Vectors for Cloning, Complementation and Protein Expression

Chapter 2. Materials and Methods

2.1. Materials

2.1.4. Vectors for Cloning, Complementation and Protein Expression

Vectors used for cloning, mutagenesis, expression of recombinant protein and complementation are described in Table 2.5, Table 2.6 and Table 2.7.

Table 2.1 - Experimental Strains used for E. coli and Enterococcus spp. experiments. Relevant charactersitics and sources of each strain are included.

Strain Relevant Characteristics Reference

Experimental E. coli

Top10 Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15 e14- ϕ80dlacZΔM15 recA1 relA1

endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC)

Grant et al., (1990) NEB5 F’ proA+_B+_lacIq_{∆(lacZ)M15 zzf::Tn10 (Tet}R_{) / fhuA2∆(argF-lacZ)U169 phoA glnV44}

Φ80Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17

NEB

BL21(DE3) F- ompT hsdSB(rB-_{, mB}-_{) gal dcm(DE3)} _{Stratagene; Studier and Moffatt (1986)}

Experimental E. faecalis

JH2-2 FusR_{, Rif}R_{, JH2 spontaneous mutant} _{Jacob and Hobbs (1974)}

JH2-2 Tn1549 VanR_{, vanB (Tn1549)} _{Launay et al., 2006}

OG1RF RifR_{, Fus}R_{, OG1 isolate from human oral cavity} _{Dunny et al., (1978)}

OG1RF::∆ireK RifR_{, Fus}R_{, ireK,} _{Kristich et al., 2007}

OG1RF Tn1549 RifR_{, Fus}R_{, Van}R_{, vanB (Tn1549)} _{This Study}

OG1RF ∆ireK::Tn1549 RifR_{, Fus}R_{, Van}R_{, vanB (Tn1549), ∆ireK} _{Goss, 2013}

Clinical and Control Strains

E. faecalis ATCC 51299 VanR_{, vanB2 (Tn1542) ant(6)-I aac(6') aph(2"")} _ATCC

E. faecium ATCC 700221 VanR_{, vanA (Tn1549)} _ATCC

E. faecium BM4525 VanR_{, vanB (Tn1549)} _{Depardieu et al.,2003}

E. faecalis ATCC 29212 CSLI Control Strain ATCC

E. coli ATCC 25922 CSLI Control Strain ATCC

Table 2.2: Media used for culturing bacteria in this study.

Name Composition Reference

Standard Media

LB Broth 10 g Tryptone, 5 g Sodium Chloride, 5 g Yeast Extract, prepared to 1 L in water and autoclaved N/A SOC 20 g Tryptone, 0.5 g Sodium Chloride, 5 g Yeast Extract, 0.2 g Potassium Chloride, 3.6 g Glucose and 1 g

Magnesium Chloride and made up to 1 L with double-distilled water and autoclaved

Hanahan (1983)

BHI Manufacturer’s Instructions (Oxoid CM1135) N/A

Media for Expression of Isotopically labelled proteins

5x M9 Salts 32 g Sodium Phosphate Dibasic, 6 g Potassium Phosphate Monobasic, 2.5 g Sodium chloride made up to 500 mL with double distilled water and autoclave

Adapted from (Cai et al., 1998)

Minimal Media 200 mL 5x M9 Salts, 2 mL 1 M Magnesium Sulphate, 100 µL Calcium Chloride Adapted from (Cai et al., 1998)

Media for antibiotic susceptibility

Muller Hinton II Broth See Manufacturer’s Instructions (BD L007475) N/A

Isosensitest Broth See Manufacturer’s Instructions (Oxoid CM0471) N/A

Media for E. faecalis competent cells

5x M17 broth 5.0 g Pancreatic Digest of Casein, 5.0 g Soy Peptone, 5.0 g Beef Extract, 2.5 g Yeast Extract, 0.5 g Ascorbic Acid, 0.25 g Magnesium Sulphate and 10.0 g Disodium-glycerophosphate and was prepared to 200 mL in water and autoclaved

