FACTOR TECNOLOGICO

Where PCR products were of low abundance, or from particularly AT-rich intergenic regions where sequence errors were likely, they were subcloned for amplification and sequencing. The TOPO-TA and Zero Blunt TOPO cloning kits for sequencing (Invitrogen) were used for PCR products generated with AmpliTaq and Pfx respectively, following the supplied protocol. Both kits insert the PCR sequence into the vector pCR4, with EcdR. I sites and sequencing primer start sites flanking the insertion.

2.7.3. Restriction Digestion

Restriction endonucleases were obtained from Roche, with the following exceptions:

Bsg I, BsrG I, Psi I, and Sal I were from New England Biolabs; RjrBR I was from Promega. All restriction digests were carried out according to the supplied protocol. Restriction fragments were separated from unwanted DNA by agarose gel electrophoresis (section 2.6.3), the ethidium bromide stained gel being visualized on a non-UV Dark Reader (Clare Chemical Research) and the band corresponding to the desired product excised. DNA was recovered from gel slices using the QiaQuick gel extraction kit (Qiagen) and eluted in 30pl lOmM Tris-HCl, pH 8.0.

2.7.4. Processing and Ligation of DNA fragments

10 X Nick-translation buffer: 0.5M Tris-HCl, O.IM MgS0 4, ImM DTT,

500pg/ml BSA in H2O, pH 7.5

Where necessary, sticky ends of DNA generated by restriction digestion were blunted by the method o f Sambrook et al (1989). The DNA was mixed with Nick-translation buffer to a final concentration o f Ix, dNTPs to 80pM, and lU o f Klenow fragment. After incubation for 30 minutes at room temperature, the reaction was stopped by adding EDTA to a concentration o f 20mM.

5’-dephosphorylation of DNA fragments was carried out using alkaline phosphatase obtained from Roche by the supplied protocol. Phosphorylation o f PCR products for direct cloning used T4 polynucleotide kinase (New England Biolabs) and the supplied protocol. When multiple processing steps were required, the DNA was re-purified after each enzyme reaction using the QiaQuick PCR purification kit (Qiagen).

Ligations were carried out using T4 DNA ligase (New England Biolabs) following the supplied protocol. The concentration of each DNA fragment was estimated, and the fragments mixed to give a vector:insert molar ratio o f approximately 1:3 (or 1:3:3, for triple ligations.) Incubation was overnight at 16°C for sticky ends or 4°C for blunt ends.

2.7.5. Transform ation of plasmids into E. coli

Luria-Bertani (LB) broth: 1% w/v Bacto-Tryptone, 0.5% w/v Bacto-Yeast Extract, 170mM NaCl in H2O, sterilized by autoclaving

LB-Agar: As above with 1.5% w/v Agar

SOB medium: 2% w/v Bacto-Tryptone, 0.5% w/v Bacto-Yeast Extract, 8.5mM NaCl, 2.5mM KCl in H2O, sterilized by autoclaving, with MgCb added to a

final concentration of 1 OmM just before use.

IB Buffer: lOmM PIPES, 15mM CaCL, 250mM KCl, 55mM M nCh in H2O, pH 6.7,

sterilized by passing through a 22pm pore filter and stored at 4°C.

TOPO cloned PCR products and ligations were transformed into INVa or TOP 10 One Shot chemically competent E. coli (Invitrogen) following the manufacturer’s protocol, and then spread on LB-Agar plates containing lOOpg/ml Ampicillin.

Where long, highly AT-rich sequences were being cloned, E. coli strain PMC-103 (a gift from D. Baker) was used instead. To make them competent, a single colony was used to inoculate 5ml of LB broth, which was incubated overnight at 37°C with shaking at 250rpm. Forty millilitres o f SOB medium was inoculated with 400pl o f the overnight culture, and incubated overnight at 18°C with shaking at 220rpm. Next the culture was chilled on ice for 10 minutes, transferred to polypropylene centrifuge tubes and spun at 3000g for 10 minutes at 4°C. After discarding the supernatant, the pellet was resuspended in 12.8ml TB Buffer, held on ice for 10 minutes, spun down as before and resuspended in 3.2ml TB Buffer. To this was added 240pl DMSO dropwise with gentle mixing, and the cells were again held on ice for 10 minutes. They were then divided into aliquots in cryotubes, fast frozen in N2(i), and stored at -80°C.

To transform competent PMC-103 E.coli, 5pi of a ligation was added to lOOpl thawed cells, held on ice for 1 hour, heat shocked at 42°C for 45s, and spread on an LB-Agar plate containing lOOpg/ml Ampicillin.

2.7.6. Screening of transform ants

Colonies generated by the transformations described above were tested to ascertain whether they contained the correct plasmid as follows: standard AmpliTaq PCR reactions were set up, using two primers within the DNA insert, or one in the insert and one in the vector sequence from a given ligation. A number o f individual colonies from each plate o f transformants were picked using a sterile loop, scraped on the inside o f a PCR tube, and then transferred to a culture of 3.5ml LB broth containing lOOpg/ml Ampicillin. Negative control PCR reactions containing no colonies were also set up, and the cultures were incubated overnight at 37°C, 250rpm. PCR products were analysed by agarose gel electrophoresis, and those colonies which gave no or incorrectly sized bands discarded.

The cultures from the remaining colonies had 0.5ml mixed with an equal volume o f 30% v/v glycerol in LB broth, and stored at -80°C. Plasmid was purified from the other 3ml of the culture using the S.N.A.P plasmid miniprep kit (Invitrogen), from which plasmid DNA was eluted in 60pl TE. These plasmids were further checked for integrity by restriction digests and/or sequencing.

2.7.7 DNA Sequencing

Sequencing Dye: Deionized formamide mixed 5:1 with 25mM EDTA, 50mg/ml Blue dextran in H2O.

