Part:BBa_K672000:Experience

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Applications of BBa_k672000

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

Experience with BBa_K672000

FIRS 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 EXPERIMANTATION

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)
 • With RFP (control)
 • Bristol BBa_K381001
 • GaTech  BBa_K672000

 Temperatures we used are:

 • 37ºC
 • Between 15ºC and 20ºC  (18ºC)

 We will study growth using the 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

1. All Colonies in LB, for references without antibiotics and without nitrate, @37ºC and @18ºC.
2. All Colonies in LB with antibiotics and nitrate @18°C and @37°C, first passing through thermal shock @10°C for 1 hour.
3. All Colonies in LB with antibiotics and nitrate @18°C and @37°C, without thermal shock, direct growing.

User Reviews

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