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Downstream expression system

In our original design, if recombination happens, the next step is to express downstream gene turned off by “double terminator” before. Out of the whole design of our project are two kinds of downstream protein expression: GFP & Int.

In terms of GFP, we played the recombination experiment mentioned before to testify if the green fluorescence is observed. Unfortunately, although we got the positive outcome of the kanamycin-resistant gene recombination, green fluorescence isn’t seen.

Speaking of Int, we also devised a testing experiment to detect the downstream expression of Int after recombination, where three distinct plasmids got involved. These plasmids are “Socket”, “Set” and “PKD46”. As mentioned before, when Socket and Set co-transformed, the bacteria expresses green fluorescence; but with successful recombination of Socket, downstream Int got open, which functions as inverting the promoter towards RFP gene and express RFP gene.

Figure 1. Socket insertion by kanamycin-resistant gene. Lane 1~6 are PCR product of positive bacteria picked up on the kanamycin plate. 1000bp fragment serves as verification of recombination; 300bp bands represent double terminator which means recombination is incomplete.

On account of deficiency of the chromosome recombination Socket.coli, we also use low copy number plasmid PSB4C5 to simulate single copy number situation of chromosome. For the substitution of chromosome for plasmid, we should introduce the chl+ plasmid PSB4C5 to support the socket device that should have be placed into chromosome. As can be seen, without the chromosome recombinational bacteria, we have to devised a rather complex experiment to validate the downstream expression of Int by bringing in Set.

Specifically, the control group is easy to carry out by co-transforming Socket and Set; nevertheless, the experimental group needs several rounds of electrically transformation and competent cells preparation. In other words, the first step is to co-transform Socket and PKD46 into E.coli and prepare arabinose-induced competent cells. Next, kanamycin-resistant gene is transformed and triggers recombination. With set of negative control and PCR affirmation, kanamycin-resistant gene inserted bacteria are sure to gain. Lastly, the positive colonies are picked up to make competent cells and be transformed by Set. Although PKD46 and Set share with the same antibiotic-resistant gene, ampicillin R, temperature sensitivity of PKD46 is made use of to inhibit the PKD46 proliferation in 45℃ and thus cells carrying with Set get huge growth advantage under LB with ampicillin. In spite of no growth of control group, we still don’t get expected fluorescence outcome.

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