Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Ramona Emig, Julika Neumann, Lara Stuehn
Date created: 2015-08-29 11:00:00
Date modified: 2015-09-01 01:44:49
pOP - protein expression vector compatible with RFC[25]
| Types | DnaRegion |
| Roles | plasmid_vector Plasmid_Backbone |
| Sequences | BBa_K1621009_sequence (Version 1) |
Description
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.
Using a modified version of pSB1C3 (plasmid map pIG15_001) 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, this new 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 new vector, 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 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. 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.
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.
Notes
The original sequence of pET22b+ was modified to make it compatible to iGEM standard cloning procedures. This includes the removal of recognition sites of several restriction enzymes as well as the insertion of RFC[25] as a multiple cloning site. Further details can be found on the main page.Source
The sequence is a modified version of the common expression vector pET22b+. Five fragments that were needed for the assembly of the whole part were synthesized by Integrated DNA Technologies.| Sequence Annotation | Location | Component / Role(s) |
| BioBrick suffix RFC[25] BioBrick prefix RFC[25] T7 terminator VR beta-lactamase ColE1 ori lacI T7 promoter RBS VF2 | 1,30 4984,5012 105,151 159,178 339,1196 1354,1973 3391,4470 4857,4873 4876,4900 4906,4924 | feature/misc sequence_feature feature/misc sequence_feature sequence_feature feature/misc feature/primer_binding primer_binding_site CDS feature/cds operator feature/operator feature/cds CDS promoter feature/promoter feature/rbs ribosome_entry_site primer_binding_site feature/primer_binding |