IGEM:IMPERIAL/2006/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

• First, grow an overnight cultures of part <bbpart>J37022</bbpart> in plasmid pSB2AK3 (Ampicillin resistant) in 2 mL of LB with 50 μg/mL Ampicillin. Keep the culture in the 37°C shaker overnight. (20 min + 8-12 hrs)
• In the morning, prewarm 40 mL LB with Ampicillin in the 37°C 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 - ________

• Reculture the overnight culture in 10 mL of prewarmed LB with Ampicillin by diluting to an OD of 0.1 by using the following formula (5 min)

<amsmath>

\frac{0.1}{\mbox{OD of culture}} \times \mbox{10 mL}

</amsmath>

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

Amount of prewarmed LB with Ampicillin to use = ________ mL (10 mL - above result)

• Split the culture into two cultures and label one with +IPTG and the other -IPTG.
• In the +IPTG culture, innoculate the culture with IPTG with 100 μL of IPTG to simulate AiiA production. (5 min)
• At 20 minute time intervals (starting at t=0, then t=20, t=40, etc):
  • Remove a 1 mL sample from the batch culture
  • Spin down the 1 mL sample, and remove the supernatant
  • Resuspend the cells into 1 mL of LB with Ampicillin and vortex
  • Spin and resuspend again to ensure a higher removal of IPTG
  • Use the ELISA assay to determine the concentration of AiiA in the suspension, do 4 repeats to ensure accuracy. ELISA protocol here JS: Someone has to check the Elisa protocol to see if it is the one we are using. I'm not sure which kit you guys ordered, so I don't have any specifics on how to use the ELISA protocol.

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.

• Why are we doing this all over again? Unclear. JS: We are not doing the same experiment over again, this is just an extension to the first experiment. In our second method that follows, the data will allow us to measure the halflife of the enzyme AiiA just as we would measure the halflife of any radioactive substance k_deg values). The method above is a completely experimental method and may not work, so we would like to test both of the protocols to see which one works the best. The first protocol, as you will have noticed, is considerably shorter in time than the second one (since we don't need to wait for the AiiA to be produced). This is the only main advantage to using the above method instead of the normal method to determine halflife. 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. What's the significance of the 3 hours?
• 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 in a 37[[:Category:{{{1}}}|{{{1}}}]] shaker for approximately 1 hour until an OD600 of at least 0.1 is reached
• Dilute the culture to and OD of 0.1 using the above formula into 20 mL of LB with Ampicillin.
• Separate the culture into 5 different white capped test tubes each containing 4 mL LB with Ampicillin (to test 4 different IPTG concentrations).
• Introduce 1μL 4 μL of 10 mM, 1 mM, 0.1 mM, 0.01 mM, and 0 mM IPTG into the respectively labeled tube 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 in a 37C shaker for approximately 4 hours to allow the production of aiiA to reach a steady state. 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.
• Measure and record the OD600 of the batch culture. JS: Depends on if we can measure the OD at the same time we are doing the ELISA protocol. If we can, then this step can be omitted.
• Remove a sample of 400μL (or as specified for the ELISA immunoassay plate)JS: as above, the four samples will act as our repeats of the experiment. The ELISA protocol only needs 100 μL to work, so that is how much we will remove if we want to do 4 repeats using the same sample.
• Place the sample into a labelled eppendorf tubes
• 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?? Spinning allows the IPTG to go into the supernatant so that we can remove it. Having IPTG remaining in the solution may cause the production of unwanted AiiA
  • Remove the supernatant
  • Add 400 μL volume of LB medium
  • 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. Do multiple repeats as allowed on the ELISA plate. 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]

JS: This section as not yet been edited. Is the biosensor ready to be used and can we use the biosensor instead of the T9002 assay. It will prove to be less tempermental to environmental constrains and give us more accurate readings.

Equipment and Materials

• Equipment
  • Fluorimeter
  • Photospectrometer
  • Centrifuge

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

Protocol

• 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).

JS: This section has not yet been altered. I think it might just be easier to do the western blot technique for this part than to follow this protocol. Again, this is in response to the tempermental T9002 we have been using.

Equipment and Materials

• Equipment
  • Fluorimeter
  • Photospectrometer
  • Centrifuge

• Materials
  • E.coli DH5α Culture containing J37022
  • LB medium
  • M9 medium (Endy Lab Protocol)
  • Ampicillin
  • 0.1M AHL

Protocol

• Culture cells with part J37022 in 2mL M9 minimal media with 50μg/mL ampicillin (or appropriate antibiotic) and 2mL LB media with 50ug/mL ampicillin (or appropriate antibiotic)
• Leave overnight in 37[[:Category:{{{1}}}|{{{1}}}]] shaker.
• Reculture (1μL in 1mL concentration) JW - therefore 5μL for each culture into two separate 5mL M9 media with ampicillin batches (one containing 1 mM ZnCl2) and one 5 mL batch of LB culture and allow to grow to an OD600 of 0.1 (in exponential phase) which takes roughly 1 hour.
• Introduce XμL of 1 mM IPTG to induce AiiA expression into all three batch cultures. The IPTG concentration (X)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 placing the cultures in a 37C shaker for about 4 hours.
• Inject a quantity of 0.1 M AHL into the solution and vortex to mix. JW - How are we working out this quantity? - trial and error? JS: I presume that we already have accurate solutions of AHL made up?
• Every 20 minutes for 1 hour, vortex the solution, measure the OD, and aliquot a 500 μL 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 |