- Bouyant Bacteria Project (BBP)
- GVP is a cluster of 14 genes involved in gas vesicle production in B. megaterium and has been succesfully expressed in E.coli. Not all genes are of critical importance and at least 8 genes are needed. Our aim is to improve the bouyant capacity of the vesicles in E.coli.
We are working on part BBa_I750016, a biobrick submitted by Melbourne iGEM 2007 team. The part consists of: gvpB, gvpR, gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, gvpT and gvpU in vector BBa_J61035. The gvpL part contains a 40bp repeat, which is repeated 10 times.
Future directions of Melbourne
- smaller functional biobrick
- → design primers for ligation strategy to knock out individual genes
- literature suggests gvpN might be a negative regulator of expression
- → knock-out might produce truly bouyant bacteria
- phenotype not affected by gvpL insert of repeat
- → why?
- gvpA and gvpC are to be included in mimimal set of 8 genes according to literature, but they are not present in biobrick
- → does gvpB have a similar function, and can it be substituted by gvpA or gvpC?
Own plan of action
- test insert length of gvp cluster in biobrick
- → E-genR-X-RBS-part-S-P in vector BBa_J61035
- check cluster for restriction sites
- → EcoRI, XbaI, SpeI and PstI are not allowed and not present in part
- → other sites might become a problem in future assembly protocols, and need to be silenced
- → promotors (constitutive, metal sensitive....)
- → RBS (substitute exsisting one)
- → add gvpA and/or gvpC, knock-out gvpN
- test initial phenotype with different constitutive promotors
(..) = dit element komt van nature niet (meer) op aarde voor.
Alle elementen met hogere atoomnummers dan 111, Roentgenium (Rg), zijn (nog) niet geratificeerd door het IUPAC.