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.

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.

• 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.
• At the appropriate time, add the purified enzyme protein. Incubate at 30°C in between treatments.
• After 48 hours and 30 minutes, transfer each overnight to an eppendorf and centrifuge at 16,000xg for 2 minutes, then discard the supernatant.
• Resuspend pellets in 200 ul ddH2O and stain with 0.03% Crystal Violet solution. Incubate at room temperature for 5 minutes.
• Centrifuge for 2 minutes at 16,000xg and discard the supernatant.
• Wash with 0.8 ml 95% EtOH, centrifuge for 30 seconds at 16,000xg, and discard the supernatant.
• Repeat EtOH wash two more times
• Resuspend the pellet in 200 ul EtOH and transfer to a 96 well microplate reader.
• Set the reader to shake for 10 seconds and then read at 540 nm.