Week 10: Week 10 — Advanced Imaging & Measurement Technology

Final Project Homework:

Identify at least one aspect of your project that you will measure.

One aspect I will measure is the spatial response of receiver cells to a signaling source, by quantifying reporter intensity as a function of distance from sender cells. Another aspect I will measure, especially for Aim 0.5, is the activation of the fat-related proxy, using eGFP fluorescence as a readout for co-expression of ‘TesA in the receiver cells. Together, these give me two measurable outputs: whether the system forms a real gradient in space, and whether that gradient is successfully coupled to a fat-related expression program.

Describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements. What are the technologies you will use?

I would like to measure the following metrics:

a. I will confirm fragment sizes during cloning and validation steps to validate and check whether the expected DNA products are present using gel electrophoresis.

b. Receiver activation by tracking eGFP fluorescence intensity over time and, when possible, as a function of distance from sender cells using video timelapse. This would let me see whether the circuit is simply on or off, and whether it produces any spatial pattern rather than a uniform response. I would compare this against controls such as receiver-only culture.

c. ‘TesA expression, since the conceptual point of Aim 0.5 is to link fluorescence to a fat-related expression program. I would ideally use a protein-level assay kit such as an 4–20% Mini-PROTEAN® TGX™ Precast Gel to check whether a protein of the expected size is being produced. In principle, mass spectrometry could also be used to confirm protein identity more precisely, although for the scope of Aim 0.5 it would likely be a more advanced method than I strictly need.

d. Whether expression of ‘TesA corresponds to any increase in free fatty acid production (FFA). Even a coarse measurement here would strengthen the experiment, because it would move the result toward actual biological activity. For this I could use a free fatty acid assay kit / colorimetric assay such as this MAK466 Sigma-Aldrich Free Fatty Acid assay kit for a crude evaluation.

e. I would monitor culture performance: growth, viability, and environmental parameters like media conditions, temperature, and possibly pH. Since both the sender and receiver rely on engineered expression, these background conditions matter because weak growth or excessive burden could distort the interpretation of fluorescence or proxy expression. I plan to use standard microbiology measurements such as optical density, growth observations, and controlled culture conditions.