Cloning techniques and vector construction

DNA cloning techniques. Standard techniques were employed for restriction enzyme digestion of DNA, ligation reactions and propagation of plasmids in E. coli.

Isolation o f DNA fragments from agarose gels. For purification of DNA from aga­

rose gels the QIAquick® Gel Extraction Kit (Qiagen) was used according to the manufacturer's instructions.

Vectors fo r expression o f MET2 and MET3 in E. coli. For in-frame cloning into the expression vector pTrcHis C (Invitrogen), the respective coding sequences were PCR-amplified from T. cruzi CL Brener genomic DNA as follows: fragment met2C was amplified with the primers TRCMET2F and TRCMET2R, and fragment met3C was amplified with the primers TRCMET3F and TRCMET3R. pTrcHis-met2 and pTrcHis-met3 were generated by digesting the pTrcHis C vector and the met2C and met3C fragments with Bam HI and Hind III, and subsequent ligation of the relevant PCR fragment with the vector. This resulted in fusion genes encoding proteins with an N-terminal 6x histidine tag for protein purification, and an epitope tag for detec­

tion with the anti Xpress antibody. Plasmid clones were identified by restriction digest analysis, and the sequences were verified b y sequencing both strands of the inserts.

Vectors fo r expression o f c-myc tagged protein in T. cruzi. Episomal expression vectors pTEX-MET2-9E10 and pTEX-MET3-9E10 (Figure 2.1) were constructed by replacing the APX coding sequence in pTEX-APX-9E10 (Wilkinson et al., 2002) with the gene of interest. The resulting proteins carried at their C-terminus the amino acid sequence of the c-myc(9E10) epitope (EQKLISEEDL). The fragments corre­

sponding to the MET2 and MET3 coding sequences were amplified by PCR from T. cruzi CL Brener genomic DNA as follows. The MET2 coding sequence was amplified using primers met20RF-lf and met20RF-lr. The MET3 coding sequence was amplified using primers met30RF-lf and met30RF-lbr. The resulting PCR fragments were cloned in pGEM-T (Promega) and sequenced. The vector pTEX- APX-9E10 was digested with Spe I / Eco RV to remove the 1 kb APX fragment. The linearised vector fragment was ligated with the MET2 or MET3 coding sequence, which had been excised from the relevant pGEM-T construct with Spe I / Nae I. The resulting plasmids pTEX-MET2-9E10 and pTEX-MET3-9E10 were sequenced, and the correct in-frame fusion of the c-myc sequence with the 3’ end of the coding sequence was confirmed.

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Figure 2 .1: pT E X -M E T 2-9E I0 andpTEX -M E T3-9E I()

Episomal vectors for expression o f MET2 and MET3 proteins with a C-terminal c-myc epitope tag.

Strong black lines indicate OAPDH intergenic sequences that facilitate transcript processing.

Vectors fo r expression o f GFP-fusion proteins in T. cruzi. pTEXeGFP was con­

structed by Martin Taylor. The eGFP gene was excised from pEGFP (Clontech) using Eco RI (overhang blunted) and Hin dill and ligated into pTEX (Kelly et al., 1992) digested with Xho I (overhang blunted) and Hind III. Vectors to express MET3-GFP fusion proteins were constructed by replacing the BPP-l-N fragment in pTEXeGFP-N (Bromley et al., 2004) with fragments of the MET3 coding sequence.

The MET3 fragments were generated by PCR, using the vector pTEX-MET3-9E10 as DNA template. Forward primers were MET3-GFPf(2), -(5), -(7), -(11) and -(14).

Reverse primers were MET3-GFPr(3), -(4), -(6), -(8), -(9), -(10), -(12) and -(13).

The resulting PCR fragments were digested either with Eco Rl / Bam HI or Eco RV / Bam HI, depending on which site was engineered into the primer. pTEXeGFP-N was

cut with Eco RI / Bam HI to remove the BPP-l-N fragment, and then ligated with the Eco RI / Bam HI digested PCR fragments. For ligation with the Eco RV / Bam HI digested PCR fragments, pTEXeGFP-N was linearised with Eco RI and the ends made blunt. The BPP-l-N fragment was then excised by digesting with Bam HI, and the vector fragment ligated with the relevant PCR fragment. To build fusion construct 7/8 (encoding N-terminally truncated MET3 with a C-terminal c-myc tag) primers MET3-9E10f(7) and MET3-9E10r(8) were used to amplify the relevant fragment from pTEX-MET3-9E10. This PCR fragment was Spe I / Hin dill digested and cloned into Spe I / Hin dill digested pTEX. The MET3-GFP and MET3-9E10 fusion constructs were sequenced. A schematic diagram of the constructs is shown in Figure 4.27. The names of the constructs indicate the primer pairs used for the amplification of the MET3 fragments.

MET2 and MET3 knockout targeting constructs. Fragments corresponding to the sequences flanking the MET2 and MET3 coding sequences were amplified by PCR from T. cruzi CL Brener genomic DNA as follows. The MET2 5’ flanks were ampli­

fied using primers MET2-5FL-F and MET2-5FL-R. The resulting fragment derived from allele 39p3 is 921 bp and contains a diagnostic Xho I restriction site. The frag­

ment derived from allele lol7 is 639 bp. The MET2 3’ flanks (2.3 kb fragments) were amplified using primers MET2-3FL-F and MET2-3FL-R. The MET3 5’ flank (416 bp fragment) was amplified using primers MET3-5FL-F and MET3-5FL-R.

