Team:CIDEB-UANL Mexico/labwork methods

Experiments

In this section, it is described all the experiments designed and performed in order to prove experimentally our modules.

Capture Module

UV Experimentation

This experiment was designed in order to know if the UV promoter is working properly.

Procedure:

1. Inoculate by streak 2 Petri dishes with NhaS DNA in pSB1C3 Red bacteria

2. Repeat step 1 but with NhaS DNA in pSB1C3 White bacteria

3. Let them grow during one day in the incubator at 37°C

4. Expose the four Petri dishes to UV irradiation (302nm) during 2 hours

5. Take photos each 10 minutes and wait for results. (You can also take video during the 2 hours instead of the photos)

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Viability in salt - Return to the top

In the research on NhaS it was said that the gene gives certain resistance to salt, but the exact percentage of resistance is unknown. There were designed 3 experiments to know the viability in salt of the NhaS transformed bacteria, also in order to know the maximum concentration in which bacteria with the capture module can be exposed to later experiment with it and find out how much salt can capture.

As in the ligation on NhaS and pSB1C3 was obtain two types of bacteria (red and white) it us uncertain if the ligation occurred properly and why some are red and some are white, through the next three experiments it can be determined which type has the gene or if both of them have it.

Experiment 1

1. Put in 27 test tubes 9 milliliters of different concentrations of NaCl (0.85%, 0.90%, 1.00%, 1.50%, 2%,

2.50%, 3.50%, 5.00% and 10.00%) to finish with 3 test tubes of each concentration.

2. Separate the different groups of bacteria: white NhaS, red NhaS and bacteria without Nhas gene (Control)

3. Separate the 27 test tubes into 3 groups (for each type of bacteria) so that each group has 9 different salt concentrations and repeat any concentration.

4. Put 1000 micro liters (1 mL) of red NhaS bacteria in each of the 9 corresponding test tubes to obtain a ratio of 1:10. Perform this step over a distance of no more than 30 cm of a Bunsen burner to avoid sample contamination

5. Repeat step 4 but with White NhaS bacteria.

6. Repeat step 4 but with the Control bacteria. At the end there will be 9 test tubes of red bacteria, 9 of white ones and 9 with the control.

7. Separating two petri dishes (LB medium must contain chloramphenicol) for each concentration of NaCl with appropriate bacteria.

8. Select one of 9 test tubes with NaCl and bacteria

9. Introduce 1 milliliter of the contents of the test tube into a petri dish and repeat it in two petri dishes. Perform this step over a distance of not more than 30 cm of a Bunsen burner to avoid sample contamination.

10. Put a glass inoculation spreader into a glass full of alcohol and remove it burning it with a Bunsen burner so that alcohol is removed from the spreader

11. Wait 5 - 10 seconds

12. 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 contamination

13. Repeat step 7 to 12 with all test tubes

14. Incubate the 54 petri dishes (9 concentrations of duplicates for each different bacteria) at 37 ºC during a day.

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Experiment 2

Procedure:

1. Prepare 5 concentrations of NaCl in mQ water (1.0%, 2.5%, 5.0%, 10.0%, 15.0%) in different flasks.

2. Get the three groups of bacteria (white, red and without Nhas gene [control]) in different test tubes.

3. In a petri dish (with LB agar and Chloramphenicol), introduce 1 milliliter of the 1.0% concentration of NaCl, repeat it for a total of two petri dishes. Perform this step over a distance of no more than 30cm of a Bunsen burner to avoid contamination

4. In both both petri dishes introduce 200 microliters of nhaS transformed red bacteria, and distribute the content with a sterile glass inoculation spreader in a distance of less than 30 cm of a Bunsen burner to avoid contamination.

5. Repeat step 3 and 4, four times but with the other four concentrations: 2.5%, 5.0%, 10.0%, 15.0%.

6. Repeat steps 3 through 5 two more times, but each time with a different group of bacteria, the white ones and the control.

7. Inoculate 3 petri dishes (must contain only LB agar) with the bacteria without the gene Nhas (control), introducing 200 microliters of it and spreading it with a sterile glass inoculation spreader.

8. Incubate all the 33 total petri dishes at 37º C during a day.

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Experiment 3

Procedure:

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

2. Put 200 uL of NhaS transformed white bacteria in the flasks with each of the five different concentrations Perform this step over a distance of no more than 30 cm of a Bunsen burner to avoid sample contamination.

3. Repeat step 2 but with NhaS transformed red bacteria.

4. Repeat step 2 bur with the control bacteria. At the end there will be 15 flasks. five of each type of bacteria.

5. Incubate the 15 flasks at 37 ºC during a day.

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Aroma module

In our project, one of the modules consists in the production of an ester with aroma (Winter Green). In order for the gene to work how it is 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, so in order to control the aroma production, we used temperature. But even if the gene is expressed, it will not have any smell. The reason is 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 more, it was performed the following experiment.

Procedure:

First part:

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

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

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

3. Prepare 6 Petri dishes with LB medium.

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

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

5. To all the Petri dishes, add 200 µL of bacteria.

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

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

Second part: 1. Choose random people to smell the bacteria and take video of they reaction:

- 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. Ask people to describe what they are smelling

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Construction plan and Protocols

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