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 project will be described.

The tested parts are the following:

Capture Module

Aroma Module

Union 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 were grown for one day in the incubator at 37°C

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

5. Pictures were taken of the bacteria cultures at 10 minute intervals during 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. Four 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
• Which of the two kinds of bacteria (red and white) gotten in the ligation, has the nhaS gene.

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

Materials:

● NaCl concentration of 1%

● NaCl concentration of 2.5%

● NaCl concentration of 5%

● NaCl concentration of 10%

● NaCl concentration of 15%

● 18 erlenmeyer flasks

● A 1000 µl micropipette

● Micropipette peaks

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

● Bacteria with nhaS gene and no reporter expression (with bacteria)

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

● 33 petri dishes

Procedure:

1. Five concentrations of NaCl in Q 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 control) 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 steps 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 #2 - Return to the Top

Materials:

●15 erlenmeyer flasks of 100 ml.

●Cultive of nhaS red transformed bacteria.

●Cultive of nhaS white transformed bacteria.

●20 ml of each one of the next concentrations of NaCl:

1.0%, 2.5%, 5.0%, 10.0%, 15.0%

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 µL 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 #3 - Return to the Top

Materials:

● 25ml NaCl 1% + 25 ml CLB + 50 µl Cm

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

● 25ml NaCl 5% + 25 ml CLB + 50 µl Cm

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

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

● 18 Erlenmeyer flasks

● A 1000 µl 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)

● 90 petri dishes

● 45 test tubes

● Peptoned water

Procedure:

First part

1. Three samples of each salt concentration were produced in order expose our three types of bacteria to each different salt concentration independently. These concentrations were made into Erlenmeyer flasks; this represents a total of 15 Erlenmeyer 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, 200µL 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 a sterile environment 30 cm near the bunsen burner in order to avoid contamination.

4. The micropipette peak was changed and 200µL 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 a sterile environment 30 cm near the bunsen burner in order to avoid contamination.

5. Last, but not least, with the micropipette and it’s respective peaks, 200µL 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 a sterile environment 30 cm near the bunsen burner in order to avoid contamination.

6. The Erlenmeyer were incubated for 12 hours.

See results of first part

Second part

1. Poured, into 45 test tubes, 9 mL of peptoned water.

2. The 45 test tubes are separated into 3 groups in order to specify 15 test tubes with peptoned water for each bacteria type.

3. 1000 µl (1ml) of a certain colony of bacteria (25ml NaCl #% (1, 2.5, 5, 10, 15) + 25 ml CLB + 50 µl Cm with 200µl bacteria (red bacteria, with bacteria and controlled bacteria)) are poured into one test tube with peptoned water making a solution 1:10.

4. 2 ml of 1:10 solution are collocated into 2 petri dishes. One ml for each one.

5. Another ml of 1:10 solution is poured into another test tube with peptone water creating a new 1:100 solution.

6. 2 ml of 1:100 solution are collocated into 2 petri dishes. One ml for each one.

7. Another ml of 1:100 solution is putted into another test tube with peptone water creating a new 1:1000 solution.

8. 2 ml of 1:1000 solution are collocated into 2 petri dishes. One ml for each one.

9. Repeat from step 9 to step 12 using all concentration of all kind of bacteria.

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Aroma module - Return to the Top

Qualitative experiments

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, but an RNA thermoswitch. which permit the synthesis of the protein. The thing is that even if the gene is expressed, it did not had any smell. The reason is because Winter Green odor is produced when the BSTM1 protein comes in contact with salicylic acid. The first experiment was designed only to prove that the protein is being produced and that the Winter Green odor can be smelled.

Test tubes aroma experiment

Materials:

●10 ml of BSMT1 opt. transformed cultive.

●8 test tubes with 3 ml of LB medium and 3 µl of chloramphenicol.

●A test tube inoculated with control bacteria (untransformed).

●Salicylic acid at different concentrations (1mM, 2 mM, 3 mM and 4 mM).

Procedure:

First part

1. 8 test tubes were inoculated with the BSMT1 opt. transformed bacteria.

2. 3 ml of salicylic acid (ratio 1:1) were added to 4 test tubes, each one with different concentrations:

1 mM of salicylic acid

2 mM of salicylic acid

3 mM of salicylic acid

4 mM of salicylic acid

3. Incubate all test tubes at 37º C during a day

Second part

1. In a rack were put: a test tube inoculated with the control bacteria, a test tube inoculated with BSTM1 opt. bacteria and salicylic acid, and a test tube inoculated with BSTM1 opt. bacteria but without salicylic acid.

