Editing BioMicroCenter:Research (section)

=== Screening yeast libraries for genes involved in DNA damage response - [http://web.mit.edu/biology/www/facultyareas/facresearch/samson.html SAMSON LAB] - [http://web.mit.edu/biology Biology]-[http://web.mit.edu/be BE]-[http://web.mit.edu/ki KI]-[http://web.mit.edu/cehs CEHS]===
A myriad of new chemicals have been introduced into our environment and exposure to these agents can have detrimental effects on biological systems. Many of these chemicals are thought to have mutagenic activity. Analysis of the cellular response to these potential toxins using ''S. cerevisiae'' can provide a description of systems level responses to environmental stress and identify new pathways of DNA damage response. High-throughput techniques have become important tools to establish and clarify toxicity-modulating pathways of potential environmental carcinogens.<BR><BR>
The BioMicro Center has worked closely with '''Laia Quiros-Pesudo''' from the Samson lab in multiple complimentary screening methods to identify the cellular systems that respond to DNA damage. The initial screening method involved performing barcode-sequencing (Bar-Seq) described by Smith et al. (Genome Res. 2009. 19: 1836-1842) on a haploid yeast knockout library. In Bar-Seq, each knockout strain is identified by two unique barcode sequences (“uptag” and “downtag” barcodes) that can be amplified from its genome and identified using Illumina sequencing, allowing the whole library to be grown together in a single vessel. The importance of each knocked out gene is then measured by comparing the frequency of the strain in the initial knockdown library pool to the frequency after subjecting the pool to an environmental stress which is summarized as the fitness defect ratio. Multiple conditions can be tested simultaneously by using a second molecular barcode added to the library that identifies the experiment. <BR><BR>
Using the Bar-Seq approach, the Samson lab was able to simultaneously analyze the frequencies of ~4,800 strains of ''S. cerevisiae'' in up to 19 treatments and doses in a single Illumina sequencing lane. The Bar-Seq method was able to reproduce previous results using a solid agar assay and the alkylating agent MMS (Begley et al, 2004). In addition, new groups of sensitive strains have been identified and analysis of these new pathways is currently underway.<BR><BR>
In addition to Bar-Seq approaches, the Samson lab is directly screening GFP-fusion libraries to identify proteins that respond to environmental stress. GFP-fusion libraries monitor both changes in protein expression and localization as a result of chemical exposure instead of survival. In these experiments, each strain is individually screened across a number of conditions, requiring significant automation to make the experiment feasible.<BR><BR>
In order to perform these screens, the Samson lab has relied on the Tecan EVO 150 liquid handler in the BioMicro Center and the Cellomics ArrayScan VTI HCS reader available through the CEHS Genomics and Imaging Core (similar instruments are also available through the Whitehead Institute and will be available through the Koch Institute).  The Tecan EVO150 performed the cellular treatment, fixation and staining of multiple GFP tagged library plates simultaneously and significantly improved the throughput of the library screen. Initial results have been promising.