Team:TU-Delft/new Standard

New Standard

In our project multiple SUMO-peptide biobricks were submitted and characterized. All these biobricks are suited to use in the novel standard approach for the production and expression of proteins that would normally be insoluble or would get caught in inclusion bodies. The novel standard approach described below makes use of one of our SUMO-peptide biobricks (BBa_K1022101/2/3) and any gene of choice where PCR can be performed on. The PCR primers should be designed in such a way the PCR product will contain an Age1 restriction site upstream and a Spe1 downstream. Care must be taken to assure the two first codons introduced by the Age1 restriction site, both coding for glycine, are in frame with the protein to be fused with the SUMO tag.
e.g. xxx.ACC.GGT.zzz.zzz. xxx representing the SUMO biobrick, zzz.zzz. showing the frame of the protein to be fused.
A double restriction digestion should now be performed on any one of the SUMO-peptide biobricks (BBa_K1022101/2/3) with Age1 and Spe1. The same has to be done for the PCR product. After ligation of the two former a SUMO-Protein hybrid will be gained which can by brought to overexpression after induction with IPTG through the pT7 promoter. Cleavage can be achieved in vivo by simultaneously expression with Ulp1 (Part:BBa_K1022113), or in vitro after HIS-tag purification through the HIS tags both SUMO and Ulp1 are equipped with.