Yeast Expression Support—Troubleshooting

Solution

The pH of Solution I or Solution III may have drifted. The pH of both
solutions should be 8.0

Check the pH of Solutions I and III. If the pH is low, increase it by adding NaOH. If the pH is high, decrease it by adding HCl. Store solutions at 4°C in order to minimize drift in pH.

Transformation reaction not mixed
during incubation

Be sure to mix the transformation reaction every 15 minutes throughout the 1 hour incubation at 30°C. Vortexing works best.

Incubation time is too short or temperature is too low

Pichia pastoris transformations may be incubated for longer periods of time (up to 3 hours) and at higher temperature (35–37°C). This may, in some instances, result in higher transformation efficiencies.

Cell density is too low (OD600 <0.6)

Resuspend cells from Preparation of Competent Cells, Step 6, page 1 of the manual, ina smaller volume (i.e.,500 μL).

Cause

Solution

No multimers or low number of multimers after transformation into E. coli

CIAP defective

Not enough insert DNA to ligate

Construct is unstable in E. coli

Multimers are too long to ligate efficiently

Use fresh CIAP.
Add more CIAP. Add 1 unit of CIAP and incubate 15 more minutes at 37°C. This is somewhat risky as CIAP can degrade the ends of your DNA.

Add more BamH I-Bgl II expression cassette to your ligation.

Decrease the number of cassettes in the vector.

Try ligating each expression cassette
Stepwise.

Recombinant vector rearranges and deletions are detected

Construct is unstable
in E. coli

Decrease the number of cassettes in the vector.

No antibiotic-resistant Pichia transformants

Integration efficiency is low

Transform using more DNA and/or
do multiple transformations with more DNA and cells.

Cause

Solution

No multimers or low number of multimers after transformation into E. coli

CIAP defective

Not enough insert DNA to ligate

Construct is unstable in E. coli

Multimers are too long to ligate efficiently

Use fresh CIAP.
Add more CIAP. Add 1 unit of CIAP and incubate 15 more minutes at 37°C. This is somewhat risky as CIAP can degrade the ends of your DNA.

Digest more pAO815 containing one copy of the expression cassette.

Use the in vivo method to isolate multimers.

Try ligating each expression cassette separately.

Recombinant vector rearranges and deletions are detected

Construct is unstable
in E. coli

Use the in vivo method to isolate multimers.

Pichia His+ transformants
do not have multimers

Vector was linearized with the wrong enzyme (restriction enzymes in the
5´ AOX1 region are duplicated when multimers are created)

Linearize your construct with Sal I or Stu I to insert the construct into
his4.
Analyze your construct for other unique restriction sites in the vector backbone that are near the 5´ AOX1 region or the 3´ AOX1 region. These sites will preserve your multimers and allow recombination with AOX1.

Cause

Solution

False positives

Not enough levels of Geneticin resistance

Colonies can appear to be Geneticin resistant, but are actually not. To prevent this, purify your putative Geneticin-resistant colonies and confirm the level of Geneticin resistance observed for each colony before proceeding further.

Low number of Geneticin-resistant colonies isolated

Not enough His+ transformants were screened

The frequency of spontaneous, multiple-integration events occurs only at a rate of 1–10%. You will need to screen thousands of His+ transformants to isolate an optimal amount of colonies.

Few recombinants with gene of interest

Not enough His+ transformants were screened

You will need to screen thousands of His+ transformants to isolate an optimal amount of recombinants with the most copies of your gene, as successive multiple insertions are more rare.

Low isolation of His+ transformants

Low transformation efficiency

Using electroporation instead of spheroplasting may increase the transformation efficiency, thereby allowing you to isolate more His+ transformants.