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

From 2012.igem.org

(Difference between revisions)
Line 5: Line 5:
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.
-
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/.  
+
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/.  
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 site 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 is a test for an ''In vivo'' assay, where we expected that this process happened 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 site 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 is a test for an ''In vivo'' assay, where we expected that this process happened inside the ''E. coli''using a Cre recombinase enzyme expressed by the same bacteria.     

Revision as of 12:31, 26 September 2012