Part:BBa_K672000

THERMOGENIC RESPONSE NUTRIENT BIOSENSOR

This device was designed to improve growth and keep the capability of meassure Nitrates/nitrites under cold shock stress (10-12 hr of 20°C to 30°C).

The Bristol BBa_K381001 encodes an expression of GFP in response of Nitrate or Nitrites that expresses fluorescent signals upon nutrient detection, its principal application is that allows farmers to quantify soil nutrient content. The BBa_K410000 is a fusion of the HydB cold shock promoter, the OmpA a signal peptide, and AOX 1 . AOXa is a alternative oxidase found in Sacred Lotus. This is going to produce a increase of heat above ambient temperature as follow:

• From 20°C to 30°C the HydB promoter express well the AOX enzyme (Alternative oxidase, that generate heat).
• Approximately at 37°C the promoter will stop his expression.

By combining BBa_K381001 and BBa_K410000 we expected our part , the BBa_K672000 , to be able to sense nitrate and nitrites even after experiencing a cold shock at 20ºC, making our device employable in other places where the temperatures can be as low as 15ºC.

SubParts:

Characterization & Functioning Experience

Experiments with BBa_K672000

We designed the following experiment in order to: test the BioBrick operation in different substrates and temperatures, characterize the expression of the promoters, determine the strongest promoter for the device operation, define the most efficient temperature and substrate for the operation of this device, provide a way to survive at low temperatures and be able to measure nitrate.

We chose 4 temperatures:

• 5-10°C

• 10-20°C

• 20-30°C

• 37°C

These temperatures were selected so we could evaluate the response to cold shock in various ranges for our BioBrick. Since the lab equipments we had available couldn’t reach temperatures between 10-20°C this temperature was not used during the experience. In the end, the lab equipment allowed us to make the experiments in: 37°C, 23°C and 8°C.

We also chose 3 different Growth Media:

• Saline Solution

• Liquid LB

• Minimal Media

These growth medias where selected to evaluate the response of our BioBricks in different metabolic pathways.

We did Spectrophotometry measurments, obtaining the following results:

LB stands for Luria Broth; MM, for Minimal Media and SS, for Saline Solution.

Which leads us to the following conclusions:

1. The best growth is obtained in Luria Broth.
   
2. Even though we obtained some growth at 23°C in the LB substrate, the Biobrick doesn't fulfill our expectations and requirements.

Note: References in Part Design Page