Team:USP-UNESP-Brazil/Plasmid Plug n Play/Results

From 2012.igem.org

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Our results suggest that this technology is possible, that the recombination system is able to circularize the PCR-product as soon as it enters in the cell, and before it is degraded by the cellular nucleases. This was also predicted in the results obtained from the mathematical model.     
Our results suggest that this technology is possible, that the recombination system is able to circularize the PCR-product as soon as it enters in the cell, and before it is degraded by the cellular nucleases. This was also predicted in the results obtained from the mathematical model.     
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Based on the report made by the igem2010 UT-Tokyo team and some papers (e.g http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/), the lox71 (BBa_I718017) from the registry was wrong, it had a cg instead a gc in the 8bp spacer sequence between the arms. Apparently, this part was corrected by the  iGEM11_WITS_CSIR_SA team (BBa_K537020), but the sequence of this group had the same error. The corrected part from iGEM11_Tokyo_Tech (BBa_K649205) was right but no DNA was available in the registry. So we decided to synthesized it and test it, using the proper sequence described  by http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/, and we submitted the correct DNA to the Registry. Our results suggest that the lox71 submitted by our group is working.     
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Based on the report made by the igem2010 UT-Tokyo team and some papers (e.g http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/), the lox71 (BBa_I718017) from the registry was wrong, it had a cg instead a gc in the 8bp spacer sequence between the arms. Apparently, this part was corrected by the  iGEM11_WITS_CSIR_SA team (BBa_K537020), but the sequence of this group had the same error. The corrected part from iGEM11_Tokyo_Tech (BBa_K649205) was right but no DNA was available in the registry. So we decided to synthesized it and test it, using the proper sequence described  by http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/. We submitted the correct DNA to the Registry and our results suggest that the lox71 submitted by our group is working.     
This was the first test of one of the six prototypes, we want to test the others in order to find out which is the best structure for developing a complete series of recombination plasmids that use this mechanism. As a prove of concept we developed a plasmid for expression in ''E. coli'', but this methodology could be use for developing plasmids for expression in another systems, like plants and yeast.
This was the first test of one of the six prototypes, we want to test the others in order to find out which is the best structure for developing a complete series of recombination plasmids that use this mechanism. As a prove of concept we developed a plasmid for expression in ''E. coli'', but this methodology could be use for developing plasmids for expression in another systems, like plants and yeast.

Revision as of 02:58, 27 September 2012