Heat-shock coli strain DHSo..
E. coli strain D H5a ™ ( l nvitrogen™) cells were used for the mai ntenance and amplification of seq uencing and expression plas m id vectors. Chemically com petent cel ls were prepared using the calcium chloride transformation protocol described by
Seidman et al. ( 1 992) modified as follows: ( 1 ) 2XL medium replaces LB broth
(section 2 . 1 . 3) (2) E. coli were grown until they had entered mid log phase (indicated
by a solution optical density between OD600 0.45 and ODsoo 0. 55) . (3) The CaCI2 solution was replaced with a filter sterilised "Transformation Buffer" containing 1 00 mM CaCb, 70 mM MnCb.4H20, and 40 m M aqueous sodium acetate , adjusted to pH 5.5 with 0 . 1 M acetic acid.
Heat shock transformation was carried out essentially as described by Seidman
et al. ( 1 992) except that the DNA ligation m ixtu re was made to a volume of 60 !JL
with additional M i ll i-Q® water before being m ixed with 1 00 !J L of thawed chem ically com petent cells. After a 30 min incubation on i ce this solution was heat shocked at 37 °C for 5 m i n , being diluted with 800 !J L of pre-heated LB broth and i ncubated for
1 00 m in at 37 oc before plating.
2.2.6.2 ELECTRO-TRANSFORMATION E. COLI.
In E. coli strains AD494 and Origam i ™ (Novagen®) , plasmid vectors were
introduced by electroporation . Electrocom petent cells were prepared and stored cryogenically (at -80 °C until required) according to the method of Seidman et al. ( 1 992).
TABLE 3 : CELL-PORATOR® ELECTROPORATION SETTINGS FOR TRANSFORMATION OF E. COLI
Parameter Setting
Capacitance 330JIF
Resistance Low
Charge Rate Fast
Voltage Booster 2 kQ
Electrotransformation of E. coli was carried out with a Ceii-Porator® 230 V Electroporation System I device equipped with a Voltage Booster. The microelectroporation chambers used had a gap width of 0. 1 5 cm and were obtained from l nvitrogen ™. The electroporation parameters used are described in Table 3 and were suggested by Peter Murphy (personal com m u n ication).
2.2.6.3 ELECTRO-TRANSFORMATION OF P. PASTORIS CELLS.
Electrocompetent Pichi a pastoris strains GS 1 1 5 and SMD1 1 68 were prepared
according to the protocol of Cregg and Russell ( 1 998) .
Electro-transformation of electrocompetent P. pastoris cells was carried out using a Gene-Pulser® transformation apparatus eq uipped with a Bio-Rad® Pulse Controller device. The electroporation chambers used had a gap width of 0. 1 5 cm. The electroporation apparatus was set using the electroporation parameters suggested for this device by Cregg and Russell ( 1 998) .
Cells were transformed with pPIC9K based vectors linearised with either Sst I or Bgl l l to help with the integration of or the tra nsplace ment by, the recombinant gene at the genomic locus of the A OX1 gene as described in l nvitrogen T M (2002) and
Romanos et al. ( 1 998). Transformed cells were d i l uted with 1 ml of cold 1 M sorbitol and aliquots of this sol ution were plated on B M D agar. The cultures were incubated at 30 °C for 2-4 days before further experimentation .
The recom binant Pichia strains were screened to establish m ethanol utilisation (Mut)
phenotype accordi ng to procedures stipulated in Romanos et al. ( 1 998) and
lnvitroge n ™ (2002) .
2.2.6.4 PLASMID PURIFICATION FROM E. COLI.
Clones.
The process of screen ing transformants for the correct plasmid was carried out according to the m ethod of Zhou et al. ( 1 990). Plasmids purified in this m anner were analysed by restriction d igestion. The enzyme and buffer combinations and i ncubation conditions recom mended by the manufacturer were used, but an
Methods and Materials.
additional 1 �L of RNase A ( 1 0 mg/ml) was added to each reaction to remove residual RNA. After having been digested, the sam ples were resolved by agarose gel electrophoresis and then analysed. When analysing pGEM-r® based plasmids, the restriction enzyme(s) used were a com bination of either Bst XI and Sst 11 or
Nco I and Pst I. 1 U of each enzyme was used per 20 � L of reaction solutio n . When analysing pGEM-T Easy® plasmids, Eco RI (1 U per 20 �L reaction) was used .
Plasmid DNA was purified from E. coli using a Q IAq uick® Plasmid Mini prep Kit as
described in the manufacturer's instructions.
Plasmid DNA that was required for plasmid construction, or enzymatic digests, o r transformation experiments was prepared using t h e alkaline lysis "mini-prep"
m ethod (Engebrecht et al. , 1 992). Plasmid DNA req u i red for sequencing reactions
was am plified by inoculating 3.5 ml of LB broth [supplemented with ampicillin ( 1 00 �g/m l)] for 16 h at 37 °C with shaking. After 1 6 h , 1 00 �g/m l spectinomyci n was added and the cultu re was incubated as before for an additional 1 6 h . At this point, the plasmid DNA was pu rified from the host bacteria using a QIAq uick® Plasmid Miniprep Kit according to manufacturer's instructions. The identity of
pKK223-3 based plasmids was established by double digestion with Sai l and Nde I
after they had been a m plified by this method. 2.2.6.5 LIGA TION PROTOCOLS.
