IGEM:IMPERIAL/2008/Bioprinter/Integration

_NOCT_

Integration in B.subtilis

General Mechanisms

Integration Mechanisms - Picture is adapted from Bacillus Genetic Stock Center

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	Sequence
ThrC	Threonine auxotropy	Sequence

Key issues

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