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Team:SDU-Denmark/Tour51
From 2013.igem.org
Cloning
The beauty of creation
To clarify the progression of our cloning, the results will not be presented chronologically, but rather in a logical manner, starting with the simplest constructs. This is not an exhaustive list, but merely a presentation to clarify our work and give insight into our thoughts. Please consult our submitted parts-page (dig deeper), as well as our comprehensive protocols to gain full insight into our work.
pSB1C3-Plac-dxs (E. coli)
The dxs from E. coli was already present in parts registry (BBa_K118000), which simplified our aim to increase expression of the gene. However, the middle part of the brick was unsequenced, so this was an obvious focal point. The full sequence has been entered into part registry. User cloning was successfully used to clone Plac together with E.coli dxs. The picture shows the colony PCR result for the cloning and subsequent transformation. The gel picture shows the expected length around 2300 bp including verification primers.
pSB1C3-Plac-dxs (B. subtilis)
The literature suggested that dxs from B. subtilis performed better than its E. coli counterpart. Therefore, a colony PCR was performed on the B. subtilis strain 168. USER cloning was used to produce the construct pSB1C3-Plac-dxs (B. subtilis). Our tests of the construct showed unfortunately two restriction sites within the coding sequence.
