Experiments

Soil Sample Investigation

Materials

• 1. 12 different soil samples
• 2. Semi-solid agar
• 3. Agar
• 4. Distilled Water
• 5. Glucose, sucrose, malate, and mannitol broth
• 6. Bromothymol Blue Indicator

Protocol

1. 1. Each of the 12 soil sample were diluted with 100 ml of distilled water.
2. 2. Diluted samples were placed on an orbital shaker at 80 RPM for 15 minutes
3. 3. Samples were inoculated into semi solid and solid agar containing bromothymol blue indicator.
4. 4. 500 µl of each sample was also added to a 25 ml mix of bromothymol blue indicator and a broth of each carbon source (glucose, sucrose, malate, mannitol).
5. 5. All samples were incubated at 25°C, and were periodically removed to be inspected and analyzed.

Fig 1.1: Preparation of diluted soil samples to be introduced to media

Plasmid DNA Cutting

Materials

• DNA
• Buffer 2.1
• 2uL of EcoRI
• water
• 1uL of Pst 1

Protocol

1. Water is combined with the chosen DNA amount.
2. 10x the amount of DNA is added in buffer fluid.
3. The restriction DNA is added to the solution.

IF Single Cut

1. 1. 80ng of the chosen DNA is added to the solution.
2. 2. 1uL of EcoRI is added to the solution.
3. 3. 10x the amount of DNA is added in buffercutsmart.
4. 4. Water is added to the solution.

IF Double Cut

1. 1. 80 ng of the chosen DNA, times by x uL.
2. 2. 1uL of EcoRI is added to the solution.
3. 3. 1uL of Pst I is added to the solution.
4. 4. 10uL of buffer 2.1 fluid is added to the solution.
5. 5. Water is added to the solution to dilute.

Electrophoresis

Materials

• Electrophoresis tray
• Comb
• Agarose
• TAE buffer
• sticky tape
• Electrophoresis chamber
• DNA samples
• pipette
• Ultraviolet light

Protocol

1. 1. Seal the edges of an empty agarose gel tray with masking tape to prevent leakage during gel preparation.
2. 2. Place a comb with evenly spaced teeth into the tray, determining the location and size of sample wells.
3. 3. Carefully pour liquid agarose (kept at around 55 degrees Celsius) into the tray up to the new top of the comb's teeth.
4. 4. Allow the agarose to solidify for approximately 20 minutes.
5. 5. Gently remove the spacing comb from the tray, leaving wells for loading samples.
6. 6. If not used immediately, store the gel at 4 degrees Celsius.
7. 7. Place the solidified agarose gel into the electrophoresis chamber.
8. 8. Pour electrophoresis buffer solution into the chamber, ensuring it covers the entire gel surface and fills the chamber to an adequate level.
9. 9. Prepare the DNA samples you wish to test. These samples may come from various sources or experiments.
10. 10. Using a syringe or a dedicated pipette, suction up each DNA sample, ensuring a clean, sterile tip for each sample.
11. 11. Carefully add the DNA samples into the wells created by the comb during gel preparation. Insert the tip of the syringe or pipette into each well to keep the DNA inside the well without floating in the buffer liquid.
12. 12. Attach the chamber's lid securely to maintain the electric field within the chamber and prevent sample evaporation.
13. 13.Connect the electrophoresis chamber to the power supply.
14. 14. Turn on the power supply unit.
15. 15. pply an electric field that causes the DNA molecules to migrate through the agarose gel. The rate of migration depends on the size and charge of the DNA fragments.
16. 16. Run the electrophoresis at a specified voltage (e.g., 100V) for a designated duration (commonly around 1 hour, but it can vary based on gel thickness and DNA fragment size).
17. 17. After the electrophoresis run, turn off the power supply and disconnect the chamber.
18. 18. Carefully remove the gel from the chamber.
19. 19. Place the gel on a UV transilluminator or gel documentation system.
20. 20. Observe the separated DNA bands, which have migrated based on their size, under ultraviolet (UV) light. Document the results as necessary.

Plasmid Extraction

Materials

• Centrifuge
• 2ml E. coli culture
• 250 µl Suspension Buffer
• 250 µl Lysis Buffer
• 350 µl Binding Buffer
• High Pure filter tube
• 500 µl of Wash Buffer I
• 700 µl of Wash Buffer II
• 100 µl of Elution Buffer

Protocol

1. 1. Centrifuge 2ml or E. coli culture at 6,000 RPM for 30 seconds.
2. 2. Discard supernatant (the leftover liquid that is not the pellet).
3. 3. Resuspend the pellet in 250 µl of Suspension Buffer.
4. 4. Add 250 µl of Lysis Buffer
5. 5. Mix gently. Incubate for 5 mins at room temperature.
6. 6. Add 350 µl of chilled Binding Buffer.
7. 7. Mix gently. Incubate for 5 mins on ice. Centrifuge for 10 mins at max speed.
8. 8. Discard the pellet by pouring the remaining liquid into a High Pure filter tube and discarding the original tube with the pellet at the bottom.
9. 9. Centrifuge at max speed for 30-60 seconds.
10. 10. Discard flowthrough
11. 11. Add 500 µl of Wash Buffer I.
12. 12. Centrifuge at 13,000 RPM for 1 minute.
13. 13. Discard the flowthrough
14. 14. dd 700 µl of Wash Buffer II.
15. 15. Centrifuge at 13,000 RPM for 1 minute.
16. 16. Discard flowthrough
17. 17. Centrifuge at 13,000 RPM for a further 1 minute.
18. 18. Discard flowthrough.
19. 19. Add 100 µl of Elution Buffer.
20. 20. Centrifuge at 13,000 RPM for 1 minute.
21. 21. The liquid in the bottom is what we want to keep.

