IGEM:IMPERIAL/Protocols/S01656/AHL reduction AHL reduction

=Part S01656 - IPTG induced AiiA production, test construct=

NB: This part has kanamycin resistance and must be grown in LB with 50 /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, J37022, 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 T9002 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 /mL Kanamycin
 * LB medium with 50 /mL Ampicillin
 * E. coli DH5a strain with part S01656
 * E. coli DH5a strain with part T9002
 * Perkin Elmer Victor 3 Fluorimeter
 * 96 well plate

Protocol

 * First, grow two overnight cultures of part S016565 in plasmid pSB2K3 (Kanamycin resistant) in 2 mL of LB with 50 /mL Kanamycin. In one of the cultures, innoculate with 20  of 1M stock IPTG (to obtain a 1 mM concentration of IPTG).  The other culture will be our control with no IPTG.  Also, prepare a 2 mL overnight culture of T9002 in LB with 50 /mL Ampicillin.  Keep the cultures in the 37°C shaker overnight. (20 min + 8-12 hrs)
 * In the morning, prewarm 20 mL LB with Kanamycin, 40 mL LB with Ampicillin, 20 mL plain LB medium in the 37C waterbath to prevent shock to the culture when diluting.
 * Remove the samples from the shaker and measure the OD of the cultures at 600 nm wavelength. (5 min)


 * OD of +IPTG = _________


 * OD of -IPTG = _________

\frac{0.1}{\mbox{OD of culture}} \times \mbox{5 mL}
 * Reculture both overnight cultures in 5 mL of prewarmed LB with Kanamycin by diluting to an OD of 0.1 by using the following formula (5 min)


 * Amount to dilute of +IPTG culture = ________ mL (amount of original culture to use)


 * Amount of prewarmed LB with Kanamycin to use = ________ mL (5 mL - above result)


 * Amount to dilute of -IPTG culture = ________ mL (amount of original culture to use)


 * Amount of prewarmed LB with Kanamycin to use = ________ mL (5 mL - above result)


 * Innoculate the culture with IPTG with another 50 of IPTG to ensure that AiiA production continues. (5 min)
 * Allow the reculture to grow for 2 hours by placing in the 37°C shaker. During this time, we are allowing the cells that are in the stationary phase from the overnight culture to get into the exponential phase.  (2 hrs)


 * Time begin: ____________________

\frac{0.1}{\mbox{OD of culture}} \times \mbox{16 mL}
 * Meanwhile, reculture the overnight culture of T9002 in 16 mL of prewarmed LB with Ampicillin by diluting to an OD of 0.1 by using the following formula (5 min)


 * OD of T9002 culture (1st Measurement): _________


 * Amount to dilute of T9002 culture = ________ mL (amount of original culture to use)


 * Amount of prewarmed LB with Ampcilillin to use = ________ mL (16 mL - above result)

\frac{0.1}{\mbox{OD of culture}} \times \mbox{5 mL}
 * Again take the OD of the two samples and dilute to an OD of 0.1. This time, dilute into a 5 mL prewarmed LB (no antibiotic!).  Using kanamycin will kill the T9002 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 in 5 mL LB (no antibiotic) and vortex to resuspend.  Use the following formula: (10 min)


 * OD of +IPTG culture = ________


 * Amount to dilute of +IPTG culture = ________ mL (amount of original culture to use)


 * OD of -IPTG culture = ________


 * Amount to dilute of -IPTG culture = ________ mL (amount of original culture to use)


 * Again, innoculate the culture with IPTG with another 50 of 1 M stock IPTG to ensure that AiiA prouction continues. (5 min)
 * Mix to ensure homogeneity of the IPTG by vortexing. (1 min)
 * Aliquot 1 mL of the +IPTG culture into 4 separate eppendorfs. Three of them will contain AHL, and the other ependorf will be a control without AHL.  To the tubes needing AHL, add in 10 uL of 10 uM stock solution to obtain a final concentration of 100 nM. (2 min)
 * Aliquot 1 mL of the -IPTG into 2 separate eppendorfs. One of them will contain AHL, and the other will not have AHL.  These will act as our controls as well. To the tubes needing AHL, add in 10 uL of 10 uM stock solution to obtain a final concentration of 100 nM. (2 min)
 * Place all of the aliquots in the 37°C shaker for two hours to allow the AiiA enzyme to work. (2 hrs)


 * Time begin: ________________

\frac{0.1}{\mbox{OD of culture}} \times \mbox{20 mL}
 * Just before the 2 hours are up, reculture again the T9002 in 20 mL of prewarmed LB with Ampicillin by diluting to an OD of 0.1 by using the following formula (5 min)


 * OD of T9002 culture (2nd Measurement): _________


 * Amount to dilute of T9002 culture = ________ mL (amount of original culture to use)


 * Amount of prewarmed LB with Ampcilillin to use = ________ mL (20 mL - above result)


 * After two hours, spin all of the S01656 cultures in a centrifuge at 13k rpm for 20 seconds to remove the cells. The AHL and the IPTG will be in the supernatant, but the AiiA enzyme will be held within the cell. (1 min)
 * Use the T9002 Protocol as the AHL assay as outlined below.

T9002 Protocol Section (slightly revised)

 * To start AHL incubation:
 * Label each of 6 small white tube with +/- AHL & +/- IPTG depending upon which test 3 x +IPTG +AHL, 1 x +IPTG -AHL, 1 x -IPTG +AHL, 1 x -IPTG -AHL
 * Add 100  of the appropriate AHL sample
 * Add 1900  of T9002 of OD600 0.1 (dilute the T9002 if necessary)
 * Vortex each tube
 * Add a 200uL sample from each tube to the 96 well plate.
 * Do this for 8 repeats This should fill only 8 x 6 = 48 wells
 * Add 4 x 200uL of growth medium to a well to act as a control. Total 52 wells filled
 * Incubate the 96 well palte in a 37oC shaker set to 100rpm for 4 hours so GFP expression can reach steady state.
 * After the 4 hours take the plate and an ependorf to BCHEM
 * Add 190ul of ultra pure water to the ependorf, together with 10ul of undiluted GFP standard solution and mix __HIDER__
 * The undiluted GFP is in the BCHM Level 6 Cold Room on our shelf. It is in a small grey plastic box. Pippettes, tips and ultrapure water are located on a shelf above the electroporation machine in the plate reader room.


 * Add 4 x 200uL of the 200x diluted GFP standard solution to the wells
 * Take a reading using the Perkin Elmer Victor3
 * Take the plate to the plate reader room
 * Use the Victor3 to measure flourescence and absorbance __HIDER__


 * Use the preprogrammed Assay under the 'Students' folder called GFP + Abs490


 * Repeat the measurment a further two times straight after each other __HIDER__
 * This to assess the variability of the machine


 * Save data file from computer. __HIDER__
 * You'll need a memory stick to save the information to. Unfortunatley the computer isn't networked


 * Copy and paste the data into a T9002 Data Spreadsheet

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: