Difference between revisions of "Team:Lambert GA/Experiments"

Lambert iGEM utilized a toehold switch and a trigger - which corresponds with RNA sequences of the ctxB1 cholera gene - in a biosensor system. If the trigger sequence is present and binds to the toehold switch, then a blue pigment produced by LacZ appears in the cells.

The 2018 Lambert iGEM team conducted the following experiments to assemble the proof the concept T7 LacZ toehold switch and Cholera switch. We ordered three potential Cholera toehold switches and triggers and proceeded to perform PCRs on each of the parts. All switches and triggers were amplified and validated through gel electrophoresis [Figure 1.]. With purified PCR products of the switches, triggers, and their correlating vectors, we ligated each of these parts into their vectors through Gibson Assembly. Dual plasmid transformations and single transformations were conducted with BL21 T7 E. coli

Click on the step to be directed to the procedure!

1. Miniprep
2. Nanodrop
3. Digest
4. Gel
5. Ligation
6. Transformation/electroporation, Plate
7. Colony PCR (Screening)
8. Gel
9. Inoculate correct colony to a liquid culture
10. Miniprep
11. Nanodrop
12. Sequence

Miniprep: grown culture, microcentrifuge, 2 1.5mL microcentrifuge tubes, mini column and collection tube, Solution I, Solution II, Solution III, HBC Wash Buffer, DNA Wash Buffer, Elution Buffer, micropipette and tips
Nanodrop: nanodrop machine, miniprepped DNA, Kimtech wipes, micropipette and tips
Digest: miniprepped DNA, dH₂O, 10X RE-Mix, standard restriction enzyme, micropipettes and tips
Gel: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA
Ligation: vector, parts 1 and 2, ligase buffer, ligase, Antarctic phosphatase, microcentrifuge tube, ice, micropipette and tips
Transformation: ice, ligation mixture, competent cells, incubator, LB media, microcentrifuge tubes, micropipette and tips Plate: agar plate, micropipette and tips, beads
Colony PCR: dH₂O, buffer, VF₂, VR, Q5 polymerase, dNTP, DNA dilution, micropipette and tips, PCR tubes, thermocycler, ice
Gel: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA
Inoculate: LB media, dilution, micropipette and tips

Miniprep (using Omega Protocol)
1.1 Grow 1-5mL culture overnight in a 10mL-20mL culture tube.
1.2 Centrifuge at 2500xg for 5 minutes at room temperature. Decant or aspirate and discard the culture media. (Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.)

• 1.2.1 Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.

1.3 Add 250uL of Solution I mixed with RNase A (pre-added). Vortex to mix thoroughly. Transfer the suspension into a new 1.5mL microcentrifuge tube.
1.4 Add 250uL of Solution II. Invert several times until you get a clear lysate.

• 1.4.1 Once Solution II is added, do not let it sit for more than 5 minutes!

1.5 Add 350uL of Solution III. Invert several times until white precipitate forms. Centrifuge at 13,000xg or 17,900rcf for 10 minutes. A compact white pellet should form at the bottom of the tube.
1.6 Insert a mini-column into a 2mL collection tube.
1.7 Transfer the clear supernatant into the mini-column using a micropipette. Centrifuge at the maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.

• 1.7.1 Be careful not to get any parts of the pellet! Tilt at an angle with the pellet at the top when micropipetting is advisable.


• 1.7.2 Think about what you are discarding versus what you want to keep!

1.8 Add 500uL of the HBC Wash Buffer diluted in isopropanol. Centrifuge at maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.

• 1.8.1 All wash buffers will be centrifuged for 1 minute.

1.9 Add 700uL of the DNA Wash Buffer diluted in ethanol. Centrifuge at maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.
1.10 Centrifuge the empty mini column at the maximum speed (13,000xg) for 2 minutes to remove the ethanol.
1.11 Transfer the mini-column to a nuclease-free 1.5mL microcentrifuge tube.
1.12 Add 50uL of Elution Buffer (or sterile deionized water). Let it sit in room temperature for 60 seconds. Centrifuge at maximum speed (13,000xg) for 60 seconds.
1.13 Store eluted DNA at -20℃.

Nanodrop
2.1 Vortex before nanodrop.
2.2 Wipe down the nanodrop machine with Kimtech wipes to make it sterile.
2.3 Set the program to analyze nucleic acids [because you are dealing with plasmid DNA].
2.4 Do a blank test to ensure that the platform is sterile.
2.5 Load 1uL of the miniprepped DNA onto the platform.
2.5.1 (Have steady hands. The sample needs to be in the center for best results.)
2.6 Click “measure” on the nanodrop for analysis.
2.7 Write down measurements for the concentration of DNA (in ng/uL), A260, A280, 260/280 (should be around 1.8), and 260/230 (should be around 2.1).

Digest
3.1 Dilute up to 1ug DNA to 17uL with dH₂O.

