Part:BBa_K3426000:Experience

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Applications of BBa_K3426000

We used the the Toulouse 2018 team part, Sirius BBa_K2668020, as inspiration for our CBM2a design. However, Toulouse 2018 first used pSB1C3 as their backbone, then cloned into pET-28a(+) before performing protein production and purification. We decided to clone the gene block into pET-28a(+) from the beginning, before protein production, using Golden Gate Cloning. We chose to use pET-28a(+) as opposed to pSB1C3 because it would allow us to transition directly into protein production after growing successfully transformed BL21 cells containing our Golden Gate product. In the process of implementing our design, we faced some difficulties in amplification of the pET-28a(+) backbone, using our designed primers with BsaI sites for Golden Gate. This could be due to the large size of it, around 5.4kb, in comparison with pSB1C3, which is only around 2kb. When more DNA needs to be amplified through the Polymerase chain reaction (PCR), it can create mutations and errors along the way that Toulouse 2018 may have not experienced with a smaller backbone. Due to this difficulty, we have considered fitting our gene block into another plasmid to more easily amplify the backbone, and improve the efficiency of Golden Gate Assembly.

User Reviews

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