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Single Insertion: Socket Feasibility Testing

Posterior to verification of the function ofλ-Red system, what we aim to do is to prove that our socket site can get easily knocked-in. Since the recombinational bacterial, Socket. Coli, wasn’t prepared, socket insertion can not be strictly attested on chromosome. Therefore a surrogate assay was performed to show if our socket circuit can be well knocked-in.

Figure 1. Validation of recombination in Single plasmid. Lane 1 represents the whole Single circuit; Lane 2 represents PCR product with primer A and B. The band of 1000 prove the recombination occurrence and the band of 300bp is on behalf of the double terminator. Lane is a negative control which only owns double terminators.

For imitating the situation where chromosome recombination occurs, we construct the designed chromosome socket circuit into a low copy number plasmid, PSB4C5, also named “Single plasmid” by us. To insert fragments into the socket circuit, we co-transform “Single” with λ-Red helper plasmid PKD46 and induce the bacteria cells with arabinose; then introduce kanamycin resistant gene segment added by “A” site and “B” site in each ends. After recombination, positive colonies are obtained and verified by PCR. Contrasting with chromosome recombination outcomes, recombination of plasmids seems to occurs easily and have higher recombination efficiency due to the higher copy number of plasmids relative to chromosome. However, PCR confirmation outcome displays that the recombination is incomplete also possibly because of plasmids’ higher copy numbers.

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