IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/05

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Revision as of 06:37, 5 September 2008



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Testing AHL degradation by aiiA

  • The product of the aiiA gene AHL-lactonase degrades AHL in many strains of Bacillus, however, it is unclear to what extent (if any) B. subtilis strain 168 degrades AHL. If 168 is good at degrading AHL this will be a problem when our signaling system is eventually moved into strain 168.

We are testing B. subtilis strain 168’s ability to degrade AHL by exposing synthetic AHL to the strain and then testing its ability to be recognized by the receiver.


  • Materials

Bacillus subtilis - protease deficient (NPR and APR) derived from strain 168 - grown up in an over night culture of LB.

Synthetic AHL – Sigma K3007 – AHL powder was dissolved in EA buffer and diluted to 10uM concentration

E. coli with T9002 AHL receiver grown up in overnight culture of LB

All plates contain soft agar with 200uL of E. coli AHL receiver mixed-in. Plates 1,2 and 4 contain Bacillus in fresh LB medium, while plate 5 contains Bacillus in it’s overnight LB. Plate 5 was used to test if an excreted product of Bacillus is responsible for the degradation of AHL. The product of the aiiA gene in Bacillus subtilis is most likely not exported.

  • Plate 1: 10uL Bacillus + 10uL AHL
  • Plate 2: 10uL Bacillus + 10uL EA buffer (negative control + does EA buffer kill bacillus?)
  • Plate 3: 10uL LB + 10uL AHL (positive control)
  • Plate 4: 10uL Bacillus + 10uL AHL incubated together for 1.5 hours prior to plating
  • Plate 5: 10uL Bacillus in overnight LB + 10uL AHL
  • Results

plates visualized 3 hours after AHL/Bacillus inoculation.....


Checking vectors

- Plasmid miniprep for ECE166 (for stock), ECE172 and ECE153

- Digest of ECE153, ECE172 and ECE162 (with plasmid miniprep from yesterday)

- Run on a gel single digest for ECE147 (from yesterday with more DNA), ECE149 (from yesterday with more DNA), ECE150 (from yesterday with more DNA) and ECE 153, ECE162, ECE172

  • Results
  • Lane3 : HyperladderI
  • Lane4 : ECE147
  • Lane5 : ECE149
  • Lane6 : ECE150
  • Lane7 : ECE151
  • Lane8 : ECE1162
  • Lane9 : ECE172
  • Lane10 : hyperladderI


Very low bands : not enough DNA!!!

Transformation of Bacillus

  • Result of Nanodrop
Vector 260/280 ng/μL
ECE112 1.75 64.6
ECE166 172 138.6

- Prepare medium A with tryptophan, and medium B

- add 5mL of medium A in 3 different tubes, inoculate each tube with colonies (1A1, IA751 ans IA771)

- Check OD every 20min

- Incubate 90min

- Add 0.45mL of medium B and 0.05mL of culture in Ependorf tubes

- Incubate 90min

- Transform : add 1μg of DNA (some with ECE112, some with ECE166) (so you need to nanodrop samples before!)

- Incubate 30min

- Pipette 200μL of solution, spread it on aech plate, wait 10min, and do it again

- Incubate 24hours

- A few glycerol tubes to stock cells : add 2/7 glycerol to cell tubes


- Transformation from glycerol stock from 30/07/2008

- Spin glycerol stocks, pipette out glycerol

- Add 0.5mL of medium B, incubate for 1 hour

- Add 10μL of ECE112 (640ng)

- Incubate 2hours

- Plate 200μL, and 10min later, still 200μL


New stocks

- Do glycerol stock of I746001 and I746101 (no sterile glycerol)

- Put IA751, IA771 in 10mL LB

- Put ECE 176 in 10mL LB + antibiotic

- Reinoculate the tube of LB from yesterday with ECE166 plate

- ECE176 replated onto Amp100 + Cm5



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