Team:Virginia/demonstrate

TRANSFOAM

Team Attributions
Description Design Experiments Model Safety
Results Future Directions Demonstrate
Parts Collection
Integrated Human Practices Public Engagement Collaborations Mid-Atlantic Meetup
Awards Medals

We plan on demonstrating the functionality of our device and model in the two weeks leading up to the Jamboree. We will be presenting these results during the competition.

Demonstrating our Device

To fully demonstrate the success of our device, we must prove that it can produce PHBs from styrene. This can be done by growing our device in styrene medium and assaying for bacterial growth and PHB production. The procedure is detailed below.

Procedure:

  1. Begin two bacterial cultures of our device with both Chloramphenicol and Ampicillin antibiotics. Cultures will be grown at 37°C for 72 hours in a shaking incubator. Culture 1: 200 mL of M9 medium; 4mL of styrene + 400 uL glucose as the carbon source, and 1mM IPTG. Culture 2: (Negative control) 200 mL of M9 medium; 400 uL glucose as the carbon source, and 1mM IPTG
  2. Observe the growth rate of the two cultures by measuring the optical density (OD) at regular intervals for a total of 72 hours.
  3. After 48 hours of growth, the cell cultures can be assayed for PHB production using Nile Red
  4. After 72 hours of growth, PHBs can be extracted using the Density Centrifugation protocol, dried and weighed to determine yield.

Demonstrating our Model's Function

To demonstrate the function of our genome-scale metabolic network (GEM), we will experimentally determine the percent yield of our device grown on M9 medium using glucose as a carbon substrate. The following procedure will determine the percent yield based on the theoretical maximum determined from our Flux Balance Analysis model and experimental yield from wetlab.

Procedure

  1. This experiment will provide the following input formation for the model: (1) growth rate of our device with glucose as the carbon source, (2) change in glucose concentration, and (3) experimental PHB yield.
  2. Begin bacterial cultures of our device with both Chloramphenicol and Ampicillin antibiotics. Culture cells at 37°C for 72 hours in a shaking incubator. Take note of initial glucose concentration.
  3. Observe the growth rate of the two cultures by measuring the optical density (OD) at regular intervals for a total of 72 hours. After 72 hours, measure final glucose concentration using Beer’s Law.
  4. Extract PHBs following the Differential Centrifugation protocol. Dry and record the mass of the PHBs.

Analysis:

  1. Input experimental glucose uptake as a constraint for the GEM.
  2. Run robustness analysis for PHB production based on glucose uptake to obtain the molar ratio for maximum PHB yield.
  3. Compare experimental and optimized molar ratios to obtain the percent yield of PHB production by the pha plasmid when grown on glucose.
  4. Repeat the previous three steps, this time including the experimental growth rate to produce a more constrained PHB percent yield.

The results obtained from this procedure will provide an experimental ratio of PHB to growth rate. This ratio further optimizes the model to produce a more robust theoretical maximum yield. The percent yield obtained from this procedure will also allow us to identify the efficiency of the pha plasmid, which will direct future improvements of our device.