Methionine Sharing Experiment

Multiple trials went underway for the growth of empty JT2 in knockout MetE media to definitively confirm that the predicted results occured in regards to CcaS/R JT2 and empty JT2 growth. As the goal of the experiment was to assure that there was no “leaking” of methionine from the CcaS/R JT2 cells when stimulated with methionine production, leaking that could support the growth of another population. The following test matrix was performed to assess the possibilty.

Test Tube #	Control	Components	Results
1	Experimental	→ M9 - met with CcaS/R JT2 removed (3 mL) → Empty JT2 (50 µL)	OD = 0.010
2	Positive	→ M9 - met with CcaS/R JT2 removed (3 mL) → CcaS/R v2.0 JT2 (50 µL)	OD = 0.820
3	Positive	→ M9 - met with CcaS/R JT2 removed (3 mL) → Empty JT2 (50 µL) → Met (150 µL)	OD = 0.799
4	Negative	→ M9 - met with CcaS/R JT2 removed (3 mL)	OD = -0.011
5	Negative	→ Stock M9 - met (3 mL) → Met (150 µL)	OD = 0.004
6	Negative	→ Stock M9 - met (3 mL) → Empty JT2 (50 µL)	OD = 0.005
7	Positive	→ Stock M9 - met (3 mL) → Empty JT2 (50 µL) → Met (150 µL)	OD - 0.812

In this chart, a negative control refers to an experiment where there was an expectation that there should be no bacterial growth as per our hypothesis, and a positive control denotes an experiment where there should have been growth expected.

As can be seen from the chart, the experimental trial, in which the CcaS/R JT2 strain was grown and removed from the methionine knockout media, had no growth once empty JT2 was introduced into it. This was an encouraging result as it suggested that there was little or no “leaking” of methionine produced by the CcaS/R JT2 during growth into the media, and thus the empty JT2 did not have any means to grow.

The minor growth of CcaS/R JT2 as a positive in control in trial 2 was expected, and was done to confirm that there would be no adverse factors from the removal of CcaS/R JT2 that were causing the introduced empty JT2 to not be able to grow, and that the composition of the media was static.

Trial 3 showed with the repliaction of experimental conditions and the addition of MetE that the reason that the growth was not happening in experimental conditions was in fact the lack of methionine in the M9 media. This showed growth as expected.

Trial 4 indicated no growth as expected. This was carried out, with the removal of the CcaS/R JT2 and addition of the antibiotic, to confirm that all the bacteria was being taken out and that no other bacterial contamination was what was being seen in the positive control tests that had reported growth.

Trial 5 similarly was carried out with stock M9 to assure no environmental contamination was in the media from the beginning, especially bacteria that required external methionine to grow.

Trial 6 was carried out as a negative control to assure that the stock media itself was not contaminated with any nutrients that could be used by the empty JT2 to grow. This was important as it is possible that trace amounts could have been consumed in the other trials by the CcaS/R JT2 bacteria and thus were not reflected in the experimental trial.

Finally, the 7th trial indicated that the empty JT2 strain that was in this bacteria was healthy and could grow in the desired conditions within the M9 media, with the methionine and the KM from stock solution.

GFP vs Non-Fluorescent Flow Test Experiment

Our comparison of the flow cytometer against pour plating started with a fluorescent and a non-fluorescent culture of roughly the same OD values. Below our initial ODs and results from both the flow cytometer and pour plating.

	Fluorescent	Non-Fluorescent
Initial OD	0.405	0.430
Flow Cytometer 480nm Laser at 10mW	52%	48%
Pour Plating Percent total CFU/mL	52%	49%

These results lead us to believe that the flow cytometer is representative of pour plating, indicating that the flow cytometer is most likely capable of giving accurate readings of a 50/50 population of fluorescent/non-fluorescent cells.

MetE Metabolic Load Experiment

Four sample tubes of JT2 CcaS/R-containing cells in M9 were made, and all were placed in the turbidostat. The samples were made by inoculating the cells off a plate into complete M9 made with casamino acids as the amino acid source.

Two samples were grown under only red light, and two under only green light. The samples were optically insulated from each other. The cells were allowed to grow for 3 hours to acclimatize them to the light conditions they were being kept under.

Unfortunately, though the doubling times for the two green-light samples were 53.8 and 55.8 minutes, the doubling times for the samples grown under red light were 45.3 and 61.2 minutes. The red-light sample doubling times were too far apart to be averaged reasonably, and therefore no conclusion could be drawn from the experiment.

Additionally, the team realized that the metabolic load of MetE may be different in media made with casamino acids versus media made with an amino acid mixture without methionine. Therefore, the experiment was not repeated and estimates of metabolic load would henceforth only be obtained by observing growth rates under different light conditions in M9 made without methionine.

See pages 77 to 79 in the online lab book for additional details.

“Robot” Experiments

In order to automate experiments and make the designed system more useable, the team set out to create a "robot" capable of measuring cell fluorescence (and hence co-culture composition). See the "Automation" page for full details.

Single Population Behaviour Experiments

The co-culture we used for our proof of concept consists of two populations: slow growing DH5alpha with GFP and light-controllable JT2 with CcaS/R. Before mixing these two populations together, we needed to determine how they each behave on their own.

What is the doubling time of JT2+CcaS/R and can we control it?

This experiment happened over 7.3 hours and aimed to determine the doubling time of JT2 with CcaS/R under full green light, no green light, and medium green light in duplicates.

NOTE: Light Set up

Full green: indicates the green light was on 100% of the 7.3 hour run. No green indicates that the green light was never on. Medium green: indicates green light was on 50% of the 7.3 hour run Red light was on for 100% of the 7.3h run for all samples

For each green light ratio, two tubes of M9 (without methionine) were inoculated with JT2 cells with CcaS/R from an overnight culture in LB. All tubes were then grown in the turbidostat under one of the three green light conditions for 7.3 hours.

The first three hours of the run were dedicated to adjusting the cultures to their designated light condition. This was done to prevent any noise from previous growing environment of the cells (such as light contamination) [1] from effecting our measurements.

Once three hours had passed we began to monitor our cultures using OD₆₀₀ measurements in intervals of 20 min for 4.3 hours. Below outlines those measurements:

The doubling time of each population was determined by plotting log OD 600 values against time in minutes and taking the slope of the linear portion of the curve. Here are the growth curves for each of the samples and the doubling times we calculated from them:

In conclusion, growth of JT2 with CcaS/R is significantly slowed under red light in comparison to its growth under full or medium green light. Interestingly, JT2 in this experiment has a faster growth rate in medium green light compared to full green light. This may indicate that metE imposes a metabolic load on our cells if overproduced. However, further experiments need to be preformed to fully confirm that this is the case. We will preform another growth curve experiment at a wider range of green light ratios in the next few days to get a better idea on why see this.

What is the doubling time of our DH5alpha?

Refer to our lab book for experiments regarding the doubling time of DH5alpha. DH5alpha will be used as our competitior strain in co-culture experiments. These co-culture experiments will be run in the next few days. Look forward to seeing our results at the Jamb!