The MET3 3’ flank (1.3 kb fragment) was amplified using primers MET3-3FL-F and MET3-3FL-R. The PCR fragments were cloned in vector pGEM-T (Promega). The resulting plasmids pGEM-T-5’MET2(lol7), pGEM-T-5’MET2(39p3), pGEM-T- 3’MET2(lol7), pGEM-T-5’MET3 and pGEM-T-3’MET3 were sequenced.

Construction o f pko-MET2(39p3)-NEO. The 5’ A/£T2(39p3) Xho I / Kpn I fragment excised from pGEM-T-5’MET2(39p3) was ligated into pko2-MPX-NEO (Shane Wilkinson, unpublished) from which the 5’ MPX fragment had been excised with Sac I / Spe I. The resulting plasmid pko-5’MET2-NEO was digested with Hin dill / Kpn I to remove the 3 'MPX fragment. The 3’MET2(39p3) Hin dill / Kpn I fragment excised from pGEM-T-3’MET2(39p3) was ligated into the linearised pko-5’MET2- NEO vector, resulting in plasmid pko-MET2-NEO (Figure 2.2A).

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Construction o f pko-MET2(lol7)-HYG. The 5’ M ET2(\o\l) Sac I / Spe I fragment excised from pGEM-T-5’MET2(lol7) was ligated into pSHYGK (courtesy of Martin Taylor) which had been linearised with Sac 1 / Spe I. The resulting plasmid pko-5’MET2-HYG was linearised with Hin dill / Kpn I. The 3’MET2(lol7) Hin dill / Kpn I fragment excised from pGEM-T-3’MET2(lol7) was ligated into linearised pko-5’MET2-HYG resulting in plasmid pko-MET2(lol7)HYG (Figure 2.2B).

Construction o f pko-MET2(lol7)-PAC. The HYG gene in pko-MET2(lol7)HYG was replaced with the PAC gene as follows. pko-MET2(lol7)HYG was digested with Eco RI / Sac I and the pko-MET2(lol7) vector fragment was isolated from an agarose gel (thereby removing the HYG ORF and the 5’ GAPDH flank). Digestion of pko-MPX-PAC (Shane Wilkinson, unpublished) with Eco RI / Sac I, allowed isola­

tion of a Eco RI / Sac I fragment containing the PAC ORF, and an Eco RI fragment containing the 5’ GAPDH flank. First, the PAC ORF was ligated into the pko- MET2(lol7) vector fragment, resulting in pko-MET2-PACl. Then the 5’ GAPDH flank was ligated into pko-MET2-PACl, linearised with Eco RI, resulting in plasmid pko-MET2(lol7)-PAC (Figure 2.2C). The correct orientation o f the 5’ GAPDH flank was verified by a diagnostic Sac II digest.

Construction o f pko-MET3-HYG. The 5 'MET3 Sac I / Spe I fragment excised from pGEM-T-5’MET3 was ligated into pSHYGK (Martin Taylor, unpublished) which had been linearised with Sac I / Spe I. The resulting plasmid pko-5’MET3-HYG was first linearised with Hin dill, the ends blunted, and then digested with Kpn I. pGEM- T-3’MET3 was digested with Xho I and the end made blunt. Then the plasmid was digested with Kpn I to excise the 3’MET3 Xho I / Kpn I fragment, which was ligated into linearised pko-5’MET3-HYG, resulting in plasmid pko-MET3-HYG (Figure 2.3A).

Construction o f pko-MET3-PAC. The HYG gene in pko-MET3-HYG was replaced with the PAC gene as follows. pko-MET3-HYG was digested with Spe I / Sal I to obtain vector fragment pko-MET3, containing the MET3 flanks and the 3’ GAPDH flank. A fragment containing the 5’ GAPDH flank and the PAC ORF was excised

from pko-MET2(lol7)PAC (see above) and ligated into pko-MET3, resulting in pko-MET3-PAC (Figure 2.3B).

The integrity o f all knockout constructs was checked by analysis of restriction pat­

terns. For transfection of T. cruzi, the fragments containing the MET2 or MET3 flanks and drug selectable marker gene were excised from the relevant vector with Sac I / Kpn I.

Figure 2.2: Targeting constructs fo r MET2 knockouts

Linear targeting fragment from (A) pko-MET2(39p3)-NEO, (B) pko-MF.T2(lol7)-HYG and (C) pko- MET2(lol7)-PAC. These fragments were used to replace the M E T 2 gene with the neomycin phos­

photransferase gene (NEO), the hygromycin phosphotransferase gene (//KG') or the puromycin acetyl transferase gene (PAC). Strong black lines indicate GAPDH intergenic sequences that facilitate transcript processing. Blue lines indicate sequences flanking the MET2 gene, used to target homolo­

gous recombination.

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Figure 2.3: Targeting constructs fo r MET3 knockouts

Linear targeting fragment from (A) pko-MET3-HYG and (B) pko-MET3-PAC. Strong black lines indicate G A P D H intergenic sequences that facilitate transcript processing. Red lines indicate sequences flanking the MET3 gene.

The Rediprime” II Random Prime Labelling System (Amersham Pharmacia Bio­

tech) was used according to the manufacturer's instructions to label DNA fragments with [32P] dCTP for use in hybridisation protocols.

Southern blotting. Genomic DNA was incubated with the appropriate restriction enzymes for >4h at 37°C and size-fractionated on 0.8% agarose gels in lx TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0), at 20 V over-night. DNA was transferred to Nylon membranes (Osmonics) in lOx SSC by standard Southern blot procedures, using capillary transfer over-night. After transfer, the DNA was cross- linked to the membrane in a UV Stratalinker (Stratagcne).