2. Random people was chosen and asked to smell the three test tubes and describe what the smelled.

Petri dishes aroma experiment - Return to the Top

Then in order to know if the RNA thermoswitch 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 bacteria were 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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Quantitative aroma experiment - Return to the Top

Materials:

● 25ml salicylic acid 2Mm + 25 ml CLB + 50 µl Cm

● 25ml salicylic acid 10Mm + 25 ml CLB + 50 µl Cm

● 25ml salicylic acid 30Mm + 25 ml CLB + 50 µl Cm

● 4 Erlenmeyer flasks

● A 1000 µl micropipette

● Micropipette peaks

● Bacteria with BSMT1 opt. gene

● 36 Petri dishes

● 9 test tubes

● Peptoned water

Procedure:

1. One sample of each salicylic acid concentration is produced in order expose bacteria to all three concentrations independently. These concentrations are made into Erlenmeyer flasks; this represents a total of 3 Erlenmeyer flasks used in the beginning of the process.

2.One Erlenmeyer flasks was previously settled with the bacteria (which contained BsMT1 opt).

3. With the micropipette and its respective peaks, 200 µl of bacteria solution were added to all salicylic acid concentrations in a sterile environment 30 cm near the Bunsen burner in order to avoid contamination.

4. The Erlenmeyer flask were incubated for approximately 12 hours.

5. Poured, into 9 test tubes, 10 mL of peptoned water in all of them.

6. The 9 test tubes are separated into 3 groups in order to specify 3 test tubes with peptoned water for each concentration with bacteria.

7. 1000 µL (1ml) of a certain colony of bacteria (25ml salicylic acid #Mm (2, 10, 20) + 25 ml CLB + 50 µl Cm with 200 µl bacteria (which contained BsMT1 opt.) are poured into one test tube with peptoned water making a solution 1:10.

8.4 ml of 1:10 dillution are collocated into 4 Petri dishes. One ml for each one.

9. Another ml of 1:10 dillution is putted into another test tube with peptone water creating a new 1:100 solution.

10. 4 ml of 1:100 dillution are collocated into 4 Petri dishes. One ml for each one.

11. Another ml of 1:100 dillution is putted into another test tube with peptone water creating a new 1:1000 solution.

12. 4 ml of 1:1000 dillution are collocated into 4 Petri dishes. One ml for each one.

13. Repeat from step 9 to step 12 using all Mm dillutions.

14. The total amount of Petri dishes are divided into two samples in order to have 2 Petri dishes of each 1:10, 1:100 and 1:1000 solution of the bacteria. This creates two groups of 18 Petri dishes.

15. The first group is saved into an incubator with a temperature above 32o C. Approximately 35o C.

16. The second group is saved into an incubator with a temperature below 32o C. Approximately 29o C.

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Union module - Return to the Top

Even if the construction of the union module was not finished, there was a planned experiment in order to characterize the module. As the function of the union module is to give E. coli the ability of binding to silica pearls the experiment was designed with the expectative of get results of how many bacteria bind to the silica in certain time, and the relation between how many silica pearls are and how many bacteria binds to it.

Materials:

● 1000mL (1L) of bacteria with L2+AIDA (silica module) in pSB1C3 plasmid cultive.

● 500ml of control bacteria (untransformed).

● 75 corning tubes of 50 ml.

Procedure:

1. Get the weight of each one of the 75 corning tubes

2. In 50 corning tubes introduce 20 ml of bacteria transformed with L2+AIDA and in other 25 tubes, 20 ml of untransformed bacteria (control).

3. Divide the corning tubes in groups (1, 2, 3, 4 and 5) of 5 control and 10 with L2+AIDA bacteria

4. Centrifugate all groups at 5,000 rpm during 10 minutes. Throw the supernatan.

5. Weight all the groups and subtract it to the previous weight (Step 1).

6. Resuspend bacteria in 20 ml of LB medium.

7. In each group, separate the corning tubes and add them the next grams of silica pearls:

0.5 g of silica pearls -> 2 test tubes inoculated with BSMT1 opt. bacteria and 1 test tube with control bacteria.

1 g of silica pearls -> 2 test tubes inoculated with BSMT1 opt. bacteria and 1 test tube with control bacteria.

2 g of silica pearls -> 2 test tubes inoculated with BSMT1 opt. bacteria and 1 test tube with control bacteria.

3 g of silica pearls -> 2 test tubes inoculated with BSMT1 opt. bacteria and 1 test tube with control bacteria.

4 g of silica pearls -> 2 test tubes inoculated with BSMT1 opt. bacteria and 1 test tube with control bacteria.

8. Wait during the next time:

- Group 1 - 1 hr

- Group 2 - 3 hr

- Group 3 - 5 hr

- Group 4 - 12 hr

- Group 5 - 24 hr

9. At their respective time separate the silica pearls of bacteria by decantation

10. Centrifugate at 5,000 rpm during 10 minutes all the corning tubes. Throw the supernatant.

11. Weight each of the corning tubes and subtract it to the initial weight (step 1).

12. Compare both weights (Step 5 and step 11).