All the constructs we cloned for this year’s iGEM competition were meant to be used for overexpression of the respective proteins, either in a cell-free expression system or in an E. coli expression strain. Usually, vectors that are optimized for protein overexpression are low- to medium-copy plasmids. In contrast to cloning purposes, this is sufficient for protein expression. When a certain copy number is reached, the expression machinery of the cell is saturated and a further increase will not result in higher expression yields. High copy plasmids rather result in lower expression rates because more metabolic capacity is shared for replication.

Many E. coli strains optimized for overexpression carry a chloramphenicol resistance gene as a selective marker. Plasmids carrying the same marker are not suitable for protein expression for two reasons. First, double selection for successfully transformed colonies on a medium with two antibiotics is not possible. Second, there is no selective pressure which makes it an advantage for the cell to even keep the plasmid.
The iGEM standard vector pSB1C3 was designed for cloning purposes and thus is provided with an origin of replication yielding high plasmid copy numbers. Moreover, it carries a chloramphenicol resistance gene as selection marker which makes it a tough task to use this vector for expression purposes.

Using a modified version of pSB1C3 (plasmid map pIG15_001) for overexpression resulted in an unsatisfying protein amount (link to an early SDS gel by ProtPur?). Thus, we decided to use a common expression vector for further experiments.
Obviously, it is a lot of work to clone every part we want to send to the registry into an expression vector and additionally into pSB1C3 which exhibits a completely different cloning site. For those reasons, we aimed to add a vector backbone to the iGEM registry which is suitable for efficient protein overexpression and compatible for cloning parts derived from the registry in a standardized procedure.

Fig. 1: pOP (plasmid for Overexpression of Proteins). Expression vector based on pET22b+ that is fitted to iGEM standards by incorporation of RFC25 and removal of forbidden restriction sites.

The improved vector, called pOP – plasmid for Overexpression of Proteins (Fig. 1) - combines features needed for efficient overexpression of proteins with standardized elements derived from pSB1C3. Based on the commonly used expression vector pET22b+, some changes had to be applied to fit all the iGEM standards.
First, one of the standard cloning sites had to be inserted instead of the original multiple cloning site. RFC[25] seemed to be the most suitable for protein expression purposes, because it allows the assembly of several coding sequences because the scar which is left between the sequences does not result in a frameshift or a stop codon. This is advantageous for expression of fusion proteins on the one hand and enables to genetically fuse the protein of interest to a specific tag for affinity purification on the other hand. Additionally, signal sequences can be added to cause secretion of the protein which is a commonly used tool for simplification of protein purification.
Next, recognition sites for restriction enzymes which are used for the insertion of coding sequences had to be eliminated in the backbone, so those enzymes (namely AgeI, EcoRI, NgoMIV, NotI, SpeI, PstI and XbaI) remain single cutters.
To allow sequence analysis in a standardized way, the binding sites of the primers VF2 and VR as they are used by the iGEM registry have been inserted in an appropriate distance to the insertion site.

The final vector was designed theoretically based on pET22b+. Because the sites where adaptations had to be applied were distributed all over the vector, a multi-step mutagenesis approach was difficult. Instead, the vector was divided into five fragments which were pieced together in two steps by Gibson Assembly. The resulting plasmid is shown in figure x.

Taken together, we designed an improved plasmid for the overexpression of proteins that is compatible with iGEM cloning standards. We are happy to provide this new backbone to the iGEM registry and help future iGEM teams to reach high expression yields in an easy way.

Link to genebank file: BBa_K1621009.gb.