A Highly Efficient Protocol for Transformation of Saccharomyces cerevisiae and Pichia pastoris by Electroporation＊

XU Zhiwu,ZHANG Ying,WANG Zhixue,CAO Yan,CU I L iang,LI Sheng,LIU Qiuyun

(1.State Key Laboratory of Biocontrol,The Key Laboratory of Gene Engineering of Ministry of Education and Biotechnology Research Center,Sun Yat-sen University,Guangzhou 510275,China;2.Guangzhou Center for Disease Control and Prevention,Guangzhou 510080,China)

Saccharom yce cerevisiaeis the workhorse of eukaryotic genetics and molecular biology.It is also widely used for high throughput screening in drug development.Electroporation is regarded as one of the most efficient transformation techniques forS.cerevisiae,particularly in the propagation of cDNA libraries,genomic libraries and random DNA libraries.However,we frequently encountered problems of yeast strain degeneration overt imeorover environmental exposure,which resulted in sharp decline of electroporation frequency in routine experiments.Pretreatment of S.cerevisiaecells with lithium acetate(LiAc)and dithiothreitol(DTT)enhanced the frequency of trans for mation by electroporation[1],but the efficiencies still varied with strains,and changed over time.Although the LiAc/single stranded carrierDNA/PEGmediated method was reported to be highly efficient[2-3],it did notwork verywell in our hands.

The yeast Pichia pastorisis widely used as a host for the expression of heterologous proteins.Most vectors must integrate into thePichiachromosome,an inherently inefficient process[4],hence transformation efficiencies were very low[5-6].Pichiais usually transfor med by electroporation and requires DNA in the microgram range.The optimized lithium acetate(LiAc)and dithiothreitol(DTT)Pretreatment protocol has been adapted toP.pastoris and yielded good results[7].However,asPichiastrains degenerate over time,transformation efficiencies drop substantially.

Single-stranded carrier DNA has been previously reported to be capable of enhancing transformations[2].As electroporations produce membrane fracture and holes,it is hypothesized that an 1 hour post electro poration reviving growth in an isotonic medium may heal these cells and substantially raise transformation efficiency.This has been shown previously[8].Here we present data in the investigations for a highly efficient electroporation procedure forS.cerevisiaeand P.pastoris.

1 Materials and methods

1.1 Stra ins and plas m ids

S.cerevisiaestrainGY2050(his3Δ200 ura3Δ0trp1Δ63leu2Δ0)was provided by Dr.Greg Prelich(Albert Einstein College of Medicine,NY).S.cerevisiaestrain INVSc1(MATa/MATa his3-Δ1/his3-Δ1leu2/leu2trp1-289/trp1-289ura3-52/ura3-52)andP.pastorisstrain GS115(His4)were products of Invitrogen(Carlsbad,California).S.cerevisiaeS150-2B(MATaleu2-3,112ura3-52trp1-289his3-Δ1)wasprovided byDr.Derek Jamieson(Heriot-Watt University,United Kingdom).The procedure was developed using the 6 kbE.coliand yeast shuttle vector pYES2/CT plasmid(Invitrogen,Carlsbad,California)as the input DNA.Pichiaplasmid p ICZαA was also a product of Invitrogen(Carlsbad,California).Plasmid DNA was prepared with the Spin Miniprep(Minipreparation)Kit(ACT·Gene,China)aspermanufacture's instruction and quantitated on a DU®530 DNA/Protein Analyzer(Beckman,Fullerton,CA).

1.2 M ed ia and single stranded DNA

The YPD medium consisted of 2.0%(w/v)Peptone,1.0%(w/v)yeast extract,2.0%dextrose.S.cerevisiaeUra+transfor mantswere selected on synthetic medium containing 0.67%Yeast Nitrogen Base without amino acids(D IFCO Laboratories,Sparks,Maryland)supplemented with 1.0%glucose,1 mol/L sorbitol,and 20 mg/L of leucine,histidine and tryptophan each aswell as 2%agar.YPDS plates contained 1 mol/L sorbitol and 100μg/mL Zeocin in addition to YPD.

Single stranded Salmon sperm carrier DNA(Sigma-Aldrich,Saint Louis,MO)was prepared by weighing outDNA and placing it in a boiling water bath for at least 5 min followed by a quick cooling in ice water slurry.Carrier DNA can be frozen after boiling and used 3 or 4 times,and re-boiled for 1 min for subsequent uses.The ss DNA is used at a concentration of 20μg/μL.

1.3 Electroporation procedure

S.cerevisiaecompetent cellpreparation was as described[1].Briefly,BothS.cerevisiae and P.pas tor is were propagated overnight to stationary phase.100μL of the culture were inoculated into 200 mL of YPD broth and grown overnight at30℃with shaking.Yeast cells were harvested at anA600nm of 1.0～1.5.The cells were pelleted by centrifugation(2 500×g,5 min),suspended in 25 mL of 0.1 mol/L lithium acetate,10 mmol/L dithiothreitol,10 mmol/L Tris-HCl,pH 7.5,1 mmol/L EDTA(LiAc/DTT/TE)and incubated at 25℃for 1 h.The cells were centrifuged at 2 500×g for 5 min at 4℃and the pellet was suspended in 50 mL ice-cold water.This step was repeated and the pellet was then suspended in 10 mL of ice-cold 1 mol/L sorbitol followed by centrifugation.Subsequently the pelletwas suspended in 800μL of 1 mol/L sorbitol which yielded 1 200μL of cell suspension in total.Twentyμg of single stranded carrier DNA and 15 ng plas mid DNA in no more than 10μL were added to 80 μL of cell suspension corresponding to around 5×108cells,which was transferred to an electroporation cuvette and electro porated at 9.0 kilovolts/cm on an Eppendorf 2 510 electroporator(Eppendorf AG,Hamburg,Germany)with a built-in time constant of 5 ms.I

