La marcha del negocio: factores en contra y a favor

For preparation of media as well as reagents, water was deionised and sterilised. Heat-sensitive solutions were filtered using Millex 0.2 μm syringe filters (Millipore, PES membrane). Chemicals were purchased from various sources, mainly Sigma-Aldrich and were at least of analytical grade.

2.1.1 Solutions, media and other reagents

Standard molecular stock solutions were prepared following standard protocols (Sambrook et al., 1989).

- 50 TAE (2 M Tris-base, 0.05 M EDTA pH 8.0, adjusted to pH 7.8 with glacial acetic acid - 10 and 40 %(w/v) glycerol stock filtered sterilised.

- DNA gel loading buffer type III (30 %(w/v) glycerol, 0.25 %(w/v) bromophenol blue and 0.25 % (w/v) of xylene cyanol, stored at 20oC).

- 100 mg/ml ampicillin stock filtered and stored at 20 oC and added to media to a working concentration of 1 μg/ml.

- 0.1 M IPTG, working concentration of 0.1 mM (E. coli blue/white screening), 0.4 mM or 1 mM (for bacterial protein expression).

- 50 mg/ml XGal, dissolved in DMSO, filtered, wrapped in foil and stored at 20 oC, added to final concentration of 40 μg/ml.

- 1 %(w/v) agarose gel for routine DNA analysis was sourced from Biogene and for DNA gel extraction low melting point Seakem LE agarose from Cambrex was used.

- 2 SDS loading dye (0.5 M Tris-HCl pH 6.8, 4.4 %(w/v) SDS, 20 %(v/v) glycerol, 2 %(v/v) 2- mercaptoethanol, and 20 %(w/v) bromophenol blue).

- 1 M potassium phosphate adjusted to pH 6 (132 ml of 1M K2HPO4 + 868 ml 1M KH2PO4) - 20 %(w/v) biotin (made fresh and filtered sterilised).

- 20 %(w/v) dextrose, filter-sterilised.

- 1 %(w/v) yeast extract -2 %(w/v) peptone for Pichia pastoris culture was disolved in water and autoclaved. Yeast extract and peptone were purchased from Oxoid.

- Luria Bertani (LB) broth and 1 % (w/v) agar for bacterial culture were purchased from the university store.

- dNTPs were purchased from Promega and aliquoted in 10 μl volume, stored at 20 oC and used only once when thawed. Molecular weight markers used were 1 kb marker and 100 bp step ladder from Promega. Ethidium bromide at 10 mg/ml for DNA visualisation was also supplied by Promega. - DMEM, fetal calf serum (FCS), trypsin and PBS were purchased form the university stores. - Optimem was sourced from Gibco.

- Cell transfection reagent PEI (40 000 Da, from polysciences) was diluted in HEPES pH 7 at 1 mg/ml at 20oC, and filter-sterilised in sterile environment and stored at 20oC. Other transfection reagents were lipofectamine 2000 (Invitrogen) and fugene 6 (Promega).

- CelLytic for cell lysis was purchasd from Sigma.

- Restore western blot stripping buffer was purchased from Thermo Scientific. - Protein marker precision plus protein standard was purchased from Biorad. - PBST solution for western blot 0.5 % (v/v) tween 20 dissolved in PBS.

2.1.2 Bacterial strains and cell lines

Escherichia coli strain used for all routine bacterial transformation was JM109, genotype: endA1,

recA1, gyrA96, thi, hsdR17 (rk–, mk+), relA1, supE44, Δ( lac-proAB), [F´ traD36, proAB, laqIqZΔM15].

