Team:UCSF/Results
EXPERIMENTS/RESULTS
PROJECT ACHIEVEMENTS
- We built a communication circuit in yeast that exhibits stable divergent populations after communication even though their responses to an initial stimulus are similar.
- We characterized several communication parameters to further optimize this output.
- We submitted and characterized BioBrick parts of almost all components of our circuit.
EXPERIMENTAL DESIGN
Our goal for our circuit is to elicit a divergent community response from two genetically similar cells by varying different communication parameters. To measure distinct community responses in our circuit, we will look at INDIVIDUAL (GFP) response versus COMMUNITY (RFP) response.
We quantify our readouts by inducing our genetic circuit with varying concentrations of doxycycline, our stimulus, and measuring the fluorescent readouts with flow cytometry at 0, 1.5, 3, and 5 hour timepoints.
BASIC CIRCUIT TESTING
To test the functionality of our basic circuit, we characterized and validated the fluorescent readouts for individual and community response to ensure they would provide accurate and robust measurements. To do so, we ran three different experiments:
- Characterization of our community response readout
- Validation of LexA transcription factor feedback sensitivity
- Stimulus induction of basic circuit with altered communication parameters
1. Characterization of Community Response Readout:
In our circuit design, our community response is read out by RFP fused with a LexA transcription factor chimera. This readout is regulated by an alpha factor responsive promoter (αFRP), either pFig2c or pAga1. It is vital that these αFRPs have a high dynamic range to distinguish between different alpha factor concentrations and see a difference between cell populations (i.e. activated vs. unactivated).
Our team characterized pFig2c (BBa_K1829002) and pAga1 (BBa_K1829005) with an alpha factor dose response to measure sensitivity to the community signal. Our testing supports that pAga1 is a better αFRP to use to measure community response in our circuit due to greater dynamic range and a higher expression peak.
