Team:Warsaw/Biosafety

=Safety=

This page is made mainly basing on questions published on Safety page. We've tried to answer any safety questions raised in case of our project.

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 ( K177026 ). 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 ( K177032 ), 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 ( K177039 and K177040 ) and MgtC promoter ( K177021, 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 taken into consideration that usage of elements from highly pathogenic organisms may cause the final product, BacInVader, to be pathogenic in special conditions. Remembering that we have implemented many control points allowing us to control whole process of bacteria invasion into eucaryotic cell. The first step of invasion is controlled by the bistable switch (more details here), switched on by IPTG introduction and heating to 42&deg;C. Expression of genes in the cytoplasmatic operon is also tightly regulated, requiring the presence of tetracycline and arabinose. Taking into account that the combination of all aforementioned elements is not likely to occur by chance, especially in a human body, we can be almost sure that, when BacInvader will be ready, it won't be dangerous until it's activated to work in eucaryotic cells. To increase the safety even further we have designed some ways of introducing a so called 'kill-switch', enabling easy induction of BacInVader's death even inside eucaryotic cells. However, opinion of some professors which got involved in our project is, that if even our system will work well, most likely there will be no doctor who would risk infecting his patient with pathogenic bacteria, even if it might lead to effective therapy of for example cancer. This is just ethic/mental issue - doctors usually stand away from any pathogenic organisms.

Environmental and public safety
We haven't found any prerequisites to assume that any of the parts prepared might be of hazard to the environment. However, a fully functional system, which is described on our Project page, may potentially be hazardous if proper precautions are not met. The same situation is in case of public safety.

Law regulations
There isn't any local biosafety group, committee, or review board at our faculty. However, every aspect of work with genetically engineered organisms is strictly regulated by acts of parliament and European Union directives.

Main act regulating work with GMO has been passed by Polish parliament on 22 June 2001, and further changed on 21 May 2003. The text of act can be found here (in Polish). One of the executive acts is the edict of Polish Ministry of Environment passed on 29 November 2002, defining the list of pathogenic organisms, their classifcation and required hermeticity. This list can be obtained here. Organisms which were source of some of parts we've used in our project (Listeria, Yersinia and Salmonella) are classified to II Category, what means that they are hazardous and might cause severe disease, although there are cures for them present.

Next source of law regulations of work with GMO are European Union directives, especially:
 * 1) directive 90/219/EWG passed on 23 April 1990, regulating limited usage of genetically  modified microorganisms
 * 2) directive 98/81/WE passed on 26 October 1998, which is changing the 90/219/EWG directive.

All of our work was in agreement with presented law acts.