Cfrench:BioBrickVectors1

Making a Broad Host Range BioBrick Vector

As part of the iGEM2007 competition, the University of Edinburgh team sought to make a broad host range BioBrick vector that could be used to transfer BioBrick constructs into hosts other than Escherichia coli. Since we were working on production of flavour and pigment compounds in yoghurt, we initially wanted a shuttle vector that could operate in both E. coli and in Lactobacillus spp. We contacted Prof. Mike Gasson at the Institute of Food Research, Norwich, and he and his colleague, Dr. Claire Shearman, kindly provided us with vector pTG262. This 5.6 kb rolling-circle plasmid was known to replicate in E. coli, Bacillus subtilis, Lactococcus and Lactobacillus spp., all from the same replication origin. This raised the question: if this replication origin works in E. coli, might it also work in other useful Gram negative bacteria such as Pseudomonas, Shewanella, Agrobacterium and Rhizobium?

Making pTG262 into a BioBrick vector

pTG262 possessed a multi-cloning site with PstI and EcoRI sites, and also a unique XbaI site between them. There was no SpeI site present. By inserting a BioBrick between the EcoRI and PstI sites, we removed the XbaI site and replaced it with a full set of BioBrick cloning sites including EcoRI, NotI, XbaI...(insert)...SpeI, NotI, PstI, thus converting pTG262 to a fully compliant BioBrick vector which we have temporarily named pTG262-BB, until it can be given a proper BioBrick vector name by the Registry. We inserted three different BioBricks in this way: two encoding the monomeric DsRed version of Red Fluorescent Protein (BBa_J04450, with a lac promoter, and BBa_I13521, with a tet promoter) and one with the lacZ' minigene (BBa_J33207). Red and blue colonies were obtained in E. coli JM109, though transformation efficiencies were not high (see below). We also transformed B. subtilis 168 with pTG262BB-BBa_I13521 and obtained chloramphenicol-resistant colonies, but RFP was apparently not produced. This may be due to a promoter issue, or DsRed may not work in B. subtilis for some reason. We are currently investigating this further.

Information about pTG262

Most of the information I have been able to find about the history of pTG262 comes from W.M. De Vos and G.F.M. Simons (1994) 'Gene cloning and expression systems in Lactococci', chapter 2 (pages 52 to 105) in 'Genetics and Biotechnology of Lactic Acid Bacteria', edited by M.J. Gasson and W.M. de Vos, Blackie Academic and Professional, London, ISBN 0 7514 0098 X, Darwin Library classmark QR 121 Gen.

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