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

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This experiment was made to test if the loxP and the lox66 were working properly, it also allowed us to measure the Cre concentration needed for an ''in vitro'' recombination reaction. The particularity of lox66 is that it has an altered sequence at the end of it's left arm when compared to loxP (natural recombination site from P1 Bacteriophage). This sequence variation reduces affinity of the Cre recombinase for the arm.
This experiment was made to test if the loxP and the lox66 were working properly, it also allowed us to measure the Cre concentration needed for an ''in vitro'' recombination reaction. The particularity of lox66 is that it has an altered sequence at the end of it's left arm when compared to loxP (natural recombination site from P1 Bacteriophage). This sequence variation reduces affinity of the Cre recombinase for the arm.
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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 lox66 (BBa_I718016) from the registry was wrong, it had a gg instead a cg in its left arm. This part was corrected by the  iGEM11_Tokyo_Tech team (BBa_K649206) and by the iGEM11_WITS_CSIR_SA team (BBa_K537019), but no DNA was available in the registry. Anyway, we needed to synthesized it and test it as part of our PCR-primers, we used the proper sequence described by http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/.  
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Based on the report made by the igem2010 UT-Tokyo team and some papers [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/], the lox66 (BBa_I718016) from the registry was wrong, it had a gg instead a cg in its left arm. This part was corrected by the  iGEM11_Tokyo_Tech team (BBa_K649206) and by the iGEM11_WITS_CSIR_SA team (BBa_K537019), but no DNA was available in the registry. Anyway, we needed to synthesized it and test it as part of our PCR-primers, we used the proper sequence described by [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/].  
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We designed two primers, one containing the lox66 and one containing the loxP sequences, these primers amplified the ORF from the kanamycin resistance gene, flanked upstream by the loxP and downstream by the lox66, using PCR. These sites should be recognized by the Cre recombinase (from NEB company), which could circularized our linear PCR product. This is important because we don't want it to be degraded when inserted in the bacteria. This ''In vitro'' assay was a test for a posterior ''In vivo'' assay, where we expect that this process happens inside the ''E. coli'' using a Cre recombinase enzyme expressed by the same bacteria.     
We designed two primers, one containing the lox66 and one containing the loxP sequences, these primers amplified the ORF from the kanamycin resistance gene, flanked upstream by the loxP and downstream by the lox66, using PCR. These sites should be recognized by the Cre recombinase (from NEB company), which could circularized our linear PCR product. This is important because we don't want it to be degraded when inserted in the bacteria. This ''In vitro'' assay was a test for a posterior ''In vivo'' assay, where we expect that this process happens inside the ''E. coli'' using a Cre recombinase enzyme expressed by the same bacteria.     
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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.     
+
Based on the report made by the igem2010 UT-Tokyo team and some papers [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  in [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.     
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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.
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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 proof 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.
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This technology could help in the screening/development of candidate genes in a high-throughput manner for developing new standard biological parts.       
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This technology could help in the screening/development of candidate genes in a high-throughput manner for creating new standard biological parts, making faster and easier to produce tools for hacking biosystems.       
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Latest revision as of 03:51, 27 September 2012