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

Adjust pH to 7.8
Add

• 10.00 g Bacto-tryptone
• 05.00 g Yeast extract

Fill to 1000 ml and autoclave

V. cholerae Biofilm growth

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

Phusion PCR (50ul reaction)

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

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

Taq polymerase PCR (50ul reaction)

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

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

Restriction Digest (50ul reaction)

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

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

Ligation

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

Incubate the reaction at room temperature for 30 minutes

Transformations

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

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).

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 stain with 0.03% Crystal Violet solution. Incubate at room temperature for 5 minutes.
5. Centrifuge for 2 minutes at 16,000xg and discard the supernatant.
6. Wash with 0.8 ml 95% EtOH, centrifuge for 30 seconds at 16,000xg, and discard the supernatant.
7. Repeat EtOH wash two more times
8. Resuspend the pellet in 200 ul EtOH and transfer to a 96 well microplate reader.
9. Set the reader to shake for 10 seconds and then read at 540 nm.