Team:BYU Provo/Notebook/Cholera - Enzymes/Protocols

Cholera Enzyme

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Protocols

• 24.00 g NaCl
• 11.90 g MgCl2-6H2O
• 02.00 g CaCl2-2H2O
• 00.85 g KCl

2. Adjust pH to 7.8
3. Add

• 10.00 g Bacto-tryptone
• 05.00 g Yeast extract

4. Fill to 1000 ml and autoclave

V. cholerae Biofilm growth

1. Start a 5 ml overnight of V. cholerae in SSW LB and incubate at 30° for 72 hours.
2. In a separate culture tube add 4 ml SSW LB, 50 ul overnight culture and incubate at 30°C for at least 48 hours.

Phusion PCR (50ul reaction)

1. For each sample add:

• 35 ul ddH2O
• 10 ul 5X Phusion buffer
• 1.5 ul 10mM dNTP's
• 1 ul each Primer (50 uM stock)
• 1 ul appropriate diluted template DNA
• 0.5 ul Phusion Polymerase

2. Set the PCR machine to run the following cycle with the middle three phases being repeated 35 times

• 98°C for 02:00
• 98°C for 00:30
• 65°C for 00:30
• 72°C for 04:00
• 72°C for 8:00

Hold at 4°C 3. Store in -20°C freezer

Taq polymerase PCR (50ul reaction)

1. For each sample add:

• 40 ul ddH2O
• 5 ul 10X TAQ buffer
• 1.5 ul 10 mM dNTP's
• 1 ul each Primer (50 uM stock)
• 1 ul appropriate diluted template DNA
• 0.5 ul TAQ Polymerase

2. Set the PCR machine to run the following cycle with the middle three phases being repeated 35 times

• 95 for 2:00
• 95 for 0:30
• 50 for 0:30
• 72 for 1:00
• 72 for 1:00

Hold at 4°C
3. Store in -20°C freezer

Restriction Digest (50ul reaction)

1. For each sample add:

• 14 ul ddH2O
• 5 ul 10X NEB buffer
• 0.5 ul 100X BSA
• 30 ul DNA sample
• 2 ul each restriction enzyme

2. Place in 37°C incubator or water bath for 1.5 hours

Ligation

1. For each ligation reaction add

• 6.5 ul H2O
• 1.5 ul 10X ligase buffer
• 1 ul T4 DNA ligase
• 3 ul vector
• 3 ul insert

3. Incubate the reaction at room temperature for 30 minutes

Transformations

1. Set two heat blocks at 42°C and 37°C respectively.
2. Thaw 25 ul of chemically competent cells on ice and add 5 ul of DNA to be cloned in.
3. Place on ice for approximately 5 minutes.
4. Heat shock at 42°C for 1 minute and immediately place back on ice for 2-5 minutes.
5. Add 500 ul LB to the reaction and incubate at 37°C for 30 minutes.
6. Plate 100 ul of cell on appropriate antibiotic plate and incubate at 37°C overnight.

Verifying Clones with Colony PCR

1. Add 1 colony to 50 uL of water in a PCR tube. Do this by using a pipet tip and by carefully picking an isolated colony.
2. Briefly vortex the PCR tube and streak the solution to a marked section of an LB plate to allow you to go back to the correct colony once the correct product is identified.
3. Boil the solution in the PCR tube for 5 minutes in the PCR machine. The DNA is now ready to use as a template.
4. Repeat steps 1-3 for seven more colonies to give a total of eight colonies.
5. Set up a standard Taq PCR reaction and run for at least 25 cycles.
6. Verify the inserts by running 5 uL of product out on agarose gel. For 2 or 3 clones that test positive for insert, clean up the PCR products and sequence.

Protein Purification

Reagents

• 10X Lysis Buffer: 0.1 M KCl, 0.2 M imidazole, 0.5 M Hepes pH 7.8
• 1X Lysis Buffer-300: Dilute 10X stock to 1X and add 1:300 PICs and 300 mM NaCl. Next add
• Phosphatase Inhibitors)- 50 mM NaF (0.21g in 100 mL) and 50 mM glycerolphosphate (1.08g in 100 mL). Add 1 mM BME (beta-mercaptoethanol) as reducing agent. Recheck Ph.
• Add PICs only to enough buffer to re-suspend pellets in.
• 1X Lysis Buffer-100: Dilute 10X stock to 1X and add 100 mM NaCl and 250 mM imidazole. Add 1 mM BME (beta-mercaptoethanol) as reducing agent. Recheck Ph.