DE3 strains for protein expression (from this laboratory stock or kind gift from Prof. Naismith laboratory)

BLl21: F– ompT gal dcm lon hsdSB (rB -

mB

-_{) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5].} BL21*: F-ompT hsdSB (rB

-

, mB -

) gal dcm rne131 (DE3)

C43: F-ompThsdSB (rB- mB-) gal dcm (DE3)

Origami: Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII phoR araD139 ahpC galE galK rpsLF′[lac+ lacIq pro] (DE3)gor522::Tn10 trxB (KanR, StrR, TetR)

Rosetta: F- ompT hsdSB(RB -

mB

-_{) gal dcm λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])}

pLysSRARE (CamR)

TUNR: F–ompT hsdSB (rB– mB–) gal dcm lacY1(DE3) BLR: F-ompT hsdSB(rB

-

mB -

Mammalian cells: fibroblast cells baby hamster kidney cells BHK21, human tumour cells HeLa (kindly provided by Prof. Elliott’s group) and human embryonic kidney cells 293T (a kind gift from Prof Randall) and bovine lung cell (EBL) from this laboratory stock were revived and maintained through continuous passing.

2.1.3 Enzymes, antibodies and kits

Routine PCR were performed with GoTaq DNA polymerase (Promega), amplification of fragments for cloning purposes were performed with platinum DNA polymerase (Invitrogen) and PCR for mutagenesis purposes were carried out using the KOD Hot Start polymerase mix (Novagen).

Enzymes for DNA restriction digests were purchased from Fermentas and New England Biolabs. Primers were ordered individually from IDTDNA and resuspended in sterile water to a concentration of 100 pM for PCR for cloning purpose, 100 ng/µl for mutagenesis purpose and 32 pM for sequencing reactions. The relevant primers for each study are mentioned in the related chapters. Sequencings were outsourced from Dundee sequencing services.

For routine DNA extraction, QIAprep Spin miniprep Kit and HiSpeed Qiagen maxi prep.

For purification of PCR fragments from gel the Wizard SV gel and PCR clean up (Promega) was selected. For thymine-adenine cloning, the pGEM-T system was selected (Promega). All other ligations were carried out using the T4 DNA ligase kit (Promega).

Cell-free systems for protein expression: T7 quick coupled transcription- translation system was used for routine analysis of novel 2A and mutants (Promega). The T7 coupled version of this system was used to incorporate analogs of proline. T7 insect cell-free system (Promega) was used to test for the viability of constructs for bacterial expression.

The Pichia pastoris PichiaPink expression system (Invitrogen) was selected for expression of 2A.

SDS-PAGE protein analyses were run with the Nu-PAGE 10 % Bis-Tris 1.0 mm, 12 wells Gel and MES buffers from Invitrogen or the RunBlue pre-cast 4-20 % , 12 wells and Tris-tricine-SDS buffer from Expedeon. ECL solutions kit for chemiluminescence was purchased from Amersham

Biosciences. BCA (Bicinchoninic acid) protein assay kit for microscale determination of protein concentration was purchased from Novagen.

Table 2.1: List of antibodies

Antibody Target protein source

Anti- His His tagged expressed proteins Sigma

Anti V5 V5 tagged expressed proteins Kindly provided by Prof. Randall

Anti eEF2 Elongation factor 2 Cell signalling

Anti 2A 2A This laboratory stock

Anti βtubulin β tubulin Roche Diagnostic

Secondary antibodies: Anti mouse or rabbit HRP

Mouse or rabbit primary antibody Dako

Table 2.2: List of equipment

Function Equipment Supplier

PCR GeneAmpPCR system 9700 Applied biosystems

DNA gel electrophoresis Horizon 11-14 Life Technologies

Allegra 21R centrifuge Beckman Coulter Cell/ bacterial cultures Sterile 96 and 6 -well plates Greiner Bio-one

Sterile flasks Sterile Petri dishes

Incubator: Hera cell 150 Thermo Electron Corporation

Microscopy Evos fl AMG micro

Delta vision Applied Precision

Electroporation Gene Pulser Xcell Biorad

Protein transfer to membrane iBlot transfer Invitrogen

microplate reader Infinite M200 Pro Tecan

Protein electrophoresis and gel analysis

XCell SureLock™ Novex Mini- Cell

Invitrogen

Model 583 gel drier Biorad

Kodak X-OMAT 1000 processor