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Team:TU Delft-Leiden/Project/Life science/EET/cloning/GoldenGate
From 2014.igem.org
Extracellular Electron Transport Module - Cloning - Golden Gate Assembly
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, 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 our BioBrick.
References
1. C.P. Goldbeck et al., Tuning promoter strengths for improved synthesis and function of electron conduits in E. coli. ACS Synth. Biol. 2 (3), pp 150–159 (2013)
