Team:UCL/Safety

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B12 riboswitch
In order to address safety issues associated with our psychobiotics, we have developed a unique B12 riboswitch-mediated
containment strategy
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Background

Biosafety assessment is an integral part of developing new genetically engineered organisms. Along engineering psychobiotics, it was of high importance to us to constantly review the possible risks of our project. As part of our Human Practice, we have identified and prioritized the safety issues that can arise during execution of our idea. Our research suggested that prevention of unintended environmental release of our engineered bacteria is a main concern that remain to be addressed. We agreed that developing an integrated biocontainment strategy tailored to the gut conditions could significantly enhance the biosafety of our GEM and, thus, aid in translation of our project into a safe treatment for future generations.

We are developing a biocontainment device based on a B12 dependence mechanism. B12 synthesis is known to occur naturally in the human small intestine and sufficient B12 intake is critical for both humans and their gut microbiota. Hooper, L., Bry, L., Falk, P. and Gordon, J. (1998). Host–microbial symbiosis in the mammalian intestine: exploring an internal ecosystem. Bioessays, 20(4), pp.336-343. [Link][1] We designed our circuit in such a way that bacteria will only survive in the presence of vitamin B12, while its absence will trigger the production of benzonase toxin. Such a mechanism turns B12 into a vitamin for E.coli! With the additional supplementation of vitamin B12 in our yoghurts, we hope to be able to implement such a strategy for our psychobiotics, which will only be able to survive throughout production, delivery to customer and consumption as well as inside the host under gut conditions but will quickly die when exposed to the external environment.

Constructs

In order to engineer this system we have taken advantage of B12 riboswitch-mediated regulatory system that has been identified in a variety of prokaryotic species. Riboswitches are a class of noncoding RNAs that bind their ligand with high specificity and affinity. Structurally, a riboswitch consists of an evolutionarily conserved aptamer domain and a variable expression platform. Ligand binding to the aptamer brings about structural changes in both the aptamer and the downstream expression platform, leading to the regulation of gene expression at either the translational or the transcriptional level. We have decided to use E.coli btuB riboswitch which operates at the translational level. In our construct, the upstream region of btuB gene, consisting of riboswitch and RBS, is fused to the coding sequence of benzonase, an endonuclease from Serrata marcenens. Furthermore, in order to prevent the export of toxin outside the cell, the N-terminal export signal was removed from benzonase CDS.

Characterization

We plan to characterize the level of B12-induced in vivo repression of downstream toxin gene by evaluating the survival of bacteria engineered with our constructs at different levels of B12 supplementation. By integrating modelling, we hope to be able to fine-tune the affinity of our engineered riboswitches to the level that would allow for responsivity to human gut conditions.



Further work

Of course, we are aware of additional potential risks of our engineered bug such as horizontal gene transfer, so we are looking into additional safety measurements to mitigate the risk of our technology.