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Team:British Columbia/Attributions
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Special Thanks
Wet Laboratory Acknowledgements The Steven Hallam lab for hosting our iGEM team, providing us with laboratory space and reagents through the 2012 competition Dr. Bob Hancock of UBC for sending us a strain of Pseudomonas aeruginosa Dr. Jonathan Van Hamme of Thompson Rivers University for sending us the Dsz operon VWR, Dr. John Church, the Annual Giving UBC Department of Microbiology & Immunology and BIOL Alumni Appeal Fund, Harry Lim Incorporated and the UBC Faculty of Applied Science for donations of reagents and resources Human Practices Acknowledgements Ian Bell from UBC UILO and Emma Macfarlane from Gowlings for discussions regarding patents Peter Wynne from Chevron Canada, Alberta Innovates Technology Futures AITF representative, Karen Budwill, Oil Sands Leadership Initiative OSLI representatives, John Vidmar and Nicolas Choquette-Levy, and Dr. Dusko Posarac of UBC Chemical and Biological Engineering for discussions regarding industrial applications of microbial consortia Team Calgary for testing out and giving feedback on our intellectual property survey during its development. Computational Modeling Acknowledgements Dr. Susan Baldwin of UBC Chemical and Biological Engineering, Gurpal Bisra and Shing Hei Zhan (UBC iGEM alumni) for advice on the consortia model Niels Hanson from the Steven Hallam laboratory for advice on the pathway model General Acknowledgements Alberta Genetically Engineered Machines (aGEM) judges and Dr. Phil Hieter of UBC for their constructive advice on our project and presentationCollaboration with University of Calgary iGEM
We thank Dr. Lisa Gieg and University of Calgary iGEM for collaborating and splitting the DSZ pathway for characterization: We wanted to collaborate with Calgary because of the friendship and hospitality they showed last year! Considering that we were both working with the same operon, we decided that Calgary would take DszA and DszB while we would take DszC and DszD. We also set up meetings to see what else we could do in collaboration. We initially wanted to see if we could do anything regarding modeling together, given that we were working with similar systems. However, Calgary was more interested in bioremediation, rather than fuel processing. We then struck on the idea of collaborating on kill switches. Calgary wanted to characterize a few new induction systems, including a rhamnose promoter and two new riboswitches for their kill switches. They have yet to have success with their rhamnose promoter. They sent us the sequence before they attempted anything with it, and we used that sequence to design primers to get the rhamnose promoter out of the genome. Noticing the paucity of standardization of kill switches on the registry, we took it upon ourselves to develop a plan to test the efficacy of any new toxic protein that might be wanted to be tested for kill switch capabilities. There was a fair amount of talk about how best to do a death assay. We suggested using Dendra2, a fluorescent protein that could be 'broken' at a specific non-cytotoxic wavelength of light, allowing us to quantify metabolic activity after supposed death after toxic protein induction by looking for further expression of the fluorescent protein. They argued that a dormant cell may not express this protein, and we wouldn't really be looking at death. We then decided that we ought to instead focus on plating assays, for this better tests the viability of cells. We both tried testing existing kill switches on the registry, but were unable to get any of them to work. Calgary postulated that this was due to the arabinose promoter, which they could never get it to work. In the mean time, we both sequenced some of the toxic genes used as kill switches in the registry, and found that everything we sequenced was indeed what it claimed to be. Calgary also sent us the gene for HPAC, an alternative NADH-FMN oxidoreductase, to be used in place of DszD. It is a native E coli enzyme, and apparently works better, though over-expression causes hydrogen peroxide formation. We had plans to see if we could get it expressed in a pET vector to look for peroxide generation, but we have not done this yet. Lastly, they sent us the yddg- strain from David's old lab at U of A, which could be used in complementation studies. Kerner et al. (2012) used the yddg part because it increases the rate of export of tyrosine by 3 times, and increases overall tyrosine production. We have created a constitutively expressed yddg part that we would ultimately like use to check for complementation (yddg also confers resistance to inhibiting concentrations of aromatic amino acids), but we have not made competent cells from this strain.Our 2012 Sponsors
