Team:CIDEB-UANL Mexico/labwork methods

Construction plan and Protocols - Return to the Top

How do we plan to get the construction of our modules? Here is shown all the construction plan that we followed with all the aspects that we considered.

Protocols and Construction Plan PDF

Experiments - Return to the Top

In this section, the experiments performed in order to test the effectivness of the predicted models for our proyect, will be described.

The tested parts are the following::

Capture Module

Aroma Module

Capture Module - Return to the Top

UV Experimentation

This experiment was designed in order to test the UV promoter's efectivness.

Procedure:

1. 2 Petri dishes were inoculated according to the "streak method" with transformed bacteria from a cultured petri dish, containing the NhaS and the RFP genes in their plasmids. (These colonies looked red due to the RFP)

2. The first step was repeated, this time with the transformed bacteria cultures containing the NhaS gene only, without the RFP gene. (These colonies looked white due to the absence of the RFP)

3. The bacteria was grown for one day in the incubator at 37°C

4. The four petri dishes were exposed to UV radiation (302nm) during a 2 hour period

5. Pictures were taken of the bacteria cultures at 10 minute intervals for the 2 hour period

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Viability test of the NhaS gene containing bacteria in salt - Return to the Top

 

In theory, the NhaS gene gives the bacteria a certain resistance to salt, but the exact percentage of increase in the resistance is unknown. Three experiments were designed in order to test:

• The viability of the NhaS transformed bacteria to survive in a salty environment
• The maximum ammount of salt in the medium which can be withstanded by the transformed bacteria
• The transformed bacteria's hability to capture sodium from the medium

In the ligation process of the NhaS gene, two different kinds of plasmids were obtained due to a possible mutation. The ones containing the RFP producing gene, and the ones that did not. Because it was uncertain the presence of the NhaS gene in the non-RFP producing bacteria these experiments were designed and performed in order to determine which one of the groups contain the NhaS gene.

 

Experiment #1

Procedure:

1. Twenty seven, 9 milliliters test tubes were filled with different concentrations of NaCl (0.85%, 0.90%, 1.00%, 1.50%, 2%, 2 .50%, 3.50%, 5.00% and 10.00%) with 3 test tubes per concentration.

2. The different groups of bacteria were separated: white NhaS, red NhaS and bacteria without Nhas gene (Control)

3. The 27 test tube were separated into 3 groups (for each type of bacteria), so that each group had 9 different salt concentrations.

4. In a sterile environment, no more than 30cm away from the bunsen burner, 1000 micro liters (1 mL) of RFP producing NhaS bacteria were added into each of the 9 corresponding test tubes, to obtain a ratio of 1:10.

5. The previous step was repeated with non RFP producing NhaS bacteria and with the non-NhaS producing control bacteria.

6. Two petri dishes, per each concentration, per each bacteria groups were filled with prepared LB medium containing chloramphenicol.

7.One of the nine test tubes with NaCl and bacteria was selected.

8. One milliliter of the contents of the test tube was introduced into the three petri dishes. This step was performed at a distance of not more than 30 cm of a Bunsen burner to avoid sample contamination.

9. A glass inoculation was placed in the spreader into a glass full of alcohol and remove it burning it with a Bunsen burner so that alcohol is removed from the spreader and 5 - 10 seconds were waited before the next step.

11. Spread the content of the test tube into the petri dish. Perform this step over a distance of no more than 30cm of a Bunsen burner to avoid any contamination

12. The previous series of steps were repeated with all of the tubes

13. The 54 petri dishes were incubated (9 concentrations of duplicates for each group of bacteria) at a temperature of 37 ºC during 24 hours.

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Experiment #2

Materials:

● NaCl concentration of 1%

● NaCl concentration of 2.5%

● NaCl concentration of 5%

● NaCl concentration of 10%

● NaCl concentration of 15%

● 18 erlenmeye flasks

● 1000 micro micropipette

● Micropipette peaks

● Bacteria with NhaS gene and reporter expression (red bacteria or RB)

● Bacteria with NhaS gene and no reporter expression (with batceria)

● Bacteria with no NhaS gene and no reporter presented (with bacteria without the gene)

● 33 petri dishes

Procedure:

1. Five concentrations of NaCl in mQ water (1.0%, 2.5%, 5.0%, 10.0%, 15.0%) were prepared in separated flasks.

2. The three groups of bacteria (RFP+NhaS, NhaS and the contro) were separated in different test tubes.

3. In a petri dish with LB agar and Chloramphenicol,1 milliliter of the 1.0% concentration of NaCl was introduced; this process was repeated in the three petri dishes and in a sterile environment 30 cm near the bunsen burner in order to avoid contamination.

4. In both both petri dishes, 200 microliters of NhaS transformed red bacteria were introduced, and distributed the content with a sterile glass inoculation spreader in a sterile environment.

5. The steps 3 and 4 were repeated four times, but with the other four concentrations (2.5%, 5.0%, 10.0%, 15.0%.).

6. The s teps 3 through 5 were repeated two more times, with a the remaining groups of bacteria.

7. Three petri dishes with LB agar were inoculated with the control bacteria, introducing 200 microliters of it and spreading it with a sterile glass inoculation spreader.