Golden Gate Assembly

Prototype A

BBa_K4735004 BBa_K4735005 BBa_K4735006 BBa_K4735009 BBa_K4735010 BBa_K4735003

narG promoter driving superfolder GFP expression.

The part should promote the expression of GFP under low oxygen conditions.

Testing indicates this does not respond very well to oxygen. It shows relatively high GFP fluorescence under all oxygen conditions.

Prototype A, will consist of :
Part 1 - lacIQ-rbs-fnr-TrrnBT1
Part 2 - T-Spacer-T7-3FNRbindingNarG (part a)

Prototype B

BBa_K4735004 BBa_K4735005 BBa_K4735007 BBa_K4735009 BBa_K4735010 BBa_K4735003

FNR under control of lacIQ promoter for testing of narK promoter driving superfolder GFP expression.

The part should promote the expression of GFP under low oxygen conditions.

Testing indicates this does respond very well to oxygen. It shows no GFP fluorescence anaerobically or aerobically but gives GFP fluorescence at 0.5% oxygen and 2% oxygen.

Part 1 - lacIQ-rbs-fnr-TrrnBT1
Part 2 - T-Spacer-T7-3FNRbindingNarK (part b)
Part 3 - RBS2-sfgfp + TrrnBT1

Prototype C

Prototype C will consist of :

BBa_K4735004 BBa_K4735005 BBa_K4735008 BBa_K4735009 BBa_K4735010 BBa_K4735003

FNR under control of lacIQ promoter for testing of dmsA promoter driving superfolder GFP expression.

The part should promote the expression of GFP under low oxygen conditions.

Testing indicates this does respond to oxygen. It shows no GFP fluorescence aerobically AND gives GFP fluorescence anaerobically at 0.5% oxygen and 2% oxygen in nitrogen.

Part 1 - lacIQ-rbs-fnr-TrrnBT1
Part 2 - T-Spacer-T7-3FNRbindingDmsA (part c)
Part 3 - RBS2-sfgfp + TrrnBT1

GFP Fluorescence Intensity under Varying Oxygen Concentrations

Materials

• LB media
• 29 tubes
• Labels for tubes
• Negative control sample
• Anaerobic chamber
• Desiccator
• Loosely screwed caps for tubes
• High vacuum grease
• Syringe
• Balloon
• Cylinder filled with water
• Nitrogen gas
• Rubber stopper
• Gas chromatography machine
• Shaker

Protocol

In this experiment, we picked 6 samples (1A, 3A, 2B, 4B, 1C, 2C) and 1 negative(L) control to grow in 4 different conditions (aerobic, 0.5% oxygen, 2% oxygen, anaerobic ). Each 50 ml falcon tube is labeled in the following way: condition(Ae, 0.5%, or 2%, An), sample name(1A, 3A, 2B, 4B, 1C, 2C, or L), LB(the media)

1. 1. Add 10 µl samples from each of the six constructs to the corresponding tubes. (eg. add 1A sample to all tubes with label 1A on it)
2. 2. Add 10 µl each sample from negative control to each of the tubes.
3. 3. Create an anaerobic chamber by vacuum sealing one chamber, and an aerobic chamber full of standard air.
4. 4. Place the 7 tubes labeled with 0.5% with loosely screwed caps into the chamber of desiccator and place 7 tubes labeled 2% with loosely screwed caps into another chamber of desiccator.
5. 5. Seal the desiccators by applying high vacuum grease on the edge of the container and the lid and valve.
6. 6. Purge the desiccators by flowing nitrogen gas through for 1 minute.
7. 7. Calculate the volume of the gas (air) to fill into the desiccator to make a 0.5% oxygen condition (using c1v1=c2v2).
8. 8. Use a syringe to measure 50 ml of air to inflate the balloon, mark the estimated volume on the surface of a big cylinder filled with water above the waterline and test the volume of the balloon by immersing the balloon in the water to observe which it reaches the mark.
9. 9. Open the valve before filling the nitrogen gas to the desiccator by connecting the tube from the gas tank to the tap of the desiccator and slide the cover slightly open to let the air out
10. 10. Squeeze the tube tightly to ensure no gas leakage and open the rubber stopper quickly and inject the air into the chamber by connecting the syringe to the balloon and closing it immediately after injection.
11. 11. To fasten the equilibrating process, use the empty syringe into the tap and purge at least 10 times
12. 12. Once equilibrated, insert the GC needle into the rubber stopper and draw 5 µl gas from the chamber and put it into the gas chromatography which will automatically calculate the actual concentration of oxygen % to ensure there is the correct amount of oxygen in the desiccator.
13. 13. Repeat steps 6-12 again for the 2% desiccator(for step 8 and 9, insert 4 times as much air as the amount inserted into the 0.5% desiccator).
14. 14. If the percentage of oxygen in the desiccator is accurate, seal the desiccators and the anaerobic chamber, incubate them and the aerobic tubes at 37 degrees with 90 RPM speed shaking overnight.
15. 15. Create two biological replicates and a technical triplicate sample for each sample under the varying oxygen concentrations.
16. 16. Using a gel reader, measure GFP intensity per OD600 to model the response of each sample to oxygen.

Fig 2.1: Purging of desiccators to create 0.5, 2% oxygen chambers