Sections of DNA o f up to 600 bases at a time were sëquenced by the dye terminator method (Sanger et al, 1977). All sequencing reagents, hardware and software were supplied by PE Biosystems. Cycle sequencing reactions contained 8pl dRhodamine terminator mix, 50-500iîg purified template DNA, and 3.2pmol o f the sequencing primer in a total volume o f 20pl. Reactions were carried out on a PTC-100 thermocycler (MJ Research) with the following program:

1. 96°Cfor45s 2. 50°C for 30s 3. 60°C for 2 minutes

4. Repeat steps 1-3 24 more times 5. Cool to 4°C

After cycling the products were purified by adding 2pl 3M NaOAc and 50pl ethanol to each reaction and microcentrifuging at top speed for 25 minutes at 4°C. The supernatants were carefully removed, and the pellets washed by adding 75 pi 70% ethanol and centrifuging at top speed for 10 minutes at 4°C. Once more the supernatants were carefully removed, and the pellets air dried for 5 minutes before resuspending in 2pl Sequencing Dye, and stored at -20°C if necessary.

The samples were loaded on a sequencing gel which was run in an ABI Prism 377 DNA sequencer by Irene Ling, and the data were analysed using ABI Sequence Analysis 3.4 software.

2.8. Analysis of Proteins

2.8.1. Protein isolation from Plasmodium falciparum

NP40 Buffer: 1% v/v Nonidet P40 (BDH Laboratory Supplies), 5mM EDTA, 5mM EOT A, ImM PMSF, Ix Complete™ protease inhibitor cocktail (Roche), in PBS

Denaturing Buffer: 1% w/v SDS, 5mM EDTA, 5OmM Tris-HCL in H2O, pH 8.0

Cultures o f parasitized erythrocytes for protein extraction were spun down as normal, the medium aspirated off, and the resulting pellets of c. lOOpl packed cells stored at -80°C if necessary. After thawing the pellets were resuspended in 1ml NP40 buffer, and left on ice for 30 minutes with occasional vortexing. They were then ultracentrifuged at 55,000rpm for 45 minutes at 4°C, at the end o f which the supernatants were removed and stored in 200pl aliquots at -80°C. The NP40 pellets were resuspended in lOOpl Denaturing Buffer, vortexed, and heated to 100°C for 5 minutes. After cooling on ice, 900pl NP40 Buffer was added, mixed and left for 10 minutes on ice. These mixtures were ultracentrifuged as before, the supernatants and pellets being stored separately at - 80°C.

2.8.2. In vitro transcription and translation

Genes were transcribed in vitro using T7 RNA polymerase, which along with all other RNase free reagents was obtained from Promega. Transcription reactions contained 20- lOOng of a plasmid template including the T7 transcription start site, 60U RNasin, 2U T7 polymerase and final concentrations of 1 x T7 transcription buffer, lOmM DTT, and rNTPs at 2.5mM o f each rNTP in a 50pl reaction. After incubation at 37°C for 2 hours, Ipl of each sample was run on a 1% w/v agarose gel to verify the production o f RNA, which was stored at -80°C.

RNA was translated in vitro using rabbit reticulocyte lysate (Promega). To radiolabel the protein, 45pCi Redivue ^^S-labelled methionine (Amersham Biosciences; lOOOCi/mmol, 15mCi/ml) was included in the reaction along with 40U RNasin, 1 pi o f a ImM mixture o f all the biological amino acids except methionine, 3pi o f the RNA transcript generated as described above, and 25 pi rabbit reticulocyte lysate in a 50pl reaction. When it was not necessary to radiolabel, unlabelled methionine was used instead. After incubation at 30°C for 1 hour, reaction products were stored at -80°C.

2.8.3. SDS-Polvacrvlamide Gel Electrophoresis

Running Buffer: 25mM Tris-HCl, 192mM Glycine, 0.1% w/v SDS in H2O, pH 8.3,

stored at room temperature

Sample Buffer: 125mM Tris-HCl, 21% v/v glycerol, 4.3% w/v SDS, 0.01% w/v Bromophenol blue in H2O, pH 6.8. Stored wrapped in foil at room

temperature, and DTT added to 0.2M just before use.

Coomassie stain: 0.1% w/v Coomassie Brilliant Blue R-250 in a Methanol:H20: Acetic Acid mix of volumetric ratio 45:45:10, filtered through Whatman no. 1 paper and stored at room temperature.

Destain: 20% v/v Methanol and 7% v/v Acetic Acid in H2O.

SDS-PAGE to separate proteins was by a modification of the method of Laemmli (1970). Separating gels were made up containing final concentrations o f 12.5% w/v acrylamide/methlyene bis-acrylamide (Bio-Rad), 370mM Tris-HCl pH 8.8, and 0.1% w/v SDS, with polymerization catalysed by adding ammonium persulphate to 0.05% w/v

and TEMED to 0.1% v/v. This mixture was poured into a vertical gel casting apparatus (Hoefer Scientific Instruments) to set for 1 hour, after which a stacking gel was made up similarly but containing 5% w/v acrylamide, and 125mM Tris-HCl pH 6.8. This was poured on top of the separating gel and a comb inserted to create wells before it set. Gel assemblies were then fixed to vertical electrophoresis apparatus and the reservoirs filled with Running Buffer.

Protein samples or prestained protein molecular weight markers (Bio-Rad) were mixed with the same volume of Sample Buffer, heated to 100°C for 5 minutes, and loaded into the wells. A constant current o f 20-25mA was applied until the blue dye had reached the bottom o f the gel, after which it was removed from the apparatus. Protein could be visualised by soaking the gel in Coomassie stain for 10 minutes and then Destain for 1 hour or more.