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Team:TU-Eindhoven/Preparation
From 2013.igem.org
Preparation
Before a start could be made on the lab work it was of course of importance that detailed protocols and plannings were made alongside other preperations. The aim of this page is to provide an overview of these initial steps leading up to the lab work.
Construct Design
To be able to start work in the lab, DNA constructs, which will form the basis of all our lab work, were designed using GenomeCompiler and oredered at GeneScript. As these constructs will be the basis for such a large part of our labwork it seemed logical for us to go into the details of their design.
General Design
In the lab we wished to compare a number of different proteins which led to the design of a general construct in which only the specific protein sequence would need to be adapted for each of the proteins we wished to test. The general constuct can be regarded as follows:
Official iGEM Restriction Sites - FNR Promoter - RBS - M - G - S - S - 6-His Tag - Thrombine Cleavage Site - NheI Restriction Site - Protein Sequence - XhoI Restriction Site - Terminator - Official iGEM Restriction Sites - HindIII Restriction Site
The general construct was devised in this way to enable us to use this construct for almost all lab experiments meaning no other constructs will have to be designed and ordered seperatly. We will use the EcoRI restriction site in the official iGEM restriction site in combination with the HindIII restriction site to transfer the entire construct from the pUC57-Simple vector in which the construct has been ordered to the pBR322 vector in which we can perform anaerobic experiments testing the FNR promoter. We can use the NheI and XhoI restriction sites to transfer the protein sequence from the pUC57-Simple vector to the standard pET28a expression vector. Within the pET28a vector the proteins will be brought to expression aerobically allowing for protein purification, quantification and finally CEST imaging. Other parts of the general construct such as the His Tag, Thrombine Cleavage Site and the Terminator sequence have been included to optimise transcription and ultimatly expression.
