Part:BBa_K672000

THERMOGENIC RESPONSE NUTRIENT BIOSENSOR

This device was designed to improve growth and keep the capability of measure 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

FIRST ROUND OF EXPERIMENTATION

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.

SECOND ROUND OF EXPERIMENTATION

We decided to do a second round of experimentation in order to sustain the operation of our BioBrick and going deeper into the characterization.

Objectives:

1. Perform further experiments with our device.
2. Improve the characterization of THE RENBO.
3. Determine the relationship between the parameters we are studying (gene expression, temperature, substrate and thermal method).
4. Obtain the proper functioning of our device.

Methodology:

-We will use 10mL of substrates (liquid LB)
-We will have controls with and without antibiotics
-We will use colonies of the following:
• JM109 (just the strain with empty plasmids, control) • JM109 with RFP (control) • Bristol BBa_K381001 • GaTech BBa_K410000 • Renbo BBa_K672000

-Temperatures we used are:
• 37ºC • Between 15ºC and 20ºC (18ºC)

-We will study growth using two thermal methods:
• Studying growth putting the colonies to grow directly at the chosen temperatures • Studying growth when the colonies have undergone a thermal shock @10ºC for 1 hour

-For each temperature and BioBrick we wanted to get (in each experiment):
• Growth curve • measurement of GFP expression • measurement of RFP expression (control)

Experiments

We designed experiments that allowed us to achieve the objectives related to Renbo`s operation, and they are:

1. All Colonies in LB, for references without antibiotics and without nitrate, @37ºC and @18ºC.

Bacterial Growth

2. All Colonies in LB with antibiotics and nitrate @18°C and @37°C, first passing through thermal shock @10°C for 1 hour.

Bacterial Growth

Bacterial Growth

GFP Expression

GFP Expression

RFP Expression

RFP Expression

3. All Colonies in LB with antibiotics and nitrate @18°C and @37°C, without thermal shock, direct growing.

GFP Expression

GFP Expression

RFP Expression

RFP Expression

CONCLUSIONS

- Perform further experiments with our device & pre-existing parts.
- THE RENBO could survive and express better than the composites it was made from.
- The cold shock method @10°C affects growth directly, kills the cells.

RENBO is Able to sense nitrate and nitrites even after experiencing a cold shock at 20ºC or below.

Note: References Part Design Page