IGEM:IMPERIAL/2008/Bioprinter/Integration
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Integration in B.subtilis
General Mechanisms
Our Strategy
We have investigated the use of double crossover events to integrate our genetic devices into B.subtilis. In addition, we want to design integration biobricks, that allow site specific integration of parts and devices into the B.subtilis chromosome. We aim to create a series of integration bricks that will direct integration into neutral endogenous genes. By inserting into neutral genes our B.subtilis will still be viable and in addition, we will have a means of phenotypic screening to identify successful integration events.
Gene | Phenotypic Marker | Sequence |
PyrD | Growth Media needs to be supplemented with uracil - Uracil needs to be added to a final concentration of 40μg/ml to allow growth. This means that we would need to do a form of replica plating and compare which colonies cannot grow without this supplement [1] | Sequence |
GltA | Growth Media needs to be supplemented with glutamate - Glutamate needs to be added to a final concentration of 500μg/ml to allow growth. This means that we would need to do a form of replica plating and compare which colonies cannot grow without this supplement [1] | Sequence |
SacA | Growth Media needs to contain glucose as a carbon source - Transformants can only grow on minimal media containing glucose as a carbon source not sucrose. This means we would need to be careful what type of media we culture our cells in [1] | Sequence |
AmyE | Disruption of α-amylase - Transformants have to be grown on plates overnight and then stained with Grams iodine solution and incubated for a few minutes. Clear rings will form around colonies containing the amylase gene.Colonies without significant zones of clearing are amylase negative, and therefore presumed to be amy knockouts [2] | Sequence |
ThrC | Threonine auxotropy Threonine needs to be added to a final concentration of 50μg/ml to allow growth. This means that we would need to do a form of replica plating and compare which colonies cannot grow without this supplement [3] (N.B.Find More e.g. Protocols) | Sequence |
Key issues
- How long homology is required
- Promoter upstream?
- Directionalilty
- Max length for one site- probably fins examples
- Assembly
References
- Middleton R and Hofmeister A. New shuttle vectors for ectopic insertion of genes into Bacillus subtilis. Plasmid. 2004 May;51(3):238-45. DOI:10.1016/j.plasmid.2004.01.006 |
- Resnekov O. Role of the sporulation protein BofA in regulating activation of the Bacillus subtilis developmental transcription factor sigmaK. J Bacteriol. 1999 Sep;181(17):5384-8. DOI:10.1128/JB.181.17.5384-5388.1999 |
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www.bgsc.org