8. The 33 total petri dishes were cultivated at 37º C for 24 hours.

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Experiment #3

Procedure:

1. In flasks of 100mL, different concentrations of NaCl in mQ water were preparated(1.0%, 2.5%, 5.0%, 10.0%, 15.0%), having a finale volume of 20mL, and were divided into 3 flasks of each concentration

2. 200 uL of NhaS transformed white bacteria solution was placed in the flasks with each of the five different concentrations in a sterile environment, no more than 30 cm away from a Bunsen burner, to avoid sample contamination.

3. The previous step was repeated with the other two groups of bacteria. At the end there will be 15 flasks. of bacteria (five of each type).

4. The 15 flasks were incubated at 37 ºC during a day.

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Experiment #4

Materials:

● 25ml NaCl 1% + 25 ml CLB + 50 ml Cm

● 25ml NaCl 2.5% + 25 ml CLB + 50 ml Cm

● 25ml NaCl 5% + 25 ml CLB + 50 ml Cm

● 25ml NaCl 10% + 25 ml CLB + 50 ml Cm

● 25ml NaCl 15% + 25 ml CLB + 50 ml Cm

● 18 erlenmeye flasks

● 1000 micro micropipette

● Micropipette peaks

● Bacteria with NhaS gene and reporter expression (red bacteria or RB)

● Bacteria with NhaS gene and no reporter expression (with batceria)

● Bacteria with no NhaS gene and no reporter presented (with bacteria without the gene)

Procedure:

1. Three samples of each salt concentration are produced in order expose all bacteria to all salt concentration independently. These concentrations are made into Erlenmeyer flasks; this represents a total of 15 erlenmeye flasks used in the beginning of the process.

2. Three Erlenmeyer flasks were previously settled with the different bacteria. Each of these three Erlenmeyer flask contained a specific group of bacteria (red bacteria, white bacteria and controlled bacteria)

3. With the micropipette and it’s respective peaks, 200uL of red bacteria solution were added to all salt concentrations (Just in one flask of the three ones from Nacl1% mix to NaCl 15% mix); this process was repeated in the three petri dishes and in a sterile environment 30 cm near the bunsen burner in order to avoid contamination.

4. The micropipette peak was changed and 200uL of white bacteria solution were added to all salt concentrations (Just in one flask of the three ones from Nacl1% mix to NaCl 15% mix); this process was repeated in the three petri dishes and in a sterile environment 30 cm near the bunsen burner in order to avoid contamination.

5. Finally but not less important, with the micropipette and it’s respective peaks, 200uL of red bacteria solution were added to all salt concentrations (Just in one flask of the three ones from Nacl1% mix to NaCl 15% mix) ; this process was repeated in the three petri dishes and in a sterile environment 30 cm near the bunsen burner in order to avoid contamination.

6. The Erlenmeyer were incubated for 12 hours.

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Aroma module - Return to the Top

Qualitative experiment

In the project, one of the modules (the aroma module) consisted in the production of a scented ester in (Winter Green). In order for the gene to work how it was supposed to work, the bacteria must be in an environment with a temperature higher than 32º C. This is because the gene has a constitutive promoter. In order to control the aroma production, temperature was used. But even if the gene is expressed, it did not had any smell. The reason was because Winter Green odor is produced when the BSTM1 protein comes in contact with salicylic acid. In order to know if the riboswitch is working and at which concentration of salicylic acid smells the strongest, the following experiment was performed .

Procedure:

First part:

1. 12 Petri dishes with an LB medium and the Chloramphenicol antibiotic were prepared.

2.3 mL of a solution containing salicylic acid and mQ water was added to four Petri dishes, with the concentration of salicylic acid being of 10 mM.

*The step noº 2, was repeated two more times changing the concentration of 10 mM of salicylic acid to 20 mM and 30 mM.

3. 6 Petri dishes with LB medium were prepared.

4. 3 mL of a solution containing salicylic acid and mQ water was added into 2 Petri dishes, with the concentration of salicylic acid being of 10 mM.

*The step noº 4 was repeated two more times changing the concentration of 10 mM of salicylic acid to 20 mM and 30 mM.

5. 200 µL of bacteriawas added into all of the petri dishes.

6. 2 Petri dishes containing Chloramphenicol from each of the three concentrations and 1 Petri dish without Chloramphenicol from each concentration were incubated at 29 ºC for one day.

7. 2 Petri dishes containing Chloramphenicol from each of the three concentrations and 1 Petri dish without Chloramphenicol from each concentration were incubated at 35 ºC for one day.

Second part: 1. Random people were chosen to smell the bacteria and a video was taken of their experience:

- A group of bacteria with the aroma module at a temperature below 32 ºC.

- A group of bacteria with the aroma module at a temperature above 32 ºC.

- A controlled group of bacteria without the gene at a temperature below 32 ºC.

- A controlled group of bacteria without the gene at a temperature above 32 ºC.

2. The people were asked to describe what they were smelling

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