mmediately,650μL cold 1 mol/L sorbitolwas added and the contents of the cuvette were gentlymixed,and transferred to 1.5 mL Eppendorf tubes.650μL 2×YPD and 1 mol/L sorbitol were then added to the mixture,and incubated at 30℃for 1 hour with gentle shaking.The cell suspensions were washed once with sterile water and concentrated by spinning at 400×g for 2 min.An aliquot was spread on plates.Ura+trans formants were selected on synthetic medium containing 0.67%Yeast Nitrogen Base without amino acids supplemented with 1.0%glucose,1 mol/L sorbitol,and 20 mg/L of leucine,histidine and tryptophan each,and 2%agar.Incubation was carried out at 30℃,andS.cerevisiae colonies were counted after 2～3 days.

P.pastoriscellselectroporated with10ng p ICZαA linearized withSacII were directly plated on YPDS/zeocin plates without washing,and incubated for 3～4 days at 30℃.

2 Results and discussion

The preparation of competent cells was essentially as described[1].Modifications were conducted at 3 levels,and all included a lithium acetate(LiAc)and dithiothreitol(DTT)pretreatment step.Initially an hour of post-electroporation growth in YPD/1 mol/L sorbitol was included.The next modification was the addition of single stranded carrier DNA into cell suspensions prior to electroporation.The final optimization included an incubation of cell and DNA mixture in refrigerator for 5 minute before pulsing.The refrigerator usually has a temperature of 6～8℃.

The results of an electroporation experiment are shown in Fig.1 .A post-electroporation growth in YPD had a clear effect on transfor mations ofS.cerevisiae with shuttle vector pYES2/CT,resulting in 3.5 fold enhancement over lithium acetate(LiAc)and dithiothreitol(DTT)pretreatment along.The presence of single stranded carrier DNA for electro poration had an effective role in the improvement of electroporation efficiency,and resulting in about 4 fold enhancement.Although occasionally 2-3 fold increase in transformation frequencieswas obtained with a 5 minute incubation in the refrigerator immediately before electroporation,only marginal effect was observed in Fig.1 .O-verall,a 13 fold enhancement was clear in Fig.1 with inclusion of ss DNA and post-electroporation growth prior to plating.Untransformed S.cerevisiaestrain failed to grow.We observed near 100 fold enhancement over lithium acetate(LiAc)and dithiothreitol(DTT)pretreatment along in electroporations of some degenerated strains of S.cerevisiaeS150-2B and INVsc1,to a level of 105transformants/μg(data not shown).

Fig.1 Logarithm of transfor mants of a 6 kb plas mid pYES2/CT electroporated intoS.cerevisiaeGY2050 cells via different treatments.(L iAc+DTT),pretreatment with L iAc+DTT.(Growth in YPD),an hour of post-electroporation growth in YPD/1 M sorbitalwas conducted in addition to a LiAc+DTT pretreatment.(ss DNA+Growth in YPD),ss DNA was added to cell suspensions prior to electro poration in addition to pretreatment and growth in YPD.(ssDNA+6C+Growth in YPD),DNA and cell mixture were incubated in a refrigerator for 5 minutes before pulsing in addition to pretreatment,growth in YPD and inclusion of ssDNA.Transformation efficiencies are shown with vertical bar in average value of triplicate electroporations with one standard deviation.Standard deviations were calculated using Microsoft Excel 2003.

Linearization of p I CZαA at site of the multiple cloning region by SacIIwasper for med before electroporation of P.pastoris.Marked enhancement in transformation by inclusion of ss DNA and an hour of post-electro poration growth prior to plating,over lithium acetate(LiAc)and dithiothreitol(DTT)pretreatment along[7],is evident from Fig.2 ,and an 114 fold increase was recorded.It has been reported that linearization using different restriction enzymes may have 30 fold variations in trans formation frequencies,and SacI was the most efficient among tested[7].

Fig.2 Logarithm of transformants of a 3.6 kb plasmid p I CZαAintoP.pastoris GS115 cells via different treatments.(LiAc+DTT),pretreatment with LiAc+DTT.(ssDNA+Growth in YPD),ss DNA was added to cell suspensions prior to electroporation in addition to pretreatment with LiAc and DTT and growth in YPD.Transformation efficiencies are shown with vertical bar in average value of duplicate electroporations with one standard deviation.Standard deviations were calculated using Microsoft Excel 2003.

The enhancement of ss DNA was attributed to its higher binding affinity to the cell wall than double stranded DNA.Thus the titration of the ds-trans for ming DNA by absorption to the cellwallwas inhibited,and it increased the entry of transforming DNA molecules into the cell[9].

In summary,inclusion of ss DNA and a postelectroporation growth period markedly enhanced the trans for mations ofS.cerevisiae and P.pastoris.The 5 minute incubation in the refrigerator prior to electroporation is optional as itwas effective in some transformations.

Acknowledgment:Xu Zhiwu,Zhang Ying,Wang Zhixue and Cao Yan have made equal contributions to thiswork.

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