-
Digest 50 uL of pPic9K with EcoRI, NotI, Cutsmart buffer, Nuclease free H2O
- Purpose: This removes the current insert in the plasmid
- Don’t need to streak plates with pPic9K before since we already have 20 uL of the plasmid DNA (it’s not in bacteria, like in the -80 freezer)
- We need to get NotI from a community project
-
Run the gel
- Purpose: Pick out the empty pPic9K
-
Gibson Assembly insert into pPic9K
- Purpose: This puts the insert into the plasmid
-
Transform E. Coli with transformed plasmid
-
(1) Molecular Cloning
- Restriction Digestion
- Restriction enzymes: NotI (have) and EcoRI (have)
- Restriction enzyme buffer
- (maybe) Bovine Serum Albumin
- Do it with current pPic9K to remove current insert
- Ligation, protocol: https://www.neb.com/en-us/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202
- T4 DNA ligase: 1 uL
- T4 DNA Ligase buffer (10X): 2 uL
- (not necessary) PCR verification — can also take ligated results, run 1 with restriction enzymes to cut and 1 without; or send to plasmidsaurus
- Polymerase (have)
- dNTP mix (have)
- Primers (find which primers span the junction and buy)
- pick a part of the plasmid sequence with low GC content (for low TM (melting point)). 15-25 bases
- (skip) Gel electrophoresis. Note: This can also be used to extract the pPIC9K backbone and purify it. Will also then need scalpel blades and blue light / UV transilluminator
- Gel electrophoresis equipment and reagents
- E. coli Transformation
- Competent E. coli cells (DH5α — we have 10beta) (Serena will this for us)
- LB agar plates with ampicillin (have)
- LB broth with ampicillin (have)
- TSOC medium (have)
- Protocol — follow Gibson Assembly
- Afterwards, do cubit. you’ll get nanograms per miroliter and you know how many microliters
- You know nanograms per microliter of the dna we order
- Transform pPIC
- Plasmid miniprep kit (have)
-
(2) Transformation into Pichia
- Protocols
- Steps
- Make electrocompetent P. pastoris
- Electroporation
- Materials
- 500 ml liquid YPD media in a 2.8 l Fernbach culture shaking flask
- H2O (1 l)
- 1 M sorbitol (100 ml)
- Appropriate selective agar plates
- 1 M DTT (Dithiothreitol) (2.5 ml)
- BEDS solution (9 ml):
- 10 mM Bicine–NaOH, pH 8.3
- 3% ethylene glycol, need this
- 5% DMSO (dimethyl sulfoxide)
- 1 M sorbitol
- 0.1 M DTT
- 1 M HEPES buffer, pH 8.0 (50 ml) (Stored in 2C-8C)
- 250 ml centrifuge tubes
- Electroporation cuvettes
- Electroporation instrument
-
(3) Methanol Induction for Expression: https://docs.google.com/document/d/1KrlE3U_joIoeFXD5T32ykHJudRM392Nj014SQTo7piM/edit?tab=t.0
- Buffered Glycerol-complex Medium (BMGY) — 75 mL total
- Yeast Extract: 1% (10 g/L)
- Peptone: 2% (20 g/L)
- Potassium Phosphate Buffer (1 M, pH 6.0): 100 mL/L
- Yeast Nitrogen Base (YNB) without amino acids: 13.4 g/L
- Biotin: 40 µg/L (need to buy)
- Distilled water: to make the final volume to 1 liter
- Glycerol: 1% (v/v)
- Buffered Methanol-complex Medium (BMMY) — 200 mL total
-
Yeast Extract: 1% (10 g/L)
-
Peptone: 2% (20 g/L)
-
Potassium Phosphate Buffer (1 M, pH 6.0): 100 mL/L
- 132 g of potassium phosphate dibasic (K₂HPO₄)
- 40 g of potassium phosphate monobasic (KH₂PO₄)
- distilled water
-
Yeast Nitrogen Base (YNB) without amino acids: 13.4 g/L
-
Biotin: 40 µg/L
-
Distilled water: to make the final volume to 1 liter
-
Methanol: 0.5% to 1% (v/v) (add after autoclaving)
- Methanol — 1,000 uL
- Liquid N2 or a dry ice/alcohol bath
-
(4) Measure Protein Expression