Difference between revisions of "Team:William and Mary/Notebook"

The other foundational limitation of genetic circuit construction addresses the inefficiency and unpredictability of the design and construction process itself. The progression from synthesizing parts into a circuit on a plasmid, to transformation and testing in vivo, is a lengthy and expensive process which furthermore is largely variable in terms of actual functionality of the final product [2].This often leads to a series of trial-and-error testing cycles whose products maintain a persistent level of uncertainty with regard to precise, predictable behavior. Although it is possible to achieve functional genetic circuits in this capacity, greater problems arise regarding the tunability of the product. The success of any genetic circuit relies on the ability to precisely tune a response to a range of input concentrations; it would therefore be desirable to obtain a reliable method for tuning circuit response, ideally without the need to rewire the internal workings of the circuit. This method would allow control over output expression to be implemented in a more rapid and predictable manner [3].

Week 1 (160529 – 160604)

✻ Resuspended parts from the kit.

✻ Resuspended gBlocks of ordered sequences.

✻ Created a functional UNS Standard Backbone, containing the UNS 2 and 3 sequences within the Prefix and Suffix.

✻ Cloned K2066001-K2066016 into UNS pSB1X3 Backbone.

✻ Performed Diagnostic PCRs to test primer design for Ribozyme / RiboJ Characterization subproject

✻ Transformed interlab devices, created glycerol stocks. Could not get IMP #1, #3, and (-) control to transform.

Week 2 (160605 – 160611)

✻ Constructed K2066024, K2066025, and K2066027 using Gibson Assembly (from K2066014, K2066015, and pTAC templates).

✻ We received training on the FACS (Fluorescence Activated Cell Sorter) Machine.

✻ Realized the need for repressor protein / fluorophore fusion proteins (LacI-mCherry, TetR-GFP, i.e.)

✻ Designed gBlocks of K2066028 and K2066029.

✻ Attempted to clone Addgene 240x TetO Sequence into UNS Standard Backbone.

✻ Attempted ICA with K2066002, K2066003, K2066004 to create a TetO w/ 8bp spacer 9-mer

✻ Constructed K2066030 and K2066031 from K2066015.

✻ Troubleshot ICA, attempted 6- and 12-mers of TetO w/ 8bp spacer.

✻ Attempted IPTG Induction of K2066014 + K2066016 cotransformations.

✻ Transformed remaining interlab devices, created glycerol stocks.