Difference between revisions of "Team:William and Mary/Binding Array"

Past iGEM teams have managed to create a dizzying number of unique transfer functions from their genetic circuits, and while altering the maximal expression level through ribosome binding sites (RBS) HYPERLINK is common among the iGEM community, there is oftentimes a need to change the threshold of a transfer function. That is, to move a transfer function horizontally. For the Circuit Control Toolbox to reach the stated goal of having orthogonal control over transfer functions we needed to find a way to orthogonally shift the threshold of a given arbitrary genetic circuit. In the end we managed to accomplish this by using molecular titration.

Molecular titration as the name implies is the process of titrating out molecules of transcription factor. That means, for some amount of transcription factor, a constant amount is taken away, such that for any given amount of transcription factor concentration, we are actually working with functionally less of said transcription factor (Figure 1).

Figure 1: Diagram showing the interactions between an activator transcription factor and decoy binding array, which as a molecular titrator. Note that the number of decoy binding sites impacts the equilibrium of free transcription factor, which in turn impacts the equilibrium of the amount bound to the promoter. Diagram adapted from Lee et al. 2012 (“A regulatory role for repeated decoy transcription factor binding sites in target gene expression”)

To accomplish this shift in E. coli we used decoy binding arrays, which are plasmids containing many repeated sequences of transcription factor binding sites, these repeated binding sites cause a large number of transcription factors to be bound to sites which produce no product, thus titrating them out, see Brewster et al. 2014 (“The Transcription Factor Titration Effect Dictates Levels of Gene Expression”). This causes a rightward shift in graph of transcription factor vs gene product. If we then graph a that same gene product versus a small molecule inducer for said transcription factor, then depending on the type of transcription factor we will either get a shift to the right (activator) or the the left (repressor). (See in depth example)

We would also like to thank the following organizations and offices which have generously provided us with the financial support necessary to carry out our project, in terms of either supplies or summer stipends.

Office of the Vice Provost for Research and Graduate Studies

William & Mary Dean of Arts & Sciences

William & Mary Roy R. Charles Center

William and Mary Office of Student Leadership Development

EXTREEMS-QED(NSF)

HHMI Undergraduate Science Education Grant

Arnold and Mabel Beckman Foundation

1693 Scholar Program

James Monroe Scholar Program

Donors to our Experiment Page