Difference between revisions of "Team:Yale/notebook"

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       <p>This week served as an orientation and planning period. The graduate mentors showed us around our lab space, and we practiced some lab techniques (such as proper aseptic technique and a transformation protocol in E. coli) with the Isaacs lab graduate students. The plan is to begin working with our rhizobia strains (Sinorhizobium meliloti 1021 and Rhizobium tropici CIAT) next week; we ordered some cyanobacterium Synechococcus sp. PCC 7002 from the Pasteur collection, but it is backordered and may take some weeks to arrive.</p>
 
       <p>This week served as an orientation and planning period. The graduate mentors showed us around our lab space, and we practiced some lab techniques (such as proper aseptic technique and a transformation protocol in E. coli) with the Isaacs lab graduate students. The plan is to begin working with our rhizobia strains (Sinorhizobium meliloti 1021 and Rhizobium tropici CIAT) next week; we ordered some cyanobacterium Synechococcus sp. PCC 7002 from the Pasteur collection, but it is backordered and may take some weeks to arrive.</p>
 
       <p>We laid out a series of experiments which would eventually allow us to express a nonspecific recombinase (beta-homolog) to incorporate foreign DNA into our organism's genome; this process is at the heart of the MAGE technique. Much of this week was spent identifying recombinases which could potentially function in our organisms through literature and BLAST searches. We also spent a significant amount of time identifying inducible promoters (either native to our organisms or within the BioBrick Registry) which we could use to express our recombinase.</p>
 
       <p>We laid out a series of experiments which would eventually allow us to express a nonspecific recombinase (beta-homolog) to incorporate foreign DNA into our organism's genome; this process is at the heart of the MAGE technique. Much of this week was spent identifying recombinases which could potentially function in our organisms through literature and BLAST searches. We also spent a significant amount of time identifying inducible promoters (either native to our organisms or within the BioBrick Registry) which we could use to express our recombinase.</p>
       <p class="text-center"><img src="https://2015.igem.org/File:Yale_iGEM_Framework_Design.jpeg"></p>
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       <p class="text-center"><img src="https://static.igem.org/mediawiki/2015/6/6a/Yale_iGEM_Framework_Design.jpeg"></p>
 
       <p>The first step in our plan is to determine optimal growth conditions, transformation protocols, and selection screens for our strains. Once this is complete, we can test the effectiveness of our identified promoters by placing them upstream of a fluorescent reporter; we will use the yellow-fluorescent citrine protein since its emission wavelength does not overlap with the autofluorescence wavelengths of our cyanobacteria. After identifying the most effective promoter (lowest leakiness and highest expression level when induced), we can replace the citrine gene in our construct with our synthesized beta-homolog genes. Testing the effectiveness of our recombinases will be a bit of a challenge; we need an assay to be implemented in each organism that will allow us to quantify mutagenesis efficiency.</p>
 
       <p>The first step in our plan is to determine optimal growth conditions, transformation protocols, and selection screens for our strains. Once this is complete, we can test the effectiveness of our identified promoters by placing them upstream of a fluorescent reporter; we will use the yellow-fluorescent citrine protein since its emission wavelength does not overlap with the autofluorescence wavelengths of our cyanobacteria. After identifying the most effective promoter (lowest leakiness and highest expression level when induced), we can replace the citrine gene in our construct with our synthesized beta-homolog genes. Testing the effectiveness of our recombinases will be a bit of a challenge; we need an assay to be implemented in each organism that will allow us to quantify mutagenesis efficiency.</p>
 
       <p>While all of this is going on, we will also need to knock out the mutS gene in each of our organisms. mutS is involved in identifying nucleotide mismatches during DNA replication. Since MAGE is founded upon such mismatches, it is in our best interest to allow them to go unnoticed by the cell's DNA proofreading systems. Silencing mutS should allow us to do this.</p>
 
       <p>While all of this is going on, we will also need to knock out the mutS gene in each of our organisms. mutS is involved in identifying nucleotide mismatches during DNA replication. Since MAGE is founded upon such mismatches, it is in our best interest to allow them to go unnoticed by the cell's DNA proofreading systems. Silencing mutS should allow us to do this.</p>

Revision as of 01:22, 16 September 2015


<!DOCTYPE html> Yale iGem 2015: Notebook

Lab Notebook