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Team:TU Delft-Leiden/Project/Life science/EET/cloning
From 2014.igem.org
Module Electron Transport - Cloning
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The final BioBricks constructed for this module were:
mtrCAB and mtrCAB-His
ccmAH
mtrCAB and mtrCAB-His
Final constructs
The two BioBricks constructed using the mtrCAB genes were BBa_K1316012 and BBa_K1316017 (figures 1 and 2).
Cloning Scheme
Making a BioBrick of the mtrCAB operon is quite challenging. First of all, the coding sequence of mtrCAB contains several illegal restrictions sites. Secondly, we need to have the operon under the regulation of an adjusted T7 lac promoter, which was found to be the best promoter to express mtrCAB in E. coli [1]. To get rid of the illegal restriction sites as well as implementing the adjusted T7 lac promoter in our BioBrick BBa_K1316012 , we ended up having 5 pieces of DNA to be ligated into pSB1C3. To work efficiently, we used the LINK Golden Gate Assembly to clone BBa_K1316012 (see figure 3).
To generate BBa_K1316017 (construct with 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.
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 was 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.
ccmAH
Final constructs
The BioBrick constructed using the ccmAH gene cluster is BBa_K1316011 (figure 4).
Cloning Scheme
The BioBrick BBa_K1316011 was built up by amplifying the best performing promoter according to Goldbeck [1] and introducing it upstream the coding region of the ccmAH cluster of the Biobrick BBa_K917006 constructed by the iGEM team Edinburgh 2012, which was already BioBrick compatible.
A more graphical representation of the cloning strategy can be found on figure 5.
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.
