Transformation into Synechocystis sp. PCC 6803
We were able to show that the transformation vector we created BBa_K1945001, can successfully be integrated into the genome. Using the counter selection method developed in the Peebles lab at Colorado State University, we were able to successfully knockout MazF and kanamycin resistance from the neutral site slr0168. After the initial transformation, every colony was re-streaked on one plate containing Ni2+ and another plate containing kanamycin. Ideally, any colony that completely segregated BBa_K1945001 across every copy of the genome will only grow on the plate containing Ni2+.
As seen in the image below, almost every colony grew on both plates except for two colonies, 17 and 19 (indicated by the red arrows). Colonies 17 and 19 grew only on the Ni2+. Therefore, we subjected those two colonies to a colony PCR to see we had in fact inserted the correct size piece in our neutral site.The expected size of the product is 1.5 kb for a successful transformation. The gel below indicates that our two colonies do in fact contain the correct size piece of DNA.