1. Grow up 500 mL of yeast expressing His-tagged PSK (Grow in PSK activating conditions Gal, Raff, etc.).
2. Re-suspend pellet in 15 mL 1X lysis buffer-300. (Make sure this has the 1:300 PICs added).
3. Lyse cells.
4. Centrifuge for 30 min. to pellet cell debris. Pour into new tubes and centrifuge for another 30 min. While cells are spinning prepare Ni-NTA agarose beads (Prepare 250 uL per sample):a. To equilibrate Ni-NTA gently pellet beads at 1,000xg for 1-2 min, mark with pen on eppendorf where liquid line is, pull off ethanol, add 1 mL water and mix, gently pellet beads again, discard water, repeat, re-suspend in1X lysis buffer-300 and mix, gently pellet again, repeat then re-suspend finally in the lysis buffer up to the marked line and let sit on ice for 15 minutes before use.
5. Transfer supernatant to blue cap tube and add 250 uL of Ni-NTA equilibrated in the 1X lysis buffer described above, rotate at 4°C for 3 hr.
6. Centrifuge in 15 mL conical tubes at 1,000 RPM for 3 min. (Wash with 15 mL 1X lysis buffer-300 and repeat).
7. Transfer resin to column and wash with 50 mL lysis buffer-300.
8. Elute with 0.5 ml 1X lysis buffer-100
9. Repeat elution (3x) to verify all protein is off the column. Elute into new tubes and keep elutions separate.
10. Flash freeze protein in sterile glycerol (15% final concentration).

Biofilm Assay Protocol

• Revised from original Biofilm Degradation Assay from Pitts, B.; Hamilton, M.; Zelver, N.; Stewart, P. A Microtiter-Plate Screening Method for Biofilm Disinfection and Removal. J. Micro Methods 2003, 54, 269-276. V. cholerae biofilms are more apt to free float rather than develop on solid surfaces.

Biofilm Prep

1. Overnight culture of V. cholerae grown in SSW LB at 30 °C for 72 hrs.
2. 20 ml of culture added to 180 ml of SSW LB for dilution
3. Fill each well of a 96-well plate with 200 ul of SSW LB
4. To inoculate wells, immerse replicator pins in the diluted bacterial suspension for 30 seconds, then lower replicator pins into wells and agitate for 15 seconds.
5. Plates are covered and incubated with shaking at 30 °C for 24 hrs.
6. Every 10-12 hrs during the incubation period, spent nutrients are pipetted from wells and replaced with fresh SSW LB

Enzyme Treatment

1. At 24 hrs, planktonic suspensions and nutrient solutions are aspirated and wells are rinsed by carefully removing and adding liquid from the side of the well using a micropipette. Wells are washed four times.
2. Enzymes are added to wells immediately after rinsing and allowed to sit for 1 hour.
3. After 1 hour, wells are washed again and the stains are applied:
   1. 0.16% filter-sterilized CTC incubated at room temperature for 2 hr, CTC solutions are prepared directly prior to staining
   2. 0.3% CV solution incubated at room temperature for 5 min
4. After incubation times, absorbed stain is eluted from attached cells with 95% ethanol
   1. Wells are rinsed four times to remove excess stain
   2. Wells then filled with 200 ul ethanol and incubated
      1. CTC incubated for 15 min at room temp
      2. CV incubated for 5 min at room temp

Absorbance Readings

1. Plates are vigorously shaken for 10 seconds, then light absorbances are read with BioTek FL600 plate reader
   1. CTC absorbance is read at 450 nm
   2. CV absorbance is read at 540 nm
2. Each raw absorbance value is then corrected by subtracting the mean of absorbance readings for the blank wells prior to statistical analysis

Final V. cholerae Biofilm Degradation Assay

• Revised from original Biofilm Degradation Assay from Pitts, B.; Hamilton, M.; Zelver, N.; Stewart, P. A Microtiter-Plate Screening Method for Biofilm Disinfection and Removal. J. Micro Methods 2003, 54, 269-276. V. cholerae biofilms are more apt to free float rather than develop on solid surfaces.

1. In a culture tube, add 50 ul overnight seed to 1 ml SSW LB in culture tube. (Set up three cultures for each time interval and concentration of enzyme to be assayed.) We chose to test 2.5 ul, 12.5 ul, and 25 ul at time = 0, time = 0 & 42, time = 42, and time = 48.
2. At the appropriate time, add the purified enzyme protein. Incubate at 30°C in between treatments.
3. After 48 hours and 30 minutes, transfer each overnight to an eppendorf and centrifuge at 16,000xg for 2 minutes, then discard the supernatant.
4. Resuspend pellets in 200 ul ddH2O and centrifuge at 16,000xg for 2 minutes and discard the supernatant. 5. Resuspend pellets in 200 ul ddH2O and stain with 0.03% Crystal Violet solution. Incubate at room temperature for 5 minutes.
6. Centrifuge for 2 minutes at 16,000xg and discard the supernatant.
7. Wash with 0.8 ml 95% EtOH, centrifuge for 30 seconds at 16,000xg, and discard the supernatant.
8. Repeat EtOH wash two more times
9. Resuspend the pellet in 200 ul EtOH and transfer to a 96 well microplate reader.
10. Set the reader to shake for 10 seconds and then read at 540 nm.