Team:Freiburg/Parts
BioBricks
Our project involved the expression of many antigenic peptides. Their immunodominant properties make them feasible for many medical purposes. As ‘Health and Medicine’ is one of the most popular tracks chosen by iGEM Teams, we want to share the sequences encoding for these peptides with the iGEM Community. Thus, future iGEM Teams have the opportunity to take advantage of our research if they are planning to work in the field of diagnostics.
Most of the sequences were obtained by paper research and have been synthesized by Integrated DNA technologies, engaging their special offer for iGEM Teams. Before ordering, we used the IDT codon optimization tool to optimize the sequences for efficient expression in E. coli. Planning to submit the sequences to the Registry, recognition sites for restriction enzymes used for standard cloning have been removed without causing amino acid changes.
The group of Prof. Dr. Stefan Dübel (TU Braunschweig) provided us with an antigen specific for Salmonella Typhimurium and a corresponding single chain variable fragment (scFv). They generated the scFv themselves after identifying the immunodominant properties of the protein. Thanks to their work we are able to provide the Registry with a specific pair of antigen and scFv.
All our BioBricks are summarized in the table below. Additionally, the table guides you to more detailed info pages as well as to the corresponding Registry pages.
Although these parts significantly expand the Registry in terms of diagnostics, none of them is our favorite BioBrick. To reach the high yields of protein expression we needed for our project we used a commercial vector optimized for inducible protein overexpression. This was unavoidable as inserting the required parts (like the lacI gene or a lacI repressible promoter) into pSB1C3 did not result in satisfying protein amounts. Most likely these problems were due to the high-copy origin of replication wasting a lot of metabolic capacity on unnecessary replication.
So far, the only plasmid backbone in the Registry with a low-copy origin of replication is pSB6A1.
Therefore, we developed an improved version of this plasmid backbone by combining it with a commercial expression vector. Uniting features required for efficient protein overexpression and elements related to iGEM standard cloning, we enable future iGEM Teams to perform highly productive protein overexpression based on our new backbone.
Working in vitro is an upcoming issue in synthetic biology. The rapid development of chemical methods for gene or RNA synthesis are only examples emphasizing the importance of in vitro experiments. On the other side, the variety of methods for in vitro protein production is rather small1) making conventional protein expression and purification still an essential method. Therefore, pOP, the plasmid for Overexpression of Proteins, is our favorite BioBrick.
Find out more about the pOP vector and check if it also meets your requirements!
| BioBrick | short description | detailed desription |
|---|---|---|
| BBa_K1621009 | Standardized plasmid backbone optimized for protein overexpression | pOP |
| BBa_K1621006 | Salmonella Typhimurium specific antigenic protein (DHAD) | pRIG15_15 |
| BBa_K1621005 | Treponema pallidum specific antigenic peptides derived from bacterioferritin (TpF1) | pRIG15_18 |
| BBa_K1621004 | Human Immunodeficiency Virus specific antigenic epitopes derived from a polyprotein called gag/tat/pol/env | pRIG15_17 |
| BBa_K1621003 | Clostridium tetani specific antigenic epitopes derived from tetanus neurotoxin (TeNT_Hc) | pRIG15_11 |
| BBA_K1621002 | Herpes Simplex specific antigenic epitopes derived from glycoprotein G | pRIG15_8 |
| BBa_K1621001 | Varicella Zoster Virus specific antigenic epitopes derived from glycoprotein E | pRIG15_7 |
| BBa_K1621000 | Rubella Virus specific antigenic epitopes derived from glycoprotein E1 | pRIG15_6 |
