Team:HKUST-Hong Kong/overview.html

1. Background

In the past decade, the number of medical cases involving multi-antibiotic resistant microbes has seen a steady increase, and misuse of antibiotics has repeatedly been mentioned as one of the major reasons for the arise of such organisms. This situtation provided the inspiration for our project, which is to attempt to improve the effectiveness of antibiotics, so that we can apply less of it to achieve the same effect as we do now.

Our project name - E. trojan - has its origins rooted in the Trojan Horse found in the tale of the Trojan War. Similar to how the Greeks destroyed the city of Troy from within using a wooden horse filled with soldiers, our E. trojan also works to destroy an E. coli population that is under antibiotic stress from within through indole quorum-sensing disruption. [Top]

2. The Battle Plan

Indole has been proposed as a key signalling molecule for E. coli in mediating resistance to environmental stress.[1] In particular, indole is found to stimulate passive resistance against antibiotics by triggering stress-response mechanisms inside cells, including increased expression of drug efflux pumps and oxidative-damage repairing systems.

In a normal E. coli popoulation containing wild type and naturally resistant mutants, the mutants will supply indole to nearby cells that are under antibiotic stress. This is considered a 'charity' action as the mutant does so at a fitness cost to itsef. Our aim is to introduce E. trojan into the population and proceed to degrade indole via an enzyme (T4MO) and remove the charity effect. If the charity effect is significant, removal of its presence will cause a notable increase in the effectiveness of antibiotics. [Top]

3. Building the horse - Engineering a novel strain for antibiotics-free selection

To properly test the effect of indole degradation on the growth of an E. coli population under antibiotic stress, our E. trojan has to remain sensitive to the antibiotic used while it degrades indole. Else as a highly resistant strain itself, E. trojan will produce indole as well, lowering the efficiency of degradation. Nonetheless, molecular cloning needed to be done in order to introduce the T4MO gene into a bacteria. So here we encounter a problem: how to perform cloning without using antibiotics resistance as the selection marker?

There are existing methods to achieve this, including but not limited to auxotrophic selectoin and those that utilize toxin-antitoxins interactions. However, they have their own drawbacks and are not suitable for our purposes in our study of bacterial population dynamics.

Thus we propose the construction of EX, which would be a new bacterial strain that in theory can be selected for plasmid while remains possesses no antibiotic resistance. Selection can be performed under a simple change in physical environment – increasing the incubation temperature. The trick behind the mechanism originates from yeast artificial chromosome. It is the removal of an essential gene nadE and its relocation to a piece of extra-chromosomal DNA that we wish to maintain. [Top]

4. The army inside - Toluene-4-Monooxygenase (T4MO)

Toluene-4-Monooxygenase (T4MO) is a three-component enzyme system that is naturally able to degrade a variety of hydrocarbons. In particular, its ability to oxidize small aromatic compounds has caught the attention of scientists looking for biological means of producing certain useful organic chemicals for industrial use, resulting in extensive research on modifying its activity and affinity.

For our project, a mutated form of T4MO (double mutations at G103L & A107G) was chosen as the candidate for indole degradation. While there were several possible candidates, this particular one was known to convert almost 50% of the indole into 7-hydroxyindole (7-HI)[2]. This is of great interest to us as 7-HI has been documented to inhibit biofilm formation in Enterohemorrhagic E. coli[3], and quite possibly for other strains of E. coli as well. Biofilm formation is a major element that reduces the efficiency of antibiotics due to reduced area of action, and hence we were keen to take advantage of this synergistic effect to improve our construct.[Top]

4.1 Soldiers need shields...maybe - Multidrug Efflux Pump (Bcr)

As our novel strain will have one of its essential genes maintained in a plasmid, a lot of uncertainties are present regarding its fitness. It is quite possible that it will be more vulnerable to antibiotics compared with wild type and thus will be selected against when cultured together with wild type E. coli. This would reduce the effectiveness of our construct and is not desirable. Hence we planed an additional component to be maintained in the plasmid, known as Bcr, which we hope will boost the resistance of E. trojan. You will find more details on its construction here.[Top]

[1] Bacterial charity work leads to population-wide resistance
[2] Mutations of Toluene-4-Monooxygenase That Alter Regiospecificity of Indole Oxidation and Lead to Production of Novel Indigoid Pigments
[3] Enterohemorrhagic Escherichia coli Biofilms Are Inhibited by 7-Hydroxyindole and Stimulated by Isatin