Difference between revisions of "Part:BBa K1621009"

Latest revision as of 20:08, 17 September 2015

pOP - a plasmid backbone for protein overexpression compatible with iGEM standard RFC[25]

Figure 1: Plasmid map of the expression vector pOP. pET22b+ was modified to fit iGEM standard cloning procedures. Protein expression under the control of a T7 promoter can be induced by IPTG. Ampicillin resistance is mediated by beta-lactamase. IPTG=isopropyl-β-D-thiogalactopyranoside.

This part contains the sequence for a plasmid backbone that is designed for efficient overexpression of proteins in Escherichia coli. 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.

Figure 2: Pelleted liquid culture pOP containing GFP coding sequence. The difference in the amount of expressed GFP with and without GFP in the pOP vector can easily be observed by the yellowish color of the pellet of the induced culture.

Figure 3: SDS-PAGE of pOP containing GFP coding sequence. GFP exhibits a molecular weight of 27 kDa, which is indicated by the arrow. SDS-PAGE was performed using 1 ml of cell culture. The enrichment of the target protein is clearly visible for the induced culture.

Using a modified version of pSB1C3 for overexpression resulted in an unsatisfying protein amount. Working with a common expression vector increased the overexpression efficiency significantly.

Obviously, it is a lot of work to clone every part you want to send to the registry into an expression vector and additionally into pSB1C3 which exhibits a completely different cloning site. For those reasons, a vector backbone was added to the iGEM registry which is suitable for efficient protein overexpression and compatible for cloning parts derived from the registry in a standardized procedure.

The improved vector backbone, called pOP – plasmid for Overexpression of Proteins - 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 (BBa_G00100) and VR (BBa_G00101) as they are used by the iGEM Registry have been inserted in an appropriate distance to the insertion site. Aditionally, these primers can be used to verify successful insertion of a part by colony PCR.

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 that have been synthesized by Integrated DNA Technologies. In the first step, two or three of the fragments were assembled by fusion PCR. The two resulting fragments were pieced together by Gibson Assembly. The resulting plasmid is shown in figure 1.

We inserted the coding sequence for GFP (BBa_I13504) into the improved expression vector pOP using EcoRI and PstI for restriciton. After ligation of the digested pOP plasmid with GFP we transformed E. coli BL21 cells with the ligation mix.
Correct clones were selected for further analysis. To show that the new improved backbone is capable of protein overexpression upon induction with IPTG, two cultures of 5 ml each were inoculated from one colony. Both cultures were incubated at 37°C until they reached an OD₆₀₀ of 0.5. At this time point one culture was induced with IPTG in a final concentration of 1 mM, whereas in the other culture protein expression was not induced. Both cultures were grown for several hours after induction. Successful expression of GFP was observed by eye (figure 2) and analyzed using SDS-PAGE (figure 3).

Taken together, we designed a new 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.