Difference between revisions of "Team:Cadets2Vets/Demonstrate"

Cadets2Vets has built a biological arsenic circuit that uses a combination of native DH5-alpha E. coli genes to detect arsenic ions in solution and genetic elements capable of producing GFP (Green Fluorescent Protein). After reading through academic literature, our team found a negative repressor that could be used as an arsenic regulator known as ArsR. Sensitive to arsenic ions, this protein will bind to an operon or promoter sequence and inhibit the transcription of the downstream gene in the presence of a transcription factor (in our case arsenic). Expressing a conformation change, this strategy allows us to control GFP expression using a protein sensitive to and capable of bonding with arsenic ions in solution.

Integrated DNA Technologies, an iGEM sponsor, provided us with free synthetic DNA to build our circuit. This sponsorship allowed us to select promoters and genes that fit our project. The molecular cloning strategy was to request two GBlocks that would contain a promoter-ribosome binding site-CDS-terminator sequence for constitutively expressing ArsR and regulating the expression of GFP via ArsR. We also added in a restriction enzyme site to both GBlocks that enabled us to combine the two GBlocks together.

Before running the actual circuit, we ran control experiments to determine whether or not the GFP would indeed function as a reporter, meaning it is expressed by the plasmid.

The first control experiment drove GFP using a constitutive promoter: meaning that the GFP would always be produced. This was essentially only used to ensure that the GFP works in the paper ticket.

The second control experiment was regulated, meaning that GFP expression was dependent on a trigger. This tested if the GFP could be controlled/ regulated.

These experiments were performed using a commercial in vitro transcription/translation kit by Promega.

The results from both of these experiments are shown in the pictures below.

Fig 4.1 and Fig 4.2 - The Positive Test Circuit (top) with first row containing 2 µL of test mixture, and the second row containing 4 µL of test mixture. Possible dose response is visible. The Negative Control Circuit (bottom), containing 2 µL of reaction mixture in first and second columns.