“A pared-down life form might serve as a useful chassis. Bolt on the right handful of genes and you could have an ecologically friendly microbe factory to produce drugs or biofuels or artificial meat.”
- James Mitchell Crow
The use of Genetically modified organisms or GMOs is widely accepted and has revolutionised our approach to real world problems. The objective of synthetic biology work is to create massive results with GMOs, without compromising the safety or security of any of the stakeholders involved in the process. But a major aspect of work with GMOs is Biosafety.
Biosafety as defined by the International Convention held in Geneva, refers to “principles, technologies, practices, and measures implemented to prevent the accidental release of, or unintentional exposure to pathogenic agents”. Hence, as iGEM’ers and synthetic biologists, the onus is on us to ensure that our modified bacteria are not only useful but also well contained, safe and have a benign impact on the user in any detrimental manner.
Biocontainment strategies involving risk based approaches by analysing the pathogen type allow us to contain the pathogen we are working with, thus protecting the workers and preventing unintended release of the pathogens from the labs
Warning: DO NOT ever fool around labs, you never know what you can do!
Depending on the risk type, pathogen type and various other factors, the required safety protocols and regulations are put in place to prevent unintentional release or exposure to the pathogen being worked with.
The objective of our project is to provide an alternative treatment to diabetes patients without impacting their health or well-being while preventing the release of our modified bacteria into the environment.
The human gut is colonised by millions of bacteria. A major concern with our proposed probiotic THE BIG PIE, is its release into the small intestine, which could enable horizontal gene transfer, resulting in lab-made plasmids interacting with other natural bacteria present in the gut microbiome. Keeping this in mind, we propose to use chromosomal integration of our parts in the final probiotic bacteria that we will introduce into the pills. The integration of our genetic circuits and cassettes into the chromosome will remove any possibility of horizontal gene transfer.
Our modified probiotic is required to be active only in the duodenum of the small intestine. To ensure this we have designed a kill switch that reduces the activity of the bacteria by the time it reaches the colon and kills it as soon as it is excreted out of the human body. (Yeah just like the mosquito racquet, the insect dies before reaching the other end of the mesh)
We propose a single gate phosphate regulated kill switch that consists of a toxin, anti-toxin and repressor system.
By using one of the most widely sought kill switch mechanisms, we have a constitutively expressed toxin and anti-toxin placed under two different promoters.
Colicin E2 DNase domain "miniColicin" BBa_K1976048, is a nicking endonuclease that can degrade Host DNA while posing little threat to the gut microbiota and is expressed under a constitutive promoter in the bacteria.
In the meanwhile, the Anti- toxin : Immunity protein (Im2) BBa_K1976027 has a strong binding affinity to E2 “miniColicin” and can suppress the activity of the toxin, thereby allowing the bacteria to survive in normal conditions. This is expressed under a cI lam promoter (derived from bacteriophage) which can be repressed by cI repressor protein.
The Repressor System
We express a repressor protein called cI repressor under a phosphate sensitive promoter PphoA. The repressor is transcribed under low phosphate conditions and is translated efficiently at phosphate conditions below 50 microMolar as found in sewage water, post excretion. The repressor binds to the cI lam promoter upstream of the anti-toxin thereby suppressing its activity.
The free toxin molecules in the bacteria environment increase the toxicity and kill the modified bacteria.