IGEM:IMPERIAL/2008/Bioprinter/Integration

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_NOCT_

Integration in B.subtilis

General Mechanisms

Integration Mechanisms - Picture is adapted from Bacillus Genetic Stock Center

Our Strategy

Gene Phenotypic Marker Sequence
PyrD Growth Media needs to
be supplemented with uracil		 Sequence
GltA Growth Media needs to
be supplemented with glutamate		 Sequence
SacA Growth Media needs to
contain glucose as a carbon source,
not sucrose		 Sequence
AmyE Disruption of α-amylase Sequence
ThrC Threonine auxotropy Sequence

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.

Key issues

  • How long homology is required
  • Promoter upstream?
  • Directionalilty
  • Max length for one site- probably fins examples
  • Assembly
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