IGEM:IMPERIAL/2008/Bioprinter/Integration

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.

Key issues
How long does the homologous sequence need to be for integration? The integration vectors that are currently available contain two regions of homology to allow for double crossover events. These regions are typically made from the whole gene sequence, i.e. the 5' homology sequence corresponds the 5' half of the gene and the 3' homology sequence corresponds to the 3' half of the gene. This means that typically the regions of homology for a vector are between 500bp to 2000bp. Because we will be using DNA synthesis to construct our integration parts we are limited to size and use of integration parts greater than a few hundred bp is unfeasible. An early paper claimed that the minimal length of homology required was only 70bp length. However, a more recent paper has shown that 150bp was the minimal length of homology required for integration length2 How do we maintain the correct orientation? For the integrated devices to be expressed we need to make sure that they are integrated correctly in the 5' to 3' direction. Because we are using the double crossover integration orientation is easy to maintain because the two flanking integration sequences ensure correct orientation of integration. What is the effect of integration site on gene expression? A study using the PyrD, GltA and ThrC integration sites has shown that there is no significant difference in the levels of gene expression between these different sites of integration sites.

Maximum length of DNA that we can integrate? There have been various estimations over the maximum length of DNA that can be stability incorporated into the B.subtilis chromosome. There estimations vary from 10kb to 16kb for double homologous cross over events length3. For us it is unlikely that our design will contain more than a few genes in tandem and so we should not exceed 2-3kb for each construct.

Transcriptional Read through