Expression and Purification of Enzymes

Solutions

Resuspension Buffer

• 400 mM NaCl
• 50 mM NaHPO4
• 2.5% (v/v) glycerol
• 15 mM Imidazole
• 0.05% Triton X-100
• pH 8.0

Taq Ligase Storage Buffer

• 10 mM Tris-HCl
• 50 mM KCl
• 1 mM DTT
• 0.1 mM EDTA
• 200 μg/ml BSA
• 50% v/v Glycerol
• pH 7.4 @ 25°C

T5 Exonuclease Storage Buffer

• 50 mM Tris-HCl
• 100 mM NaCl
• 1 mM DTT
• 0.1 mM EDTA
• 50% v/v Glycerol
• 0.1% Triton® X-100
• pH 7.5 @ 25°C

Transforming the Synthesized Plasmid

1. The genes for Taq Ligase and T5 exonuclease were designed synthesized in a factory plasmid by GeneArt
2. Prepare 100 mL stocks of BL21(DE3) competent cells (stored at -80°C)
3. Allow to unthaw, and add 2 uL DNA (using 2uL water as a control) to competent cell cultures.
4. Let sit on ice for ten minutes
5. Heat-shock cells for 45 seconds in 42°C water bath
6. Return cells to ice for two minutes
7. Allow cells to recover by adding 900 uL LB and putting into 37°C shaking incubator for 1 hour
8. Plate 100 uL on Kan(50) LB agarose plates and incubate for 16 hours at 37°C
9. Determine which colonies have successfully taken up plasmid by cPCR

Colony PCR Protocol

Each tube:

• 25uL PCR tube contents:
• 1uL of Forwards and backwards Primers at 10uM concentration
• 10.5uL Nuclease Free H20
• 12.5uL goTaq
• Cell Culture

Method:

1. Pick a colony from the cell culture using a sterile toothpick
2. Streak out on Kan(50) lysogeny broth (LB) agarose
3. Place in the 25uL Reaction Mix and spin toothpick for a few seconds

PCR Block Settings:

• 1. Initial Denaturation: 2.5 min @ 95°C
• 2. Denaturation: .5 min @ 95°C
• 3. Annealing: .5 min @ primer annealing temperature
• 4. Extension: 1 min @ 72°C
• Repeat steps 2-4 30 times
• 5. Final Extension: 3 min @ 72°C
• 6. Final Hold: indefinitely @ 4°C

Starting Overnights of Cultures

• A single colony selected from each cPCR Streak plate was used to inoculate a 5 mL Kan(50) LB culture.
• Grow for ~16 hours at 37°C (Ensure that the proper antibiotic (kanamycin in this case) is added to the cultures)
• Two cultures containing each plasmid will be grown out (T5 exonuclease, Taq Ligase, and the empty pET vector)

Culture Growth

1. Overnight cultures are to be diluted using LB to an OD600 of 1.0 in order to use 2mL of diluted culture to inoculate 200mL Terrific broth
2. This is then grown at 37°C while checking the OD600 of the culture every hour or so.
3. Upon reaching an OD600 of 0.700, the culture is to be induced with IPTG, to a final concentration of 500 uM.
4. The cultures are then to be shaken for 16 hours at 20C°.

Preparation of Columns

1. Clean column using washes of EtOH and 2%SDS
2. 3 final rinses with Deionized water
3. Fill 15mL column with resuspension buffer, add 1mL Ni-NTA resin beads
4. Rock on ice for 20 min
5. Allow buffer to flow through column until it is mostly empty (Note: Never allow the Ni-NTA layer to be dry or disturbed)
6. Column is ready to bind protein