Terzaghi and Sandine (1975)

M17Glu 1x M17 Broth and sterile 0.5% Glucose Shepard and Gilmore (1995)

SGM17 1x M17 Broth, sterile 0.5 M Sucrose, sterile 2% Glycine and sterile 10% Glycerol pH 7.0 Shepard and Gilmore (1995)

SM17MC 1x M17 Broth, sterile 0.5 M Sucrose, sterile 10 mM Magnesium Chloride and sterile 10 mM Calcium Chloride pH 7.0

Shepard and Gilmore (1995)

Table 2.3: Buffers used for protein purification, storage and assays.

Name Composition

Buffers used for competent cells

TFB1 30 mM potassium acetate, 10 mM CaCl2, 50 mM MnCl2, 100 mM RbCl and 15% glycerol

TFB2 10 mM MOPS, pH 6.5, 75 mM CaCl2, 10 mM RbCl and 15% glycerol

General Buffers

TAE 40 mM Tris acetate, 1 mM EDTA

Purification of recombinant IreK domain proteins

Buffer A 20 mM Sodium Phosphate, 500 mM Sodium Chloride, 10 mM Imidazole and 10% Glycerol, pH 7.4

Buffer B 20 mM Sodium Phosphate, 500 mM Sodium Chloride, 200 mM Imidazole and 10% Glycerol, pH 7.4

Gel Filtration Buffer 20 mM Sodium Phosphate, pH 7.4

Purification of Recombinant TEV protease

Buffer A 100 mM Tris, 20 mM Imidazole, 500 mM Sodium Chloride, 10 mM Magnesium Chloride pH 8.0

Buffer B 100 mM Tris, 500 mM Imidazole, 500 mM Sodium Chloride, 10 mM Magnesium Chloride pH 8.0

Dialysis Buffer 10 mM Tris, pH 8.0, 300 mM NaCl, 1 mM TCEP, 10 % glycerol

Purification of Recombinant Atl Amidase

Buffer A 10 mM Tris pH 8.0, 20 mM Imidazole, 500 mM Sodium Chloride

Buffer B 10 mM Tris pH 8.0, 500 mM Imidazole, 500 mM Sodium Chloride

Gel Filtration Buffer 10 mM TrisHCl pH 7.0 and 1 mM Calcium Chloride

Buffers used in Biophysical Experiments

NMR Buffer 20 mM Sodium Acetate pH 4.5

CD and AUC Buffer 10 mM Sodium Phosphate pH 7.4

SEC-MALS and SEC SAXS Buffer 10 mM HEPES pH 7.4 and 150 mM Sodium Chloride

SPR Running Buffer 10 mM HEPES pH 7.4, 150 mM Sodium Chloride 0.005% (v/v) Tween-20

SPR Regeneration Buffer 6M Guanidine Hydrochloride

Table 2.4: Plasmids used for recombinant protein expression in E. coli.

Plasmid Features Content Cloning (Type and Features) Source and Primers Reference Vector

pAT816 T7-6x His-VanSB KD (AmpR) E. faecalis Tn1549 VanSB KD N/A N/A Depardieu et

al., 2003

pET28/16

pLG226 T7-6x His-IreK KD (AmpR) E. faecalis IreK Soluble KD N/A N/A Goss, 2013 pET Duet

pLG234 T7-6x His-IreK KD (AmpR) E. faecium IreK Soluble KD N/A N/A Goss, 2013 pET Duet

pLG370 T7-IreP-6x His (AmpR) E. faecalis IreP FL N/A N/A Goss, 2013 pET Duet

pCWT001 T7-Strep-HRV3C-IreK FL-

Thrombin-10His (AmpR)

E. faecalis IreK FL Synthetic, codon optimised for E. coli expression, NcoI and XhoI

GenScript This Study pET52b

pCWT002 trc-TEV-6x His-IreK PASTA 1,2,3,4 and 5 ED (AmpR)

E. faecalis IreK PASTA 1, 2, 3, 4 and 5 ED