Team:Freiburg/Parts
Biobricks
Since our project involved expression of many antigenic peptides we decided to share those antigen-sequences with the iGEM community. We obtained most of the sequences via paper research and we would like to give special thanks to the group of Prof. Dr. Michael Hust (TU Braunschweig) who provided us with expression plasmids for the Salmonella Typhimurium antigen and a corresponding single chain variable fragment. The codon optimization tool from Integrated DNA Technologies was used to improve most sequences for expression in E. coli. We also removed all restriction sites that are not allowed in RFC[10], so that all sequences are compatible with the submission vector pSB1C3. When we started working with our first plasmids for this project, we decided to use a specific nomenclature. Every plasmid name starts with “pIG15” which is short for “plasmid igem 2015”. According to this, we named the plasmids containing our biobricks in the shipping backbone pRIG15 (“pRIG” as in “brick”) In the table below we listed all our biobricks and our biobrick improvement, pOP. We decided on pOP as our favorite biobrick because it provides the iGEM communitiy with an iGEM conform backbone for protein expression. When we started our project we faced the problem of pSB1C3 being a plasmid designated for cloning colliding with our need of a plasmid for expression of proteins. We think that expression of proteins is a rather big topic for future iGEM teams, since working in-vitro is an upcoming issue of synthetic biology. (Beispiele? kommt noch) Expression of proteins is usually done in specialized expression strains, that in the case of E. coli often carry a resistence against chloramphenicol. That is why plasmids used for expression should have an additional resistance making selection of positively transformed clones possible. The iGEM standard backbone pSB1C3 only contains a chloramphenicol resistance making it difficult to use in combination with expression strains like E. coli BL21 or Rosetta. Since protein expression sometimes has negative consequences for the cell expressing the protein it is useful to have a tool, which allows induction of protein expression at a certain timepoint. One system which is often used is a lac inducible promoter system,that can be started with IPTG. pSB1C3 does not have any default promotor inserted at all, making protein expression more difficult because an appropriate promotor has to be inserted every time. One additional disadvantage of pSB1C3 is the high copy ori which is really useful for cloning, but disturbs protein expression. Therefore an iGEM backbone adapted for protein expression seemed to be a quite good idea. We improved the pSB1C3 standard vector in a way that it can be used to express proteins in E. coli but still exhibits all the properties of an iGEM standard vector. Details on which parts of pSB1C3 we changed and how we did this can be found here.
| biobrick | short description | detailed desription |
|---|---|---|
| BBa_K1621000 | Rubella Virus specific antigenic epitopes derived from glycoprotein E1 | pRIG15_6 |
| BBa_K1621001 | Varicella Zoster Virus specific antigenic epitopes derived from glycoprotein E | pRIG15_7 |
| BBA_K1621002 | Herpes Simplex specific antigenic epitopes derived from glycoprotein G | pRIG15_8 |
| BBa_K1621003 | Clostridium tetani specific antigenic epitopes derived from tetanus neurotoxin (TeNT_Hc) | pRIG15_11 |
| BBa_K1621004 | Human Immunodeficiency Virus specific antigenic epitopes derived from a polyprotein called gag/tat/pol/env | pRIG15_17 |
| BBa_K1621005 | Treponema pallidum specific antigenic peptides derived from bacterioferritin (TpF1) | pRIG15_18 |
| BBa_K1621006 | Salmonella Typhimurium specific antigenic protein (DHAD) | pRIG15_15 |
| BBa_K1621007 | scFv binding specifically to the Salmonella Typhimurium derived antigen | pRIG15_13 |
| BBa_K1621009 | Standardized plasmid backbone optimized for protein overexpression | pOP |
