Bacterial Strains and Plasmids 55

4.2.1. E.coli strains

DH5αF‟ F‟/endA1 hsdR17 (rk-, mk+) glnV44 thi-1

recA1 gyrA (NA1r) relA1Δ (lacIZYA-argF)

U169 deoR (φ80dlacΔ(lacZ)M15)

BL21(DE3) Gold (Stratagene)

B strain, F-dcm+The ompT hsdS (rB-mB-)

gal l (DE3) endA TetR

4.2.2. Plasmids

GroEL and all chaperonin mutants were constructed inpCH vector backbone (Chang et al., 2005) inserted via NdeI and NheI sites. Synthetic oligonucleotides encoding wild-type or

mutant C-terminal extensions of GroEL were introduced into the pCH-ELΔC or SR-ELΔC plasmids between the NheI and HindIII sites. EL-KKK2, SR-EL KKK2, ELGGA, SR-EL GGA were constructed in a pCH vector backbone by Yun Chi Tang (Tang et al., 2006). MBP cysteine mutants were generated by site-directed-mutagenesis in pCH vector on thebackground of the DM-MBP. SM-MBP (Y283D), DM-MBP (V8G, Y283D), WT-MBP were generated by Yun Chi Tang (Tang et al., 2006) and DM-MBP A52C/P298C, DM-MBP K175C/P298C were made by Shruti Sharma (Sharma et al., 2008). GroES was constructed in a pET11a vector inserted via NdeI and BamH1 sites (Brinker et al., 2001).

Plasmid Promotor/Origin Selection Marker

GroEL and GroES

pCH-EL T7/ColE1 Ampicillin

pCH-SR T7/ColE1 Ampicillin

pET11a-ES T7/ColE1 Ampicillin

pCH-EL GGD T7/ColE1 Ampicillin

pCH-EL GGG T7/ColE1 Ampicillin

pCH-EL GGI T7/ColE1 Ampicillin

pCH-EL GGK T7/ColE1 Ampicillin

pCH-EL GGQ T7/ColE1 Ampicillin

pCH-EL GGS T7/ColE1 Ampicillin

pCH-EL GGY T7/ColE1 Ampicillin

pCH-SR GGD T7/ColE1 Ampicillin

pCH-SR GGG T7/ColE1 Ampicillin

pCH-SR GGI T7/ColE1 Ampicillin

pCH-SR GGK T7/ColE1 Ampicillin

pCH-SR GGY T7/ColE1 Ampicillin

DM-MBP mutants

pCH-DM-MBP N18C/D296C T7/ColE1 Ampicillin pCH-DM-MBP D184C/K362C T7/ColE1 Ampicillin pCH-DM-MBP N18C/D296C/D184C/K362C T7/ColE1 Ampicillin pCH-DM-MBP A21C/V37C T7/ColE1 Ampicillin pCH-DM-MBP E22C/A292C T7/ColE1 Ampicillin pCH-DM-MBP D65C/M330C T7/ColE1 Ampicillin pCH-DM-MBP A109C/L299C T7/ColE1 Ampicillin pCH-DM-MBP D164C/K251C T7/ColE1 Ampicillin pCH-DM-MBP D184C/Q365C T7/ColE1 Ampicillin pCH-DM-MBP A206C/A351C T7/ColE1 Ampicillin pCH-DM-MBP N218C/S238C T7/ColE1 Ampicillin pCH-DM-MBP V244C/R316C T7/ColE1 Ampicillin pCH-DM-MBP P254C/K326C T7/ColE1 Ampicillin pCH-DM-MBP K189C/D358C T7/ColE1 Ampicillin pCH-DM-MBP K14C/K6C T7/ColE1 Ampicillin pCH-DM-MBP D15C/K362C T7/ColE1 Ampicillin

The following positions were not active upon mutation and could not be purified via affinity purification: pCH-DM-MBP A21C/V37C, pCH-DM-MBP E22C/A292C, pCH-DM- MBP D65C/M330C, pCH-DM-MBP A109C/L299C, pCH-DM-MBP D164C/K251C, pCH- DM-MBP D184C/Q365C, pCH-DM-MBP A206C/A351C, pCH-DM-MBP N218C/S238C, pCH-DM-MBPV244C/R316C,pCH-DM-MBPP254C/K326C, pCH-DM-MBP K189C/D358C, pCH-DM-MBP K14C/K6C, , pCH-DM-MBP D15C/K362C.

4.3. Molecular Cloning Methods

4.3.1. Preparation and Transformation of E. coli Competent Cells

For preparation of chemically-competent E. coli cells, a single colony was used to inoculate 500 ml LB medium (including antibiotic, if applicable) and grown to an optical density (OD600) of 0.25-0.5 at 37°C. The cells were then chilled on ice for 15 min and

harvested at 5000 x g for 10 min at 4°C. The cell pellet was washed with 80 ml ice-cold Ca/glycerol buffer (10 mM PIPES, 60 mM CaCl2, 15 % glycerol; pH 7.0, adjusted with NaOH, and filter-sterilized) once and incubated with additional 80 ml Ca/glycerol buffer on ice for 30 min. Finally, the cells were pelleted and resuspended in 6 ml of Ca/glycerol buffer. 100 μl aliquots were frozen in liquid nitrogen and stored at –80°C.

For transformation, ~50 μl competent cells were mixed with 0.05 - 0.2 μg plasmid

DNA or 1-5 μl ligation reaction and incubated on ice for 15 min. The cells were heat-shocked at 42°C for 45-90 s and subsequently placed on ice for 2 min. 1 ml of LB medium was added and the cells were shaken at 37°C for 1 h. The cell suspension was then plated on selective plates and incubated at 37°C, until colonies had grown (typically 10-16 h).

Alternatively, electroporation was applied to improve the transformation efficiency. Electrocompetent cells were prepared as follows: 500 ml bacterial culture was grown to an optical density (OD600) of 0.8 in LB medium at 37°C. The cells were washed carefully with

250 ml ice-cold sterilized water for two times and finally the cells were pelleted and resuspended in 2 ml of ice-cold 10% glycerol. 40 μl aliquots were frozen in liquid nitrogen and stored at –80°C. For electroporation transformation, competent cells (40 μl) were mixed with 1-2 μl plasmid DNA (or ligation product) and transferred into a 0.2 cm Gene Pulser cuvette. The electroporation was done at 2.5 kV, 25 μFD and 200Ω settings with a Gene Puser