Team:Cornell/Sandbox/notebook

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Revision as of 04:07, 5 March 2013

Notebook

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March

We began the summer by holding a synthetic biology bootcamp in the DeLisa Lab. The purpose of this bootcamp was both to introduce new members to techniques in molecular biology and to get a running start on the cloning work for our project. During bootcamp, we successfully constructed both versions of our arsenic reporter, and attempted a Gibson assembly of a naphthalene-degrading plasmid. In late June, we transitioned from bootcamp to our permanent bench space in Dr. Archer’s lab in Weill Hall. After spending a few weeks setting up the lab space troubleshooting general issues, we successfully constructed both versions of our salicylate reporter and began an alternative approach to construct a plasmid with a naphthalene-degrading (nah) operon. In parallel, we realized that electroporation efficiency for Shewanella transformation is less than optimal—to say the least. However, we were able to conjugate our constructs into Shewanella using a protocol provided by Dr. Gralnick. As we transitioned into the fall semester in late August, wetlab work was divided into subprojects that could be accomplished in parallel. Subproject leaders independently worked on (1) characterizing our engineered Shewanella strains using reactors in the Angenent lab, (2) characterizing inducible promoters via qPCR of mtrB transcript in response to analyte, (3) characterizing inducible promoters in Shewanella via fluorescent reporters,(4) appending His-tags to MtrB in order to perform immunoassays, (5) performing site-directed mutagenesis on the nah operon to delete BioBrick cutsites, (6) constructing our final naphthalene-degrading plasmid to be conjugated into Shewanella, and (7) confirming that strains carrying the naphthalene-degrading plasmid can actually eat naphthalene.

April

From June 12th-22nd, the DeLisa lab kindly hosted a synthetic biology ‘bootcamp’ for our team members. Dr. Didi Waraho assisted in the instruction of new team members, while Taylor Stevenson helped more experienced members get acquainted with the Gibson assembly method. Didi’s group worked on the construction of our arsenic reporter plasmids (now referred to as p14k and p16k). This work involved PCR steps to append non-BioBrick cutsites (AscI and BamHI) to an existing mtrB BioBrick (BBa_K098994) and an arsenic-sensing region (BBa_J33201) in order to introduce modularity for easy promoter switching. By the end of the bootcamp, we confirmed via Sanger sequencing that both versions of our arsenic reporter had been successfully cloned into DH5a, electrocompetent E. coli cells. The next step would be to get the plasmids into our Shewanella strain lacking mtrB on the chromosome (JG700). Taylor’s group had one goal: To get internal PstI and NotI cutsites out of our 10kb naphthalene-degrading (nah) operon while constructing the final plasmid to be transformed into Shewanella. By using mutagenic PCR primers that would introduce silent mutations to get rid of the non-BioBrick compatible internal cutsites, the group planned on ripping the nah operon apart, so to speak, and putting it back together again and into a pBMT-1 backbone via Gibson assembly. Using the NAH7 plasmid (containing nah operon) fromPseudomonas putida G7 (kindly provided by Dr. Gene Madsen), Taylor’s group completed all PCR steps necessary for Gibson assembly, incubated the PCR products with Gibson master mix, and transformed the Gibson products into DH5a—yielding three transformants. At the end of bootcamp, the group ran supercoiled plasmid (miniprepped from transformants) on a gel—as an initial screen for successful construction of our naphthalene-degrading plasmid. As shown below, the gel showed distinct bands for all three lanes. However, further confirmation was necessary, so the next step was to digest the Gibson products to check for correct fragment lengths, and to submit DNA for Sanger sequencing.

May

We have successfully created working arsenic and salicylate sensing constructs and have shown that they are functional in Shewanella and are continuing to characterization of our arsenic and salicylate constructs. Although the response in our reactors is not as sensitive as we would hope, we have various hypotheses that we hope to test. Furthermore, we are using RT-qPCR, western blots, and fluorescence measurements to quantify transcription and translation of mtrB levels in response to varying concentrations of arsenic and salicylate. We also plan on running time courses to determine when mtrB is expressed in the presence of analyte. We are still continuing work on the nah operon. By using site-directed mutagenesis, we aim to make our nah part BioBrick compatible, and we still need to confirm successful conjugation of nah into our Shewanella strains.