Hi everyone,

A while back a lithium acetate transformation of a triple knock out mutant Candida albicans strain was performed yielding several transformants. The transformants, however, integrated the genes at the wrong locus: the genes belong to a multigene family with high homology (~89%). The transforming DNA was prepared using a restriction digestion reaction because the DNA is integrated into the genome via homologous recombination. The protocol that was used to obtain the transformants is currently being used again, but this time no transformants have been identified. Additionally, the cells take several days to grow up (4-5 vs the recommended 2-3), exhibit only a few colonies per plate (2-4 colonies and sometimes none) and consistently exhibit growth on the negative control. (This transformation has been performed several times with the same results every time)

Any ideas as to what the problem might be, or suggestions for how to approach this problem? Any advice is greatly appreciated. Thank you!

The transformation was performed according to the protocol below.

Transformation protocol:

Grow up 4 ml YPD starter culture at 30 deg. C to OD600 ~ 0.8.

Inoculate 50 ml YPD overnight culture with 250 ul of starter culture.

Inoculate 50 ml YPD 4-6 hour culture to OD600 ~ 0.2

Harvest cells when OD600 ~ 0.8.

Centrifuge culture at 3500 RPM for 5 min, remove supernatant.

Wash pellet with sterile water, pellet again.

Re-suspend pellet in 0.5 ml TELiAc.

To two empty microfuge tubes add 100 ul of cells.

Add 5 ul of 10mg/ml ss DNA to both tubes.

Add 5 ug of transforming DNA to one tube, other is DNA control.

Incubate at room temperature for 30 min.

Add 0.7 ml of PLATE mix (LiAc + TE buffer + 50% PEG) to both tubes.

Incubate at room temperature overnight.

Heat shock cells at 42 deg. C for 1 hour.

Centrifuge cells at 5000 RPM for 3 min.

Re-suspend pellet in 0.2 ml sterile water.

Plate on selective media. (300 ug/ml CloNat)

Incubate at 30 deg. C for 2-3 days.