IGEM:IMPERIAL/2006/Protocols/S01656rev

Part <bbpart>S01656</bbpart> - IPTG induced AiiA production, test construct - revised protocol

NB: This part has kanamycin resistance and must be grown in LB with 50 μg/mL Kanamycin

The purpose of this protocol is to test the ability of AiiA to degrade AHL. This is the closest part to our actual testing construct, <bbpart>J37022</bbpart>, since it has the LVA degradation tag. The addition of the FLAG tag to our actual construct may or may not affect the activity of AiiA, but the purpose of this experiment is just to qualitatively measure the activity of AiiA. This protocol works in conjunction with <bbpart>T9002</bbpart> to measure the AHL concentration after a certain period of time.

Equipment/Materials

• Falcon tubes or similar
• Photospectrometer
• Gilson pipettes
• Eppendorf tubes
• Centrifuge
• Plain LB medium
• LB medium with 50 μg/mL Kanamycin
• LB medium with 50 μg/mL Ampicillin
• E. coli DH5a strain with part <bbpart>S01656</bbpart>
• E. coli DH5a strain with part <bbpart>T9002</bbpart> or <bbpart>J37016</bbpart>
• Perkin Elmer Victor 3 Fluorimeter
• 96 well plate

Protocol

• Grow 2 overnight cultures of part <bbpart>S01656</bbpart> (plasmid pSB2K3 - Kanamycin resistant) in 2 mL of LB with 50 μg/mL Kanamycin:

1. Inoculate with 20 μL of 1M stock IPTG (to obtain a 1 mM concentration of IPTG)
2. Do NOT add IPTG (this is the control)

• Prepare a 2 mL overnight culture of <bbpart>T9002</bbpart> or <bbpart>J37016</bbpart> in LB Amp (following T9002 protocol)

Keep the cultures in the 37°C shaker overnight

Next day, in the morning:

• Prewarm 20 mL LB with Kanamycin, 40 mL LB with Ampicillin, 20 mL plain LB medium in the 37[[:Category:{{{1}}}|{{{1}}}]] waterbath (to prevent cold shock to the culture when diluting)
• Remove the cultures from shaker and measure OD of the cultures at 600 nm wavelength
• Reculture both overnight cultures of S01656 in 5 mL of prewarmed LB with Kanamycin; diluting to an OD of 0.1 by using the following formula

<amsmath>

\frac{0.1}{\mbox{OD of culture}} \times \mbox{5 mL}

</amsmath>

• Inoculate the culture with IPTG with another 50 μL of IPTG (to ensure that AiiA production continues)
• Place diluted culture into the 37°C shaker for 2 hours (During this time, the cells that are in stationary phase from the overnight culture get back into exponential phase.)

• Reculture the overnight culture of <bbpart>T9002</bbpart> in 16 mL of prewarmed LB with Ampicillin; diluting to an OD of 0.1 by using the following formula

<amsmath>

\frac{0.1}{\mbox{OD of culture}} \times \mbox{16 mL}

</amsmath>

and place in shaker for 2hrs (same as T9002 testing protocol)

After 2hrs in shaker:

• Measure the OD of the two samples and dilute to an OD of 0.1:
  • IMPORTANT: Dilute into 5mL prewarmed LB (no antibiotic!) (Using kanamycin will kill the <bbpart>T9002</bbpart> cells since they are only resistant to Ampicillin.)
  • Before the LB is added, spin down the cells using the large centrifuge for 3 minutes at 3000 rpm and discard the supernatant
  • Add 5 mL LB (no antibiotic!) and vortex to resuspend
• Inoculate the +IPTG culture with another 50 μL of 1 M stock IPTG (to ensure that AiiA prouction continues)
• Vortex culture to mix content
• Aliquot 1 mL of the +IPTG culture into 4 separate 5mL tubes:

• 1.-3. Add 10 μL of 100 μM stock solution (to obtain a final concentration of 1 μM)
• 4. No AHL added (control)

• Aliquot 1 mL of the -IPTG into 4 separate 5mL tubes:

• 1.-3. Add 10 μL of 100 μM stock solution (to obtain a final concentration of 1 μM)
• 4. No AHL added (control)

• Place all of the aliquots in the 37°C shaker for 2 hours (to allow the AiiA enzyme to work)

After another 2hrs in shaker:

• Take S01656 out of shaker and spin all of the S01656 cultures in a centrifuge for 20 seconds at 13k rpm to remove the cells(The AHL and the IPTG will be in the supernatant, but the AiiA enzyme will be held within the cell)
• Reculture again the <bbpart>T9002</bbpart> into 25 mL of prewarmed LB with Ampicillin; diluting to an OD of 0.1 (follow T9002 testing protocol)
• Use the T9002 Protocol or the J37016 Protocol as the AHL assay as outlined below

<bbpart>T9002</bbpart> or <bbpart>J37016</bbpart> Protocol Section (slightly revised):

• To start AHL incubation:
  • Label 6 small white 5mL tubes with +/- AHL & +/- IPTG respecively; depending upon which test 3 x +IPTG +AHL, 1 x +IPTG -AHL, 3 x -IPTG +AHL, 1 x -IPTG -AHL
  • Add 800μL of the appropriate AHL sample
  • Add 1200 μL of T9002/J37016 of OD600 0.1 (dilute the T9002 if necessary)
  • Vortex each tube
• Place tubes in 37°C shaker for 4 hrs (in order to let GFP expression reach steady state)

After 4hrs in shaker:

• Add a 200uL sample from each tube to the 96 well plate.
• Do this for 8 repeats
• Add 4 x 200uL of growth medium to a well each (as control)
• Add 4 x 200uL of 200times diluted GFP to a well each (as control) - (Add 190ul of ultrapure water and 10uL of GFP standard solution to an eppendorf. Then vortex to mix)
• Take 3 repeat readings using the Perkin Elmer Victor III

(Use the Victor III to measure flourescence and absorbance, only deselect columns 10, 11, and 12, even though G9 and H9 will be empty)

• Copy and paste the data into a S01656 Results Spreasheet and copy the results into the S01656 Results Page

Results (Averages)

S01656 Results Page

• Fluorescence of +IPTG +AHL

Tube 1

Tube 2

Tube 3

• Fluorescence of +IPTG -AHL

Tube 1

• Fluorescence of -IPTG +AHL

Tube 1

• Fluroescence of -IPTG -AHL

Tube 1

• Comparison between +IPTG +AHL and -IPTG +AHL:

% Difference =

Conclusion: