II. Mixed Culture MIC Tests
Phase 1 - Wild type (RR1) with RFP-labelled kanamycin resistance strain (RFP) (99:1)
Experimental Design and Aim:
As metioned previously, when E. coli cultures are subjected to antibiotic selection pressure, a small number of naturally resistant individuals, at some cost to themselves, provide protection to other more vulnerable cells by producing indole, resulting in an overall enhancement of the survival capacity of the population in stressful environments. To mimic this naturally occurred phenomenon, a kanamycin resistant strain, which represents the mutants, was introduced into the RR-1 at 1:99 ratio. This kanamycin resistant strain was labeled with RFP for easy recognition. The ratio of kanamycin resistant strain, KanR/RFP, to RR-1 was recorded for later comparison with that of later mix culture assays.
Results:
We can clearly see the effect of the charity work from our result. Even under kanamycin concentration of 25µg/ml, which is half of the working concentration of kanamycin and almost 3 folds of RR-1 MIC, RR-1 is still growing rapidly and maintain the majority of the overnight culture. The OD600 result didn’t show a clear co-relation with kanamycin concentration and is floating around 1.1.
The column chart also shows that the ratio of RFP to RR-1 falls between ½ and ⅓ after overnight culture. This ratio may change when the kanamycin concentration approach its working concentration. We plan to prove this in our future testing.
Phase 2 - Wild type (RR1) with kanamycin resistance T4MO (GRP)
Experimental Design and Aim:
In order to interfere the indole charity work and obtain a more efficient selection, we introduce a plasmid which encodes Toluene-4-Monooxygenase (T4MO), an enzyme that catalyzes the oxidation of indole into indigo. To test its effect, we design a T4MO/KanR and RR-1 (1:1) mixture culture MIC test. In this test, we assume that the indole degradation rate of T4MO will be close to the indole producing rate of itself along with the mutated minority in RR-1, which means a lower MIC of RR-1 will be observed in comparison of the KanR/RFP and RR-1 mixculture due to the absence of the indole charity work. The ratio of T4MO/KanR to RR-1 was also kept in the testing for later comparison with that of the 3 way mix culture assay.
Results:
Since the charity work is simply being weakened by the introduced enzyme, RR-1 is still growing quite well under kanamycin concentration of 25µg/ml. However, the ratio of the two strain, T4MO/KanR and RR-1, is different from former result from mixed culture of RFP/KanR and RR-1. Under lower kanamycin concentration, RR-1 still remain to be the majority of the culture and the difference is not very obvious. When kanamycin concentration exceeds 10µg/ml, we can see that T4MO out-competed RR-1 and became the majority of the overnight culture. Comparing with the ratio got from RFP&RR-1 mixed culture, we can draw the conclusion that the charity work of is weakened and the efficiency of ( )
Phase 3 - Wild type (RR1), RFP-labelled kanR strain and GFP-Labeled KanR T4MO (98:1:1) [???]
It has been proved in the phase 2 that T4MO does interrupt the indole charity work. So in the next step, we plan to practice our model, which is introducing a T4MO strain into the environment predominantly consisting of RR-1with few kanamycin resistant mutants. By comparing the resulting ratio of RR-1 to the antibiotic resistant strain to that of the T4MO and RR-1 Mix culture, we may observe again the strong effect of indole; by comparing the resulting ratio of RR-1 to the antibiotic resistant strain to that of the KanR/RFP RR-1 Mix culture without T4MO, we would be able to tell how T4MO takes effect.
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