[BBG03-P07] Improvement of substrate-induced gene expression (SIGEX) method for exploring gene function in subseafloor sedimentary metagenomes
Keywords:gene function, metagenome
In this study, we attempted to improve the steps in constructing SIGEX library to obtain genome library with a larger number of inserted sequences and longer fragment size. The environmental DNA extracted from subseafloor sediment sample taken at offshore Shimokita Peninsula, Japan, was inserted into the SIGEX vector conferring evoglow gene that can form matured green fluorescent form of protein in both aerobic and anaerobic condition . By using topoisomerase adapted vector and optimizing ratio of vector to insert, as well as the conditions for electroporation, we could obtain SIGEX library with approximately 1×106 clonal variation with >800 bp of inserted DNA sequences. We performed gene-induction incubation by adding various organic compounds including halogenated phenol mixture, which showed induction of 0.078% of clones at 0.5 mM in concentration. In the presentation, we will show the detail of methodological improvements together with the sequence analysis results obtained from induction-positive clones.
 Uchiyama et al. (2005) Nat. Biotechnol., 23, 88-93  Drepper et al. (2007) Nat. Biotechnol., 25, 443-445