Part:BBa_K3738005:Experience

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Applications of BBa_K3738005

Our first step with these constructs was to transform them directly into our competent cells, and then to plate them on media containing the appropriate antibiotic for primary selection. This failed and no growth occurred, and we now know that this was because we were attempting to use the original constructs as delivered to us, which were still in the original pUC-IDT cloning vectors, so the bacteria were not able to express the antibiotic resistance genes included, and could not grow.

After this, we attempted to run an SDS PAGE on our transformed bacterial cells which had been incubating in media containing antibiotics. Because the bacteria were able to survive and grow in the presence of the correct antibiotics, we assumed that meant the transformation was successful. The SDS PAGE showed no bands of the size we would expect, and the conclusion we reached was that the bacteria had developed antibiotic resistance that was unrelated to the one attached to our constructs.

After the unsuccessful initial transformation attempts, we concluded that the reason it was not working was because of the vectors the constructs were in, and attempted to change the constructs from the pUC-IDT cloning vectors to pUC-19 expression vectors.

The first step of any attempts to work with our constructs were PCRs, in order to amplify the amounts of DNA we had to work with. We used Q5, Q5 high fidelity, and PFU PCR protocols, and of these, the Q5 gave us the most consistent results. Though the PCRs were largely successful, we still changed some of the parameters in order to increase our chances of success, such as the volumes of dNTP and water (raising the former, lowering the later).

After our PCRs, we needed to use a restriction digest to cleave our construct from the cloning plasmid. We initially used Tango for this, but we also used DnpI, a class IIM endonuclease. Our digestions often did not work, and when we ran agarose gels on the results we would only find bands corresponding to the full plasmids. After multiple unsuccessful digests, we concluded that the reason it was not working was because our Q5 PCR buffer was inhibiting DnpI activity. The final digests we preformed on our constructs after using a spin column to clean them so we could resuspend them in an appropriate buffer were successful, confirming our theory.

After our successful digest, we planned to preform a T4 ligation to insert our genes of interest into a Puc19 expression vector plasmid, which would then be transformed into bacterial cells. Since our constructs (and the associated antibiotic resistance genes) would then be on expression vector plasmids, we would theoretically be able to plate them on media containing the appropriate antibiotic and see growth. After that, we would preform an overexpression in LB media, and confirm the presence of our proteins of interest with an SDS PAGE.

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