Difference between revisions of "Team:Ionis Paris/Cloning Strategy"
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Revision as of 10:59, 18 October 2016
In order to obtain synthesized DNA fragments without error, we chose to synthesis our big biosensor part of 3,305 bp in 3 smaller parts between 500 bp and 1,800 bp that we planned to then assemble together in the pSB1C3 backbone. Figure 1: Biosensor Part 1: (532bp) Figure 2: Biosensor Part 2: (1 785bp) Figure 3: Biosensor Part 3: (1 215 bp) We began with the amplification of this parts by PCR in order to increase our stock. We used primers A12 and A13, that respectively bind the prefix and the suffix. Figure 4: Primers A12 and A13 Figure 5: Part inclusion in pSB1C3 The second step was to store our 3 parts in plasmids and then bacteria. For this we digested and ligated our 3 parts in pSB1C3 (forming BB1, BB2, BB3), and transformed these biobricks in E.Coli DH5 alpha in order to amplify them. After a check of correct clones thanks to colony PCR, we purified our correct biobricks and sequenced them. Figure 6: Assembly of Biosensor Part 1 with Biosensor Part 2 The third step was to assemble Part 2 with Part 1.For this we digested and ligated Part 2 and BB1, and transformed the obtained biobrick (BB12) in E.Coli DH5 alpha in order to amplified them. After a check of correct clones thanks to a PCR colony, we purified our correct biobricks and sequenced them. Figure 7: Assembly of Biosensor Part 3 to the Biosensor Part 1+2 The fourth step was to assemble Part 3 with Part 1 and 2. For this we digested and ligated Part 3 and BB12, and transformed the obtained biobrick (BB123) in E.Coli DH5 alpha in order to amplify them. After a check of correct clones thanks to a PCR colony, we purified our correct biobricks and sequenced them. Please note that all parts and primers can be found in the "Parts and Characterization" page, along with the primer sequences and the sequencing files
Step 1: Synthesis of DNA parts
Part 1 (532 bp)
Part 2 (1,785 bp)
Part 3 (1,215 bp)
Step 2: Storage of our 3 parts in pSB1C3 => BB1, BB2, BB3
Step 3: Assembling of BB1 with P2 => BB12
Step 4: Assembling of BB12 with P3 => BB123 (Whole Biosensor)