• 3.1.1 Take concentration of DNA from nanodrop and convert from ng/uL to ug/uL. Next, set up a proportion to find out how many uL you need to get 1 ug of DNA.
• 3.1.2 20uL (total reaction) - 2uL RE-Mix - 1uL standard enzyme = uL dH₂O

3.2 Use a microcentrifuge tube to put the reaction in. Put in the contents in this order: water, DNA, enzymes.

• 3.2.1 Add 2uL of the 10X RE-Mix and 1uL of the standard enzyme.
• 3.2.1.1 E and X = 10X RE-Mix
• 3.2.1.2 S and P = standard enzymes

3.3 Incubate at 37℃ for 1 hour for standard enzymes, then at 80℃ for deactivation.

Gel
4.1 Set up the chamber and put in the gel. Make sure the wells of the gel is at the end of the chamber so that the DNA runs to red.
4.2 Pour the TAE buffer evenly to completely cover the gel.
4.3 Using a micropipette, put 3uL of DNA in each well and 6uL for the ladder [if using a thin gel]. Thicker gels will require more DNA to be put in each well.
4.4 Connect the electrodes by closing the box and connecting them to the power supply. Make sure the power supply is set for 120 volts and 60 minutes.
4.5 Turn on the power supply and make sure bubbles are rising on the sides of the chamber.

Ligation
5.1 Use Antarctic phosphatase on the backbone to increase the likelihood of part insertion and decrease backbone closure.Make calculations using a 3:1 molar ratio of insert to backbone. Refer to the two tables below.
5.2 Put in each component in a microcentrifuge tube while on ice. They should be pipetted into the tube in this order: water, DNA, ligase buffer, ligase.
5.3 The ligase buffer should be thawed and resuspended at room temperature.

• 5.3.1 Gently mix by pipetting up and down and microfuge briefly.

5.4 Incubate at room temperature for 1 hour at 37℃

Transformation
6.1 Thaw materials on ice for 5 minutes.
6.2 Put 10uL of ligation mixture into 100uL competent cells in a microcentrifuge tube.
6.3 Flick the tube to mix. 6.5 Add 200uL of LB media.
6.6 Incubate at 37℃ for one hour.
6.7 Plate 150uL of cells onto a plate. Make sure plate has the correct antibiotic (based on vector backbone)! Grow overnight.

Electroporation
(for 400 ml culture, adjust as appropriate for smaller volumes)

Preparing Electrocompetent cells:
6.1 Grow overnight 5 ml culture
6.2 Dilute 1:100 in fresh media
** preparation: chill big centrifuge to 4C, chill autoclaved water and 10% glycerol soln
6.3 Grow to OD of about 0.6 (isolating in exponential phase most important)
6.4 Pour cells into 50 ml conical tubes, on ice
6.5 Keep on ice for 10 min
6.6 Centrifuge cells (all spins done at 2500 xg, 6 min), discard supernatant
6.7 Add 13 ml of ice cold sterile water to each tube, resuspend by pipetting up and down
6.8 Combine into two total tubes.
6.9 Centrifuge, repeat ice cold water resuspension
6.10 Do same thing twice with 25 ml of ice cold 10% glycerol
6.11 Resuspend in 4 ml (concentrating 100x from initial culture) of ice cold 10% glycerol
6.12 Aliquot into microcentrifuge tubes
6.13 Store in -80 freezer

Electroporation:
6.14 Take 50 ul of electrocompetent cells, and 10-100 ng of PCR product (don’t add more than 2ul) for 6.15 knockouts, 0.1-10 ng of plasmid
6.16 Flick to mix
6.17 Transfer to chilled electroporation cuvette
6.18 Electroporate (machine in Anton’s lab downstairs)

• 6.18.1 (first setting), target time constant: greater than or equal to 5 ms
• 6.19 Add 1ml of prewarmed LB to cuvette

6.20 Transfer cell/LB mixture to microcentrifuge tube
6.21 Recover with shaking in incubator for 1.5 hr

Colony PCR
7.1 Pick colonies with a combination of phenotypes i.e. large/small, red/white. Dilute each colony in 40uL dH₂O, 1uL DNA from ligation if transformation is successful.

• 7.1.1 If necessary, do a quick spin to make sure all the liquid is at the bottom.

7.2 Make the following master mix on ice in this order: 63uL dH₂O, 20uL buffer, 5uL VF₂ primer, 5uL VR primer, 2uL dNTP, 1uL Q5 polymerase.
7.3 Aliquot the master mixes into PCR tubes, then add 1uL of the DNA dilution.

• 7.3.1 Make sure PCR tubes are labeled properly and carefully!

