Team:Duke/Description

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Project Description

Overview

When a cell makes protein, it is fairly simple to leverage the protein itself to see if the gene to make it is present. However, in all genes, the common component is the genetic code that makes it up. Is it possible to leverage the ease and predictability of nucleic acid interactions to better screen for proteins? Is it possible to signal for non-coding and knocked-out DNA sequences. In dCas9, we saw such a system.

With dCas9, Duke iGEM 2014 saw an effect we termed decoy binding where a fixed concentration of dCas9 will be lured away from a reporter gene by the presence of the same sequence elsewhere in the cell. As the number of decoys increases, the repressive force of dCas9 weakened on the reporter gene. In this phenomenon, we saw rudimentary signally based not on protein concentration but on DNA concentration. We saw that this behavior was worth investigating both at a basic level and then for use in applications.

We at Duke iGEM saw an opportunity of leverage the phenomenon of dCas9 decoy binding, studied as last year’s project, to directly signal the presence or absence of a set genetic sequence into the induction of a gene on a different operon. We hope that this construct allows for more versatility in recognizing a gene or non-coding region in the creation of more complex gene circuits. In order to highlight the capabilities of the circuit, we wanted to use it in the context of one of the most dangerous gene developments in modern history: antibiotic resistance.

One of the greatest discoveries since the germ theory of disease was the discovery of chemicals that could counter them. Penicillin and other drugs helped save countless lives and revolutionized the way we saw sickness. However, in 1947, just four years after the initial mass-production of penicillin, the first resistances began to appear. Already certain drugs like penicillin have fallen out of medical utility. The problem is only expected to become worse because fewer antibiotics are being developed and resistance is occurring for these drugs much more quickly than in the past. The CDC reports MRSA (multidrug-resistant tuberculosis) and other antibiotic resistant bacteria kill 23,000 a year^[1] in the US alone.

The reason for this spread is obvious, with each dose of antibiotic putting massive selective pressure on the antibiotic gene. Especially when an antibiotic regiment is left uncompleted, those bacteria resistant to the antibiotic will survive to repopulate. With each prescription of antibiotics, this effect is repeated until full populations are filled with the resistance. Non-medical antibiotic usages within agricultural waste and cleaning products have only exacerbated the problem.

But what if the game of growth rates was shifted to favor the non-resistant bacteria? Naturally, the favored solution would be to also kill antibiotic resistant during treatment; until that is possible, the next best solution may be a weaker and continual pressure targeting these genes themselves. This pressure will give the antibiotic susceptible bacteria an opportunity to regain a majority of the population between antibiotic treatments. These slow “resets” could buy time for the development of new drugs or allow for the recycling of old drugs previously left ineffective.

We attempt to provide a negative selective pressure through the induction of programmable cell death genes. We hope to produce a plasmid to transform, conjugate, and in the future transfect into bacteria that provides the necessary machinery for limiting the populations of only antibiotic-resistant bacteria. In this first stage of our project, we find a suite of programmable cell deaths in order to find a gene both effective on bacterial populations while maintaining complete safety for eukaryotic and especially human cells.

Cell Death Screening

We created adorable CELL DEATH GENES to terrify our ANTIBIOTIC RESISTANT BACTERIA.

Thermocycler

Because we thought ANTIBIOTICS were too 1955.

Antibiotic Detection

The MRSA were getting too big for their britches.

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• Imperial
• UC Davis
• SYSU Software