Team:Warsaw/Biosafety

Overwiew

As it can be found at our Resources page, we have cloned, or attempted to, some interesting parts from enteropathogenic bacteria, like Salmonella enterolitica ser typhimurium LT2, Yersinia tuberculosis O:8 (8081) and Listeria monocytogenes EGD-E. As pathogenic microorganisms all their derivatives (and of course just themselves) are potentially hazardous.

Biohazard organisms

All steps of isolation of DNA from pathogenic organisms like Yersina tuberculosis O:8 (8081) or Salmonella enterolitica ser. typhimurium LT2 were done under the supervision of people trained and responsible for work with this hazardous material. Team members having contact with Salmonella or Yersinia cultures were special trained and have worked in specially allocated room. Genomic DNA was isolated using standard protocol and then treated like standard genomic DNA sample.

Potentially hazardous parts

We have managed to prepare listeriolysin from Listeria monocytogenes EGD-E as a biobrick part (). Due to its involvement in pathogenicity of Listeria and as it can be possible that bacteria expressing just listeriolysin may be able to invade eucaryotic cells (although literature data suggest that it requires other proteins like invasin or internalin), special safety measures should be taken in account, especially when working with bacteria transformed with this part. It includes obligatory use of laboratory gloves, and avoidance of any contact with transformed bacteria. All waste contaminated with bacteria expressing listeriolysin should be autoclaved. DNA containing listeriolysin should be also well protected because accidental transfer to other bacteria may lead to rise of potentially pathogenic organism.

Described measures concerns also other parts, which we didn't manage to finish. E.g internalinA from Listeria monocytogenes EGD-E () , especially co-expressed with listeriolysin may pose a serious danger, enabling bacteria expressing it to easily invade eucaryotic cells.

We have also attempted to make parts from Salmonella enterolitica ser. typhimurium LT2: PhoP/PhoQ ( and ) and MgtC promoter (, which are responsible for pathogenicity of Salmonella in low pH/Mg²⁺ conditions. Since they are just regulatory elements they do not require special treatment.

Regulation of bacteria invasiveness

It should be remembered that usage of elements of highly pathogenic organisms causes that final product, BacInVader, may also be pathogenic at special conditions. Remembering that we have implemented many control points allowing us to control whole process of bacteria invasion into ecuaryotic cell. First step of invasion is controlled by bistable switch (more details here), switched on by IPTG induction and heating to 42°C. Expression of genes in cytoplasmatic operon is also tightly regulated, requiring presence of tetracycline and arabinose. Taking an assumption that combination of all listed is not likely to occur together, especially in human body, we can be almost sure that, when BacInvader will be ready, it won't be dangerous until we modify it to work in eucaryotic cells without special induction. For this procedures we have designed some ways of inducing so called 'kill-switch', enabling easy induction of death of BacInVader inside eucaryotic cell.

Environmental safety

We haven't find any prerequisities to assume that any of prepared parts may be hazardous for environment. However, fully functional system, which we have described on our Project page, may be potentially hazardous if not treated well.