IGEM:IMPERIAL/Protocols/J37022

Halflife

We can determine the concentration of aiiA by assaying the FLAG immunotag. If we aliquot samples from a batch culture, then we can plot a graph of aiiA concentration vs. time, hopefully yielding the characteristic exponential curve by which we can determine the half-life.

Equipment and Materials

• Equipment
  • ELISA plate and ELISA materials
  • Fluorimeter
  • Photospectrometer
  • Centrifuge
  • Magnetic Stirrer

• Materials
  • E.coli DH5α Culture containing J37022
  • LB medium
  • Ampicillin
  • Dilution series of AHL

Protocol

• Culture cells containing part J37022 in 2 mL LB medium with 50 μg/mL Ampicillin.
• Leave overnight to grow
• Reculture (1 μL in 1 mL concentration) and grow until an OD600 of 0.1 is reached. This will take approximately 1 hour.JW/DA- Is it important for this OD to be reached?
• Introduce 1μL of 1 mM IPTG into the system to begin production of AiiA. /TH: The stock we have is 1M, therefore use 1ul for every ml of culture
• At 20 minute time intervals (starting at t=0, then t=20, t=40, etc), remove samples of the batch culture Amount? , measure OD, then assay the AiiA using ELISA method. ELISA protocol here

• In order to be more accurate, remove four (4) Why four? aliquots of 100 uL (or as specified for the ELISA immunoassay plate) from the batch culture and spin down the cells (1 min at 13k rpm) to quench the IPTG reaction. Is this done as an alternative to the above method? Should we just not do the most accurate one?
• Remove the supernatant and resuspend the cells in the same volume of LB by vortex. Repeat to ensure a higher removal of IPTG./*VR:is it enough to remove all the IPTG ? same problem as decay at the end of the day*/
• Then use the ELISA assay to determine the concentration of AiiA in the suspension. ELISA protocol here /*VR: link to Elisa method*/

The above method determines the half-life with the production of AiiA. If we want a direct measurement, we can still use a similar protocol. In fact, we can do both of these protocols to compare our results. What do you mean? /*VR: Show us how you use experimental data to compute half life - link to wiki page*/

• Why are we doing this all over again? Unclear. Once we reached 3 hours, spin down the entire culture for 5 minutes at 3000 rpm if using 50 mL falcon tubes (or 1 minute at 13k rpm if using eppendorf tubes) and discard the supernatant.

• Again resuspend the cells in LB and spin down again (5 minutes at 3000 rpm or 1 minute at 13k rpm) to ensure that all of the IPTG is removed. Repeat the spin down and resuspension to ensure as much IPTG is removed as possible. This will quench all AiiA production.
• From this, we can again measure the OD before aliquoting 100 uL samples of AiiA and measure the concentration at 30 minute intervals. We measure the OD to normalise the skewed results we will get from the raw data due to growth of the cells.
• The solution should be well mixed, so putting the mixture of a stirrer should help keep them happy.
• [Is there any way we can lyse the cell to release the AiiA?] /*VR: maybe lysis is included in Elisa method ...*/ JS: ELISA protocol just gets the tag, does not lyse the cells.

Once the raw data is obtained, see the J37022 Part Page for how to calculate the halflife.

Steady State AiiA Concentration vs. Initial IPTG Concentration

We would like to test the theory that by increasing the initial IPTG concentration, we will obtain higher AiiA concentrations. In theory, a higher IPTG concentration (assuming that it does not saturate the promoters on the DNA) will induce the transcription of more AiiA, leading to a higher concentration of AiiA. This will unfortunately take time for the concentration of AiiA to reach a steady state. This can be easily taken into account by leaving the culture for several hours, depending upon how fast the system gets to steady state, which can be found by considering the previous experiment to measure the half-life of AiiA.

