Editing BioMicroCenter:Research (section)

=== Identification of mutations in Caulobacter crescentus - [http://web.mit.edu/biology/www/facultyareas/facresearch/laub.html LAUB LAB] - [http://web.mit.edu/biology Biology]===
A major challenge in bacterial genetics is the identification of the molecular targets and pathways affected by newly discovered genes. Toward this end, one powerful technique involves the unbiased selection for mutations that are able to suppress the deleterious effects of gain- or loss-of-function mutations in the gene of interest. However, finding the genomic locations of these suppressor mutations by traditional mapping methods can be time and labor intensive. The BioMicro Center has worked with the Laub lab to bypass the need for genetic mapping by sequencing the entire genomes of mutant bacterial strains.<BR><BR>
The Laub lab, working in the bacterium ''Caulobacter crescentus'', has recently characterized a novel gene, sidA, which inhibits cell division in response to DNA damage. To identify the protein targets of sidA, the Laub lab performed a suppressor screen for mutations allowing cells to form colonies despite sidA overproduction. By directed sequencing of candidate genes, most of these mutations were mapped. However, one suppressor strain did not contain mutations in any of the known cell division genes and was directly sequenced to find the mutation.<BR><BR>
In order to generate libraries from this strain, the BioMicro Center piloted a new protocol using the Nextera tagmentation system. Standard approaches using fragmentation had appeared to be unsuccessful, possibly due to the high GC percentage of Caulobacter. The tagmentation system uses a Tn5 transposase to insert sequence tags into intact genomes, both tagging them and fragmenting them at the same time. This reduces the number of operator steps and avoids the need for sonication. With the suppressor samples, the Nextera system was directly compared to sonicated DNA prepared with the SPRI-TE. The sequencing data showed that the Nextera system was able to produce very even and consistent coverage of the genome and is now being offered as a service through the BioMicro Center.<BR><BR>