Cloning and transformation techniques

Plasmid DNA was extracted from overnight bacterial cultures using QIAprep Spin Miniprep Kit (Qiagen) according to manufacturer’s instructions. 30 µl or 50 µl of MilliQ H2O was used to elute DNA.

2.4.2 Restriction enzyme digestion

DNA was digested with restriction enzymes (New England Biolabs) in the appropriate NEB buffers at 37ºC for at least 2 hours.

2.4.3 Agarose gel electrophoresis

DNA fragments were separated on 1% agarose gels in 1x TBE buffer (UltraPure™, Invitrogen) containing 1x SYBR Safe™ DNA gel stain (Invitrogen). Samples were mixed with 6x DNA loading dye (Fermentas) at ratio of 1:5 prior to loading onto the gel. Gels were run at 140 volts for 30-60 minutes. Lambda DNA/EcoRI+HindIII marker (Fermentas) was also loaded to estimate the size of DNA fragments. Visualization of DNA was undertaken using UV light generated from a High Performance Transilluminator (UVP, LLC) and gels were photographed using DigiDoc-It™ Imaging System with Doc-It LS Analysis Software (UVP, LLC).

2.4.4 Extraction of DNA from agarose gel

DNA samples were subjected to agarose gel electrophoresis. Desired bands were excised from the gel and purified using a QIAquick Gel Extraction kit (Qiagen) following recommendations from the manufacture.

2.4.5 DNA ligation

Insert DNA and vectors at the molar ratio of 3:1 were mixed with 2 µl of 5x ligation reaction buffer and 1 µl (1 U) of T4 DNA ligase (Invitrogen). MilliQ water was added to a final volume of 10 µl. Reactions were incubated at 16ºC overnight.

2.4.6 pCR8/GW/TOPO® cloning

To facilitate cloning of PCR products into destination vectors, PCR products were

firstly cloned into pCR8/GW/TOPO® _{vectors using the pCR8/GW/TOPO}® _Cloning

Kit from Invitrogen (Auckland). The desired insert DNA was then excised from

pCR8/GW/TOPO® _{vector by restriction digestion and subcloned into the desired}

destination vectors.

2.4.7 Preparation of electrocompetent E. coli cells

Escherichia coli strains stored in glycerol-saline at -80ºC were streaked out on a LB

plate and grown at 37ºC overnight. A single colony from the plate was inoculated into 5 ml of LB and grown at 37ºC with shaking overnight. 5 ml of overnight culture was

added into 200 ml of fresh LB broth and grown at 37ºC with shaking to an OD600 of

0.6-0.7. Cells were first chilled on ice for 20 minutes and then harvested at 4,000 g for 20 minutes at 4ºC. The supernatant was discarded and the cell pellet was resuspended in 100 ml of ice-cold 10% glycerol. Cells were centrifuged at 4,000 g for 20 minutes at 4ºC and the supernatant was discarded. The cell pellet was further washed with 50 ml of ice-cold 10% glycerol twice. Cells were centrifuged at 4,000 g for 20 minutes at 4ºC and the supernatant was discarded. The cell pellet was resuspended in 2 ml of ice- cold 10% glycerol. 50 µl of aliquots were stored at -80ºC.

2.4.8 Preparation of chemically competent E. coli cells

E. coli strains stored in glycerol-saline at -80ºC were streaked out on a LB plate and

grown at 37ºC overnight. A single colony from the plate was inoculated into 5 ml of SOB and grown at 37ºC with shaking overnight. 5 ml of overnight culture was added

to a 1L flask containing 200 ml of SOB and grown at 37ºC with shaking to an OD600

of 0.6-0.7. The cell culture was collected in four 50 ml sterile tubes and chilled on ice for 15 minutes. Cells were harvested at 4,000 g for 20 minutes at 4ºC. The cell pellet

was resuspended in 16 ml of ice-cold transformation buffer I and then incubated on ice for 15 minutes. Cells were centrifuged at 4,000 g for 20 minutes at 4ºC and the supernatant was discarded. The cell pellet was resuspended in 4 ml of ice-cold transformation buffer II. 50 µl of aliquots was stored at -80ºC.

Transformation buffer I (250 ml) RbCl 3.0 g MnCl24H2O 2.5 g Potassium acetate (1M, pH 7.5) 7.5 ml CaCl22H2O 0.4 g Glycerol 37.5 ml

Adjusted pH to 5.8 with 0.2 M acetic acid and sterilized by filtration through a 0.22 m filter. Transformation buffer II (250 ml) MOPS (0.5 M, pH 6.8) 5.0 ml RbCl2 0.3 g CaCl22H2O 2.75 g Glycerol 37.5 ml Sterilized by filtration through a 0.22 m filter.

2.4.9 Preparation of electrocompetent P. fluorescens cells

P. fluorescens strains stored in glycerol-saline at -80ºC were inoculated in 5 ml of LB

broth and grown with shaking at 28ºC overnight. 1.5 ml of cell culture was centrifuged at 13,000 rpm for 1 minute. The cell pellet was washed with 1 ml of sterile, ice-cold glycerol/HEPES solution (10% glycerol and 1mM HEPES) three

times. Cells were resuspended in 50 µl of glycerol/HEPES solution and ready for

electroporation.

2.4.10 Transformation of E. coli or P. fluorescens by electroporation

5 µl of ligation products or plasmid DNA (100~200 ng) was added to 50 µl of

electrocompetent cells that had been thawed on ice. Cells were placed between 0.1 cm electrodes of a pre-chilled gapped Gene Pulser® _{cuvette (Bio-Rad) and electroporated}

using Electroporator 2510 (Eppendorf) at 1.8 kV. 250 µl of SOC medium was

immediately added and the cells were transferred to a 1.5 ml sterile eppendorf tube.

fluorescens with shaking for 1 hour. The culture was spread onto LB plates containing

appropriate antibiotic and incubated overnight (E. coli) or 2 days (P. fluorescens).

After incubation, transformants were inoculated into 5 ml of LB containing the appropriate antibiotic and grown 37ºC (E. coli)or 28ºC(P. fluorescens) with shaking

overnight.

S.O.C medium

2% Bacto Tryptone 10 mM MgCl2

0.5% Yeast Extract 10 mM MgSO4

10 mM NaCl 2.5 mM KCl

Sterilized by autoclaving and added sterile glucose to 20 mM.

2.4.11 Transformation of E. coli by heat shock method

Chemically competent E. coli cells were thawed on ice and 2 µl of plasmid DNA or

ligation products was added. Cells were incubated on ice for 30 minutes. DNA was transformed into cells by heating at 42ºC for 30 seconds. 250 µl of SOC medium was immediately added and cells were incubated at 37ºC with shaking for 1 hour. The culture was spread onto LB plates containing appropriate antibiotic and incubated at 37ºC overnight.