Team:HKUST-Hong Kong/overview2.html

2.1. Theory

0. Introduction
In order to quantitatively demonstrate the effect of indole charity as well as our construct’s ability to negate it, we have decided to perform a series of minimum inhibition concentration (MIC) tests, where we subjected different strains and mixes of E.coli to an antibiotic gradient and cultured overnight (18 hours). The OD600 readings of each test were recorded afterwards and will be shown in later sections.

I. Wild Type (RR1) MIC Test
Phase 1 - Kanamycin MIC test

Experimental Design and Aim:
RR1 is a derivative from the common strain K12 and is not known to have any antibiotic resistance other than for streptomycin. Hence it was arbitrarily chosen as the non-resistant ‘wild type’ for our tests. A simple MIC test was conducted for RR1 to serve as a benchmark for comparison with later experiments, and kanamycin was opted as the antibiotic of choice. This was primarily for two reasons:

First, the kanamycin resistance gene incorporated into our selection plasmids functions through producing a mutated ribosomal protein that is insensitive to kanamycin. Unlike some other forms of resistance where antibiotic molecules are directly inactivated, this method ensures that the antibiotic levels remain relatively constant throughout the experiment, as well as prevents the appearance of satellite colonies during plating.
The other reason for choosing kanamycin is because, being an aminoglycoside, it acts by inhibiting protein synthesis through binding irreversibly to the 30S ribosome. This causes it to be bacteriostatic at low concentrations while bactericidal at high ones.

Results:
However, since we are using another untested K12 strain, namely RR1, and our growth condition is normal LB liquid culture, RR-1 MIC was carried out in the first place to confirm the MIC value for the RR-1 source we obtained.

The result of the MIC turned out to be around 7~9µg/ml, which is slightly smaller than the result indicated by the paper. The difference in their genotype could be the dominant reason while the less nutritive culture we used may affect the testing result as well.(right or wrong????need to be proved)

Phase 2 - Kanamycin MIC test with indole supplement

Experimental Design and Aim:
Indole has been proposed as a key signalling molecule produced by unstressed (high resistant) E. coli as a form of ‘charity’ that grants stressed (low resistance) cells passive immunity against antibiotics. This enables such stressed individuals to continue to survive and proliferate. Indole functions by inducing the expression and activity of multidrug efflux pumps to expel antibiotics and toxins, as well as activating oxidative-stress protective mechanisms to minimize DNA damage.[1] In an attempt to prove and quantify this effect, we repeated the kanamycin MIC test, this time supplementing the LB medium with different concentrations of indole, ranging from 300µM to 2mM.

Results:
The effect of indole on the MIC for RR-1 various under different concentration. Naturally, the indole production of E.coli is around 300µM while under antibiotic stress, the production will decrease to undetectable level.[1] We think the indole concentration of low and high resistance mixed culture should be around 300µM as well. However, the optimal concentration for charity work is still unknown.

For the testing under indole concentration of 300µM and 500µM, we can see that the MIC for RR-1 increased to ( )which is in consistence with the result of our Mixed culture MIC posted later. The RR-1 is able to survive under kanamycin concentration of ( ) .

On the other hand, we also did some 1mM and 2mM indole MIC testing, which aims at finding out whether the over dosage of indole could kill the population instead of protecting them. The result shows that indole did have a killing effect at higher concentration and the MIC did decrease compared to the result of 300µM indole MIC.