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Team:TU Delft-Leiden/Project/Life science/EET/cloning
From 2014.igem.org
Extracellular Electron Transport Module - Cloning
Final constructs
The final BioBricks constructed for this module were:mtrCAB and mtrCAB-His
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mtrCAB and mtrCAB-His
The two BioBricks constructed using the mtrCAB genes were BBa_K1316012 and BBa_K1316017The template used to obtain the mtrCAB genes was the Ajo-Franklin plasmid [1], from which 5 different pairs of primers were designed to eliminate the main iGEM illegal restriction sites by means of the Golden Gate Assembly method.
(LINK TO GOLDEN GATE)
For the generation of the mtrCAB-His, the last reverse primer was designed in a way that a 6 x His tag was introduced at the C-terminus of the MtrB protein. This tag aims to be able to easily detect and purify this protein.Once the 5 fragments were PCRed, they were assembled together via the Golden Gate Assembly method. The Golden Gate primers were designed so that, due to the used BsaI enzyme, at the end of this assembly the 5' end of the assembled construct were compatible with a fragment restricted with EcoRI and the 3' end with a fragment restricted with SpeI. Consequently, the Assembled fragment was compatible with an iGEM backbone restricted with the enzymes EcoRI and SpeI. This way, the assembled construct was introduced into the pSB1C3 iGEM backbone.
Later on, the mtrCAB BioBricks (BBa_K1316012 and BBa_K1316017) were introduced into pSB3K3, because it is a backbone with a much lower copy number than pSB1C3, and in the literature the mtrCAB genes were found to be quite toxic for Escherichia coli.
Important note
It is important to notice that the actual assembly used the 5 fragments PCRed from the original Ajo-Franklin plasmid containing the mtrCAB genes. In the mtrCAB and mtrCAB-His figures it might seem that this happened in two steps, but that is due to the Software used to check the assembly "in silico" (SnapGene), which was limited to an assembly of four fragments.
ccmAH
References
[1] C.P. Goldbeck, H.M. Jensen et al., “Tuning Promoter Strengths for Improved Synthesis and Function of Electron Conduits in Escherichia coli”, ACS Synth. Biol. 2, 150-159, 2013.
