13. 1 µM triclosan agar plates, plated with *E. coli* DH5-alpha transformed with BBa\_2725003 (left) and BBa\_K2725004 (right)

What's Next?

* The triclosan resistant backbone should be recoded to replace all serine codons with amber stop codons, so that it is compatible with the semantic containment system
* Triclosan resistance is due to a higher than normal expression of Fab proteins, or more specifically, it is due to an increased number of triclosan binding sites. It may, therefore, be possible to decrease the metabolic burden on the cell or increase resistance by trimming the FabI or FabV protein down to just the domain which binds to triclosan. This would further optimise this system of triclosan resistance

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