BFS GOTChAs, April 2010

This page contains some GOTChA charts for possible rotation projects in the lab this summer.

Device Projects

The following GOTChAs are for projects that involve building new device technologies that can be used to push biological circuit design forward.

Integrated Load and Context Compensation (RMM)

Goals Design a transcriptional regulator that uses feedback to compensate for changes in load and changes in context and thus provides consistent performance independent of downstream loading or cellular context (cell strain, growth media) Example: A -| B, designed so that transfer curve is maintained across changes in downstream reactions involving B, cell strain, growth media

Technical Challenges

Objectives Characterize the performance of existing transcriptional regulators across 3 cell strains, 3 growth media and 3 loading conditions. Design a compensation circuit that measures output level and regulates device performance to compensate for uncertainty Idea: use RNA-based feedback in 5' UTR sequences to measure transcription and compensate Note: feedback dynamics should be 5-10X regulatory dynamics to allow for use in non-equilibrium circuits Measure the performance of one or more compensation circuits and compare to existing regulators Regulation circuitry should be "internal" to device, so that inputs and outputs are the same as traditional regulator

Approach

Fast Mechanisms for Biomolecular Feedback (RMM)

Goals Design a set of devices for implementing feedback circuits that have timescales measured in seconds instead of minutes

Technical Challenges

Objectives Identify the mechanisms and timescales of various feedback mechanisms in natural systems Example: feedback mechanisms involved in chemotaxis Use models to estimate the bandwidth of regulators based on transcriptional feedback, post-transcriptional feedback, translational feedback, post-translational feedback, and covalent modifications Formulate a test setup that can be used to characterize the speed of response of one or more circuits using different mechanisms Implement a fast-feedback mechanism using one of the following methods: RNA-based feedback using anti-sense interference RNA-based feedback using secondary structure Programmable scaffolds for phosphorylation cascades Measure the response speed of one or more regulation mechanisms and demonstrate seconds-timescale response

Approach

Cell Division Tracker (ES)

Goals Design a genetic construct that changes state upon cell division (i.e. Cells start expressing GFP, after cellular division cells now express YFP)	Technical Challenges Frequency of recombinases not well characterized Might need to use a combination between two irreversible recombinases or a reversible recombinase and an irreversible one If recognition sequences are not compatible may need to evolve recombinases to recognize different sequences that are compatible
Objectives Characterize different recombinases (example: reversible vs. irreversible, frequency of recombinase activity) Find a cell-cycle dependent promoter to use Organize recognition sites and proteins of interest in an operon Test the operon in cells (E coli or Yeast) Measure system characteristics such as % errors	Approach Use recombinases The simplest approach would be to use a reversible recombinase (like fin and hin [1]) under a cell cycle dependent promoter If we see too many double inversions need to device a more complex mechanism using a combination of recombinases

Robustness Projects

CAGEN (RMM)

Goals Design, synthesize and characterize a circuit that provides robust transcriptional regulation Win the 2011 CAGEN competition	Technical Challenges
Objectives	Approach

Consistent Gene Expression Level (ES)

Goals Design, synthesize and characterize an inducible gene circuit that will express a constant level of a protein regardless of inducer concentration	Technical Challenges Might have issues designing an ACR circuit synthetically
Objectives Design a circuit that exhibits a constant concentration of gene of interest (probably an xFP at steady state) we might be able to use ACR theory [2] Construct and characterize designed circuit	Approach Incorporate feedback into current inducer systems, possibly tie into a gene that is known to be tightly controlled