Difference between revisions of "Team:Ionis Paris/Cloning Strategy"

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Revision as of 13:21, 10 October 2016

Step 1: Synthesis of DNA parts

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.

Part 1 (532 bp)

Part 2 (1,785 bp)

Part 3 (1,215 bp)

We began with the amplification of this parts by PCR in order to increase our stock. We used primers O12 and O13, that respectively bind the prefix and the suffix.

Step 2: Storage of our 3 parts in pSB1C3 => BB1, BB2, BB3

The second step was to stock 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 amplified them. After a check of correct clones thanks to a PCR colony, we purified our correct biobricks and sequenced them.

Step 3: Assembling of BB1 with P2 => BB12

The third step was to assemble the part 2 with the part 1. For this we digested and ligated the 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.

Step 4: Assembling of BB12 with P3 => BB123 (Whole Biosensor)

The fourth step was to assemble the part 3 with the part 1 and 2. For this we digested and ligated the part 3 and BB12, and transformed the obtained biobrick (BB123) 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.