Equipment and Materials

• Equipment
  • ELISA plate and ELISA materials
  • Fluorimeter
  • Photospectrometer
  • Centrifuge
  • Magnetic Stirrer

• Materials
  • E.coli DH5α Culture containing J37022
  • LB medium
  • Ampicillin
  • Dilution series of AHL
  • Dilution series of IPTG

Protocol

• Culture cells containing part J37022 in 2 mL LB medium with 50 μg/mL Ampicillin.
• Leave overnight to grow
• Reculture (1 μL in 1 mL concentration) and grow for approximately 1 hour until an OD600 of 0.1 is reached.DA- Is it important for this OD to be reached? JW: In shaker?
• Introduce 1μL of 1 mM IPTG into the system to begin production of aiiA. TH: The stock we have is 1M, therefore use 1μL for every ml of culture
  • Repeat the experiment with 4 different initial concentrations of IPTG. JS: Not sure which concentrations we can use, so will have to look up in literature to make sure we aren't saturating the system (literature on LacI promoters that are IPTG induced).
• Leave the culture for approximately 4 hours to allow the production of aiiA to reach a steady state. JW:Just leave it there or incubate?? We are assuming here that four hours is enough time for the production of AiiA to reach steady state. This might have to be altered depending upon the results from the experiment calculating the half-life.
• Remove 4 samples of 100μL (or as specified for the ELISA immunoassay plate JW:????) from the batch culture and measure their OD600 JW:Do we record this?
• JW:Put into eppendorfs?
• IPTG removal
  • Spin down the cells (1 min at 13k rpm) to quench the IPTG reaction. JW:What do u mean by stopping the reaction - does spinning stop IPTG inducing aiiA production??
  • Remove the supernatant
  • Add the same volume of LB medium JW:What volume - 100μL?
  • Resuspend by vortexing
• Repeat "IPTG removal"" to ensure increased removal. /*VR:is it enough to remove all the IPTG ? same problem as decay at the end of the day*/
• Use the ELISA assay to determine the concentration of AiiA in the suspension. ELISA protocol here

Using this data, we can then plot an average AiiA concentration with initial IPTG concentration. This plot will allow us to decouple measuring the AiiA concentration so that we can obtain a value of Vmax (as per the activity section) and k2 in the Michaelis-Menten equation since from the derivation, it is easily seen that Vmax = k2*[E0]. Vmax will vary depending upon the concentration of enzyme we use; however, k2 should not change since it is the rate constant determining the production of product from the enzyme-substrate complex.

Activity

We would also like to test the activity of the enzyme to determine its Vmax and Km values. Once we get these values, we can compare them to the values stated in the literature. Literature from 2004 states that the Km values obtained is between 1.43 to 7.51 mM. We would ideally like to get a Km value somewhere between these values to validate our experiment. Since Vmax varies depending upon the concentration of AiiA, no literature value was found for Vmax. However, for the AHL which we will use, 3-Oxo-C6-HSL (3OC6HSL), AiiA has a Km value of 2.96 mM. [1]

Equipment

• E.coli strain DH5a
• LB medium
• Ampicillin antibiotic
• Various pipettes and tips
• Fluorimeter
• Photospectrometer
• Centrifuge
• Various AHL concentrations

Protocol outline

• First, culture cells with part J37022 for testing overnight in 2 mL LB medium with 50 ug/mL Ampicillin.
• Reculture (1 uL in 1 mL concentration) and grow until an OD of 0.1 is reached. This will take approximately 1 hour.
• Inject 1μL of 1 mM IPTG into the system to begin production of AiiA.
• Split the culture into 2 x 8 1.0 mL batches in eppendorf tubes to test different AHL concentrations. /*Do you really need to test so many AHL concentrations ? What is the volume of the culture in the eppendorf ? */ JS: Yes, we would want to get as accurate a curve as possible when we plot 1/v vs. 1/[AHL], volume of culture is 1.5 mL, enough for 4 readings of 200 uL each (from 0 to 60 mins every 20 mins)
• Inoculate the batch cultures with the appropriate AHL concentration. /*what are the concentrations of AHL used ?*/ JS: the AHL concentrations will be those used for the T9002 experiment
• Vortex the tube to ensure that the solution is well mixed. /*Do you put them back into shacker ?*/ JS: I don't think the solution needs to be kept in the shaker, as long as we vortex before each reading. But please correct if necessary...
• Every 20 minutes for 1 hour, vortex the solution, measure the OD, and aliquot a 200 uL sample from each culture. The OD is measured to normalise the flourescent readings that we obtain from the T9002. One hour should be sufficient time to degrade the AHL. We might even need to reduce the time period to 10 minutes if the AiiA enzyme works very fast.
• Once you have removed the aliquot, place the eppendorf with the aliquot on ice to prevent degradation of AHL
• Spin down the solution (1 minute at 13k rpm) to remove the cells (hopefully with AiiA within them). /*VR: aiiA could come from lysed cells, but if you keep them growing and happy it should be minimal*/
• This time, transfer the supernatant into another labelled tube and use this in conjunction with T9002 (AHL assay) to determine the concentration of AHL remaining in the solution. See the T9002 protocol for further information on how to use the part.T9002 Information Page, T9002 Protocol page