T-Tailed Vectors.
Ligation reactions were carried out on the appropriate linearised or T -tailed vector using T4 Ligase and buffers supplied with the pG EM-r® and pGEM-T Easy® kits. I n each reaction , the molar concentration of insert was always three times that of the vector and the reactions were incubated overnight at 1 0 °C for m aximum efficiency. Otherwise, the reactions were carried out according to the manufacturer's instructions.
Construction
The EAS isoform I cDNA was cloned into the pPIC9K expression vector between the Xho 1 1 193 bp site and the Eco Rl1223 bp to create a plasmid called pP9KEASF1 .
The Xho 1 1 1 93 bp is not unique in pPIC9K (see Figure 1 0), making it necessa ry to
carry out a 3-way ligation reaction in order to link the cDNA with the two separate fragments of pPIC9K.
FIGURE 10: PPIC9K. Ss! (209) pPic9K
\
Xhol (5710) EcoRI (1223)The eas cDNA sequence was synthesised by RT-PCR using m R NA extracted from conidiating N. crassa
StA as the template and o ligo-dT as the RT -PCR primer for the 1 st strand reaction (sections 2.2 .2.2 and 2.2.3. 1 ). The 5'-pP I C9KEASF1 and 3'-p PI C9KEAS PCR primers described in Chapter 4 were to prime the second stran d RT-PCR reaction. The final RT-PCR product was ligated
into pGEM-T Easy® to simplify amplification (sections 2 . 1 .3 and 2.2 .6) and verified by DNA seq uencing (section 2.2.7.3).
Concentrated solutions of each plasmid were created by pooling plasmids extracted from three 5 ml bacterial cultures (section 2.2 .6) and then passing the resulting solution th rough DNA-binding columns supplied with the QIAqu ick® kit. The columns were used according to the m anufacturer's recommendations.
The 984 bp (Sst l209 bp-Xho l 1 193 bp) and 8,262 bp (Eco R I 1 223 bp-Sst l2o9 bp) arms of pPIC9K and the 261 bp (Xho ls bp-Eco R l 266 bp) EAS cDNA fragment were excised from their respective plasmids. The plasmid (3 1-Jg) was digested with the appropriate pair of enzymes at 0. 1 U/1-J L. Each fragment was gel purified as described in section 2.2.5.3.
The EAS cDNA fragment and each arm of p P I C9K were ligated together using T 4 Ligase and 1 OX ligation buffer as recommended by the m anufacturer (Promega™). The molar ratio of the DNA fragments added to the reaction was 3 : 1 : 1 (insert: each arm of pPIC9K) . The ligation reaction was incubated overnight at 1 0 oc
to m aximise the efficiency of the ligation reaction .
Plasmids extracted from ampicillin resistant E. coli recombinants (section 2 .2.6),
were screened first by Sa/ I digestio n (p9KEASF1 is cut twice by Sa/ I ) and then by
DNA-seq uencing with pPIC9K-specific sequencing primers , 5' AOX 1 and 3' AOX1 , supplied by l nvitrogen™ (section 2.2.7.3).
Reactions Used to Construct and
For reasons outlined in Cha pter 4, I cloned the cDNA encoding EAS isoforms I and II into the prokaryotic expression vector pKK223-3 (Figure 1 1 ).
FIGURE 11: PKK223-3. EcoAI (4583) RBS tac Psn (25) 55 p K K223-3 4584 bp rRNB T1T2 Amp" pBR322 ori
Methods and Materials.
The cDNAs were inserted between the
Eco R l4sa3 bp and Pst l2s bp sites, and two plasm ids were created: p PKKEASF1 and pKKEASF2. To created these plasmids, the pKK223-3 plasmid was amplified a n d purified from E. coli (as described in sections 2 . 1 .3 and 2.2.6.4), and then digested with Eco RI and Pst I .
The digested pKK223-3 plasmid was
dephosphorylated with Shrimp
Alkaline Phosphatase (l nvitrogen™) because this step was found to enhance the efficiency of subseq uent ligation reactions. Both digestion and dephosphorylation reactions were carried out according to the enzym e manufacturer's recommendations. T h e 4,559 bp fragment derived when pKK223-3 was digested was gel-purified and ligated to cDNAs encoding each EAS isoform . These cDNAs had previously been digested with Eco RI and Pst I and then gel purified in the same m anner as the pKK223-3 fragment. EAS cDNA seq uences were amplified and cloned in the same manner described for p P I C EAS F 1 , except that different PCR primers - as outlined in Chapter 4 - were used for the second strand RT- PCR reaction . The reaction conditions used to ligate the cDNAs to the pKK223-3 fragment were the same as those used for the T -tailed vector ligation reactions described above .
The fidelity of the pKKEASF1 and pKKEASF2 plasmids constructed in this m anner were checked by digesting each with Pvu I and Pvu 11 and then comparing the size of the fragments by gel electrophoresis.