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Team:Valencia UPV/Project/modules/methodology/parts construction
From 2014.igem.org
Project > Modules > Methodology > Parts Construction
Our team used a Golden Gate based DNA cloning system, called GoldenBraid in order to construct the different DNA parts of our project. Unluckily, GoldenBraid 2.0 and BioBrick standard parts are different. The parts of the two systems have different flanking recognition sites and the overhangs obtained upon digestion are also different. Therefore, we needed a strategy to convert GoldenBraid standard parts to BioBricks.
We sent 7 Parts to the registry. Four of them, the coding sequences from AtrΔ11, HarFAR and EaDAcT (Biosynthesis) and the TA29 promoter (Biosafety) were converted to the Biobrick standard in collaboration with the NRP-UEA-Norwich team, who very kindly provided us with their Mo-Flippers. These were modified pSB1C3 plasmids altered to include GoldenBraid restriction enzymes recognition sites.
In addition, we designed a new vector that can be used to convert GoldenBraid multigenic assemblies into the BioBrick standard. This new vector is a modified pSB1A3 plasmid with two BsmBI recognition sites leaving upon BsmBI digestion overhangs that match the GoldenBraid 2.0 grammar (Figure 1). We called this new vector "omega undercover" as it works as the GoldenBraid 2.0 omega vector with a pSB1A3 backbone. This vector was sent to the Registry, so it can be used for future teams using Golden Gate based cloning strategies.
Figure 1."Omega undercover" vector.
Using the omega undercover plasmid we constructed the remaining 2 Parts that were sent to the Registry (Figure 2). These were two biosafety devices that consisted of two modules, a male sterility module (See Biosafety) and an identity preservation module, expressing either yellow or blue chromoproteins.
Figure 2.Construction of parts/devices BBa_K1554004 and BBa_K1554005.