• We can use these to plot graphs of concentration of AHL vs. time, and from this, we can determine the initial rate of reaction of the enzymatic activity.
• We can then plot a Lineweaver-Burk plot of 1/velocity vs. 1/substrate concentration to determine the Km and Vmax values of AiiA.

/*VR: you will have Km and Vmax for a unique value of [aiiA], is it what we need ? In our system [aiiA] will change over time*/ We have literature values for Km which should not change with different AiiA concentrations. The value of Vmax will reflect the concentration of AiiA, but this will not be necessary to characterise our oscillator, and may not be useful to quantify if it will continuously change over time.

Zinc Hypothesis

MIT’s 2004 results have showed no difference between inducing the I13207 test construct with and without arabinose using M9 minimal media. Recent studies on literature have shown that the AiiA enzyme is an AHL-lactonase (N-acyl homoserine lactone) which has a conserved zinc binding motif in its active site. This motif is shared by all AHL-lactonases. [2] Having analysed all of the components in M9, there is no evidence of zinc ions in the solution, suggesting that the reason for MIT’s not having produced any statistically significant results is that they neglected to add zinc in their solution. However, this literature has only come to light in 2005, allowing us a fuller understanding of the enzymatic structure and activity of AiiA. We would like to test this hypothesis by using the J37022 test construct (or a similar construct).

Equipment

• E.coli strain DH5a
• LB medium
• M9 medium (Endy Lab Protocol)
• Ampicillin antibiotic
• Various pipettes and tips
• Fluorimeter
• Photospectrometer
• Centrifuge
• AHL

Protocol outline

• Culture cells with part J37022 in 2 mL M9 minimal media with 50 ug/mL ampicillin and 2 mL LB media with 50 ug/mL ampicillin overnight.
• Reculture (1 uL in 1 mL concentration) into 2 5 mL separate M9 media with ampicillin batches (one containing 1 mM ZnCl2) and 1 5 mL batch of LB culture and allow to grow to an OD of 0.1 (in exponential phase).
• Inject a quantity of 1 mM IPTG to induce AiiA expression into all three batch cultures. The IPTG concentration will be determined by the second experiment in which we test the AiiA production in terms of IPTG initial concentration.
• Allow the AiiA production to reach almost a steady state value by waiting about 3 hours.
• Inject a quantity of 0.1 M AHL into the solution and vortex to mix.
• Every 20 minutes for 1 hour, vortex the solution, measure the OD, and aliquot a 500 uL sample from each culture into a labelled eppendorf tube. The OD is measured to normalise the flourescent readings that we obtain from the T9002. One hour should be sufficient time to degrade the AHL. We might even need to reduce the time period to 10 minutes if the AiiA enzyme works very fast.
• Spin down the solution (1 minute at 13k rpm) to remove the cells (hopefully with AiiA within them).
• This time, transfer the supernatant into another labelled tube and use this in conjunction with T9002 (AHL assay) to determine the concentration of AHL remaining in the solution. See the T9002 protocol for further information on how to use the part.T9002 Information Page, T9002 Protocol page

Our hypothesis would predict that the cultures grown in M9 with zinc ions as well as the LB will show a decrease in AHL concentration over time. The culture grown in our control volume without zinc ions should not show any significant decrease in AHL concentration over time.

1. Wang LH, Weng LX, Dong YH, and Zhang LH. Specificity and enzyme kinetics of the quorum-quenching N-Acyl homoserine lactone lactonase (AHL-lactonase). J Biol Chem. 2004 Apr 2;279(14):13645-51. DOI:10.1074/jbc.M311194200 |
2. Kim MH, Choi WC, Kang HO, Lee JS, Kang BS, Kim KJ, Derewenda ZS, Oh TK, Lee CH, and Lee JK. The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase. Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17606-11. DOI:10.1073/pnas.0504996102 |