If we want to significantly modify the expression of any genes, we need to modify the majority of chromosomes in each organism, a more difficult task than modifying one. This is because a deletion of a gene on one chromosome will not delete it from the entire genome, and likewise attempting to overexpress a gene on one chromosome will not consistently cause overexpression. Therefore we decided to use a replicative plasmid. Unlike integrative plasmids, replicative plasmids are not taken up by the genome and are preserved across several generations because of the strong selection pressure for the antibiotic resistance they contain. Unfortunately our use of a replicative plasmid meant that we had to constantly grow the transformed Synechocystis in the presence of Chloramphenicol to preserve the plasmid.
The plasmid we chose to use was pDF-lac due to its high rate of natural transformation between E.coli and Synechocystis. Although this allowed us to successfully transform Synechocystis, this plasmid is not bio-brick compatible, forcing us to perform an extra step in our project. We ligated our parts with the standard plasmid backbone pSB1C3 to submit to the registry, and then we had to perform digests again to ligate with the pDF plasmid. Many thanks to the University of Turku in Finland for providing this plasmid.
The biggest issue with using the pDF plasmid was that it was a low-copy plasmid. This meant it took far too long for any genes we were using to be expressed, and we would always see lower than expected expression. This, in conjuction with the lack of any biobrick compatible Synechocystis plasmid meant working with cyanobacteria was very difficult. If we were to continue with this project, or wanted to do continue working with this organism, we would work on creating a biobrick compatible dual host vector plasmid for Synechocystis and E.coli.
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