Team:Waterloo/Results
Results
Multiple trials went underway for the growth of J2T in knockout MetE media to definitively confirm that the predicted results occured in regards to NCM/J2T growth. As the goal of the experiment was to assure that there was no “leaking” of methionine from the J2T cells when stimulated with methionine production, the following test matrix was performed:
Test Tube # Control Components Results 1 Experimental → M9 Dropout with NCM removed (2 mL) → Added JT2 (25 uL) → KM (2 uL) No growth 2 Positive → M9 Dropout with NCM removed (2 mL) → Stock culture NCM (2 uL) Minor growth* 3 Positive → M9 Dropout with NCM removed (2 mL) → Added JT2 (25 uL) → KM(2 uL) → MetE (100 uL) Growth 4 Negative → M9 Dropout with NCM removed (approx. 2 mL) → KM (2 uL) No growth 5 Negative → Stock M9 Dropout (2 mL) → KM (2 uL) → MetE (100 uL) No growth 6 Negative → Stock M9 Dropout (2 mL) → Added JT2 (25 uL) → KM (2 uL) No growth 7 Positive → Stock M9 Dropout (2 mL) → Added JT2 (25 uL) → KM (2 uL) → MetE (100 uL) Growth
//will figure out how to format this table soon
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 NCM strain was grown and removed from the methionine knockout media, had no growth once J2T was introduced into it. This was a positive result as this suggested that there was little or no “leaking” of methionine produced by the NCM during growth into the media, and thus the J2T did not have any means to grow.
The minor growth of NCM 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 NCM that were causing the introduced J2T to not be able to grow, and that the composition of the media was static.
Trial 3 showed with the repliaction of expirimental conditions and the addition of MetE that the reason that the growth was not happening in exsperimental 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 NCM 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 JT2 to grow. This was important as it is possible that trace amounts could have been consumed in the other trials by the NCM bacteria and thus were not reflected in the experimental trial.
Finally, the 7th trial indicated that the 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.