7.4 Transfer the PCR tubes to a PCR machine and begin thermocycling.

• 7.4.1 Initial Denaturation: 98℃ for 30 seconds
• 7.4.2 25-35 Cycles: 98℃ for 5-10 seconds, 50-72℃ for 10-30 seconds, 72℃ for 20-30 seconds/kb
• 7.4.3 Final Extension: 72℃ for 2 minutes
• 7.4.4 Hold 4-10℃

Gel
8.1 Set up the chamber and put in the gel. Make sure the wells of the gel is at the end of the chamber so that the DNA runs to red.
8.2 Pour the TAE buffer evenly to completely cover the gel.
8.3 Using a micropipette, put 3uL of DNA in each well and 6uL for the ladder [if using a thin gel]. Thicker gels will require more DNA to be put in each well.
8.4 Connect the electrodes by closing the box and connecting them to the power supply. Make sure the power supply is set for 120 volts and 60 minutes.
8.5 Turn on the power supply and make sure bubbles are rising on the sides of the chamber.

Inoculate Liquid Culture
9.1 Get the remaining 39uL of colony dilution.
9.2 Get LB media and make sure to use the appropriate antibiotic resistance.
9.3 Mix the colony dilution into the media.
9.4 Grow overnight.

Water Collection

Prepping the Bottle
1.1 Obtain 4 nalgene bottles (22.75 cm in length, 5.5 cm in width).
1.2 Drill a 1.9 cm (preferably 2 cm) hole in the bottom of the bottle with a ¾ in drill bit.
1.3 Run the bottle through tap water to get any plastic bits out of the bottle. Spray the interior of the bottle with isopropyl alcohol. Rinse with soap and water to remove any alcohol.
1.4 Allow the bottles to dry by placing them on a drying rack.

Prepping Muslin Cloth

1. Obtain 90 cm by 90 cm muslin cloth.
2. Cut the muslin cloth into 6 cm. by 6 cm pieces.
3. Rinse with soap under flowing tap water.
4. Allow the muslin cloth to dry.
5. Fold in half.
6. Place the muslin cloth into an ice bottle.
7. Fill the bottle ¾th of the way with the cloth

Prepping Cheese Cloth

1. Obtain a 5.5 meter by 19.4 cm.
2. Cut the cheese cloth into 20.5 cm by 15 cm.
3. Rinse the cheesecloth in running tap water and soap.
4. Create 3-4 folds and pack the ice bottle.
5. Use a long rod to help push the cloth into the bottle and make the bottle compact.
6. Fill the bottle ¾th of the way with the cloth.

Prepping T-shirt Cloth

1. Obtain 1 t-shirt
2. Cut into 5 cm by 11 cm strips
3. Fold strips 2 times and pack ice bottle
4. Fill bottle ½ way with the cloth

Prepping Coffee Cloth

1. Obtain a pack of large coffee filter
2. Microwave filters to sterilize
3. Cut filters in ½
4. Fold pieces 1-2 times and pack ice bottle
5. Fill bottle ¾th of the way with the filters

Prepping Carb Cloth

1. Add 500 ml of dH20 and 17.5g of agar to a stock jar
2. Autoclave solution
3. Add 500µl of carb
4. Pour plates as usual

Creating Liquid Culture

1. Add 5ml of LB to a 15ml culture tube
2. Inoculate a few colonies of cells

Obtaining Water

1. Obtain 1000 ml of creek water from any nearby water body by placing a stock jar within the water and allowing the water to flow into the stock jar.

Filtration

1. Run distilled water through all the filters until all filters are wet.
2. Get five 600 ml beakers
3. Combine the 5 ml liquid culture of E.coli to the 1000ml creek water. Stir the stock jar until the liquid culture is thoroughly mixed with the creek water.
4. Measure 100ml of the creek water spiked with E. Coli.
5. Run the 100 ml of creek water through the muslin filter bottle. Make sure a labeled 600 ml beaker is placed under the bottle to catch filtered water.
6. Repeat steps 4 and 5 with the negative control (no filter), cheesecloth, t-shirt, coffee filters.
7. Run the creek water spiked with E. coli through the Micron Filter (Positive Control)
1. Pour 100 ml of the creek water spike with E. Coli to the top cup of the micron filter.
2. Attach the filtration vacuum to the side of the micron filter. Turn on the vacuum so the water is filtered through the .2 micron filter.
3. Allow the water to be filtered through the micron filter and stop the vacuum when all the water has filtered to the bottom container of the micron filter.

Serial Dilution and Plating

1. Obtain 6 centrifuge tubes.
2. Plate 150 microliters of each of the filtered solutions in the 6 600ml beakers into corresponding carb plates. Use glass beads to spread the cultures throughout the carb plates.
3. Fill a beaker with 10 mL of distilled water.
4. Pipette 15 microliters of the filtered water that ran through the muslin and dispense in a centrifuge tube. Label the centrifuge tube, muslin.
5. Pipette 185 microliters of distilled water and dispense in the same centrifuge tube.
6. Repeat steps 12 and 13 with the negative control (no filter), cheesecloth, t-shirt, coffee filters, and the micron (positive) filter.
7. Plate 150 microliters of the solutions from the microcentrifuge to the corresponding carb plates. Use glass beads to spread the cultures throughout the carb plates.
8. Place the plates in the incubator and incubate at 37 degrees Celsius for 24 hours.
9. After 24 hours observe the results and note use colony forming units.