pc2 Safety Manual | Certificate for pc2
Our bacteria will be engineered with potential to remediate sites contaminated with stubborn BTEX compounds; Benzene, Toluene, Ethylene and Xylene. Through proteomics, novel degradation pathways will be identified and cloned into our bacteria. Further, single enzymes of the degradation pathway will be investigated through enzyme studies to characterise their kinetics. An optimised BTEX uptake and degradation system based on the enzyme studies will then be introduced into our bacteria for improved degradation. Finally, a fluorescent biosensor to assess degradation efficiency will be constructed and introduced into the bacteria for live tracking of BTEX compounds and their degradation.
The overall aim is to integrate an optimised BTEX degradation pathway into E. coli. Existing BTEX degradation clusters and transporters will be isolated from Rhodococcus and Acinetobacter sp. introduced into laboratory E. coli strains. Once we have successfully engineered E. coli strains we will test its degradation efficiency by growing the organism in the presence of a variety of BTEX compounds, including but not limited to benzoate, xylene and toluene. Degradation of the BTEX compound will be tracked through GC-MS and growth curves of the E. coli strains. We will also test the specificity of the putative transporter found associated with the degradation pathway. The transporter will be introduced into a strain of E. coli that does not contain any functional transporter for BTEX transportation. Testing transporter efficiency will be done by transport assays and HPLC, after extraction of internal BTEX compounds. Novel degradation pathways will be identified in Rhodococcus and Acinetobacter strains through proteomics. Comparative proteomics will be carried out on organisms grown in the presence and absence of BTEX and through LC-MS/MS. Proteins showing increase abundance in the presence of BTEX will be identified as they may be involved in BTEX catabolism. These more abundant proteins will be investigated further by the established protocols mentioned above to confirm their involvement in BTEX degradation. After constructing the biosensor it will be tested in a fluorescence spectrophotometer with a range of benzoate and catechol concentrations. The sensitivity of the biosensor will be optimised through mutagenesis of its promoter and transcription regions.
None of the microorganisms or parts we are working with present immediate hazards. The ultimate goal of this project is to create bacterial strains that are optimised for the degradation of compounds found in oil and fuels. However, we believe it to be extremely unlikely that they could persist in high concentrations of these substances, therefore it is not foreseeable that they would threaten oil reserves or supplies. Still to prevent the unintentional release of these organisms all work will be conducted under the guidelines of the Australian Office of the Gene Technology Registrar (OGTR). Most of the mentioned BTEX compounds are carcinogenic and are highly flammable. These will be handled with appropriate precautions and will be monitored closely by our advisors.
BTEX compounds are known to be carcinogen and pose a serious health risk. The risk could occur if the BTEX compounds are mishandled or if accidental spillage occurs. As of now, there is no reason to move engineered organisms to another location. However, if it is the case there is the risk of accidental spillage and hence release engineered bacteria into the environment. Otherwise, if the internal safety system breaks down, the engineered organisms can escape the laboratory and get into nature. Hence, every time we work with these, we will be working in either a biosafety cabinet and/or using aseptic technique to decrease the risk.
Our ultimate goal would be to create a product which can be used for on-site bioremediation of BTEX contaminated sites. We envision that the engineered model organism would be released in the contaminted environment for bioremediation. However, it is not unthinkable that a native organisms could be taken from the site and engineered with our degradation pathway. Another potential use would be in factories and manufactering plants, here the engineered cells are envisoned to be in an enclosed device, where contaminated equipment will be placed. For the biosensor, we envision that it will be in an enclosed device for on-site tracking of degradation of BTEX