Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in sp. PCC 7002.

BACKGROUND

sp. PCC 7002 (henceforth ) is developing into a powerful synthetic biology chassis. In order to streamline the integration of genes into the chromosome, validation of neutral integration sites with optimization of the DNA transformation protocol parameters is necessary. Availability of BioBrick-compatible integration modules is desirable to further simplifying chromosomal integrations.

RESULTS

We designed three BioBrick-compatible genetic modules, each targeting a separate neutral integration site, A2842, A0935, and A0159, with varying length of homologous region, spanning from 100 to 800 nt. The performance of the different modules for achieving DNA integration were tested. Our results demonstrate that 100 nt homologous regions are sufficient for inserting a 1 kb DNA fragment into the chromosome. By adapting a transformation protocol from a related cyanobacterium, we shortened the transformation procedure for significantly.

CONCLUSIONS

The optimized transformation protocol reported in this study provides an efficient way to perform genetic engineering in . We demonstrated that homologous regions of 100 nt are sufficient for inserting a 1 kb DNA fragment into the three tested neutral integration sites. Integration at A2842, A0935 and A0159 results in only a minimal fitness cost for the chassis. This study contributes to developing as the prominent chassis for future synthetic biology applications.