Team:USP UNIFESP-Brazil/Results
AlgAranha Team USP_UNIFESP-Brazil
Although we had to do a lot of unexpected troubleshooting, we managed to achieve some satisfying results at iGEM 2016! Check our list of main results here or go through our Lab Notebook for a REALLY complete view!
Doing iGEM in labs without an established molecular biology line was challenging, and although some features may seem small, they were surely fruits of hard work!
Positive results
- We were able to design and clone in pSB1C3 a protein domain part (BBa_K2136002) and a working gene expression cassete for microalgae (BBa_K2136010) !
- We could efficiently transform and characterize the expression of a codon optimized version of mCherry in Chlamydomonas reinhardtii by many methods, such as fluorescence spectrum, FPLC elution spectrum, DIY fluorescence and plate reading throughout cell growth! You can check this all in our Description and Proof of Concept pages!
- We could also multimerize our synthetic MaSp1 Type2 construct, 2-mer, at the DNA level, achieving constructs with 4-mer and 8-mer ! This multimerization was done twice trhough simple “cut-and-paste” reactions with restriction and ligation. You can read more about this at the Experiments page!
Negative results
- We tried to multimerize our synthetic MaSp1 Type2 construct through USER fusion(Nour-Eldin, Fernando, and Halkier), but were not able to get proper clones with multimers, as expected in our Experiments page. We had difficulties regarding the primers, having to wait until September before having proper ones available here, so we lacked the time for proper troubleshooting. However, we still believe it is a valid option for the assembly of multiple fragments (and we will continue to try it at our lab!).
- We could not clone and detect the enzybiotics in a expression plasmid, although we tried it repeatedly, as can be seen in in our Lab Notebook . This could be due to some toxicity of the parts as well as high background/inefficiency of cells. A good method for screening enzybiotics that we did not have the time to try would be testing the E. coli lysate or the C. reinhardtii supernatant against Staphylococcus aureus cultures in 96 well plates. Efficient clones would increase cell lysis and decrease the OD600.
References:
Nour-Eldin, Hussam H., Geu-Flores Fernando, and Barbara A. Halkier. “USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories.” Methods in Molecular Biology. N.p., 2010. 185–200. Print.
