First stage of cloning

3.2.3.1 Cloning of ECE1 into pCR®2.1 vector

To ligate ECE1 into the pCR2.1 vector (Figure 3.1), the PCR reaction to amplify the target gene was repeated to obtain a fresh product using the optimal annealing temperature (57°C). Then, the ligation constituents shown in Table 3.4 were mixed and

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incubated for 5 min at room temperature. Subsequently, 2 µl of the ligation product was transformed into chemically competent TOP10F’ E. coli strain supplied by the kit and then the transformed cells were plated on LB agar containing 50 µg/ml of ampicillin, 40 µg/ml X-gal, and 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for blue/white colony selection.

Table 3.4 Cloning conditions of ECE1 into the pCR2.1 vector

Components Volume

Fresh PCR product 1.5 µl

Salt Solution 1µl

Nuclease-free water Add to total volume 5 µl

TOPO® pCR2.1 vector 1 µl

Total volume 6 µl

3.2.3.2 Chemical (heat shock) transformation

Chemically competent TOP10F’ E. coli cells were obtained from Invitrogen (Thermo Fisher) and stored at -80ºC. The cells were thawed on ice for 15 min, mixed with up to 10 µL of a DNA (ligation product) and incubated on ice for a further 30 min. The cells were then heat shocked in a 42ºC water bath for exactly 90 s, followed by cooling on ice for 2 min. The SOC broth was added to a final volume of 1 mL and the cells incubated, shaking at 37ºC for 1 h. Aliquots of 100, 200 and 300 µL (to ensure that at least one plate has well-spaced colonies) of transformed cells were plated onto LB plates containing 50 µg/ml ampicillin, 40 µl of 40 mg/mL X-Gal and 40 mg/mL of IPTG and incubated overnight at 37°C. To perform blue-white screening for the insert, IPTG was added to plates to express LacZα as TOP10F´ does express the lac repressor (lacIq), which will repress transcription from the lac promoter.

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3.2.3.3 Transformants analysis Blue-white screening

The blue-white screening method was adapted from Sambrook and Russell (2001). Due to the light sensitivity of X-gal, plates were incubated overnight at 37°C wrapped in foil. Plates were then transferred to incubation at 4ºC for 2 h to allow proper colour development before white colonies were selected for analysis. Cloning into plasmid pCR®2.1 enabled this method of screening and IPTG was included for blue-white screening as TOP10F’ was used to express the lac repressor (Sambrook and Russell 2001).

Isolation of cloned topo pCR®2.1

Ten white colonies were picked for plasmid isolation and restriction analysis. These colonies were grown overnight in 5 mL LB broth containing 50 µg/mL of ampicillin. Topo pCR®2.1 plasmid was purified using Isolate II Plasmid Mini Kit (Bioline) according to the manufacturer’s instructions. Briefly, to harvest the bacterial cells 5 ml of 10 F’ cells were pelleted for 30s at 11,000 x g and the supernatant was discarded. Cells were lysed by resuspending in 250 µl of Buffer p1 and vortexed until no cell clumps remained. 250 µl of buffer P2 was added to the mixture and cells were mixed gently by inverting the tube 6-8 times, then 300 µl of neutralisation buffer P3 was added to the mixture and mixed by inverting the tube 6-8 times followed by centrifugation for 5 min at 11,000 x g. To bind DNA, 750 µl of clear supernatant was pipetted into an Isolate Mini Spin Column and centrifuged for 1 min at 11,000 x g and the flowthrough was discarded. The silica membrane was washed with 500 µl using buffer PW1 followed by 600 µl of buffer PW2 provided by the kit and centrifuged for 1

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min at 11,000 x g after each buffer was added and the flowthrough was discarded. To dry the silica membrane and remove residual ethanol, the column was centrifuged for 2 min at 11,000 x g then the column was transferred to a new 1.5 Eppendorf tube. The resulting DNA was eluted in 50 µl of elution buffer after incubation at room temperature for 1 min and centrifugation for 1 min at 11,000 x g.

Figure 3.1 pCR®2.1. 3.9 kbp PCR cloning plasmid, AmpR, KmR, lacZ, 3’ T-overhang PCR amplification

A conventional PCR was performed to amplify ECE1 using the isolated cloned topo pCR®2.1 plasmid as DNA template. Forward and reverse primers designed for cloning in addition to M13 forward and M13 reverse primers provided with the kit were used to confirm that ECE1 had been cloned into the topo 2.1 vector. Conventional PCR (in duplicate) was performed in a PCR thermal cycler machine (G-Storm, Australia). The total reaction mixture volume of 50 µl consisted of 10 µl of 5 x MyTaq™ reaction buffer (Bioline), 1.0 µl (1.0 µM) of each primer and 1.0 µl (100 ng/µl) of cDNA and

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0.2 µl (2 U) Taq DNA polymerase. The PCR parameters were as follows: initial denaturing at 95°C for 2 min to activate the Platinum Taq DNA polymerase followed by 40 cycles of 15 s at 95°C, 30 s at 57°C and 1 min at 72°C and a final extension of 7 min at 72°C.

Restriction enzyme digests

The restriction enzymes selected were XhoI and NcoI which recognise (C▼TCGAG) and (C▼CATGG) sequences respectively. A junk nucleotide (A) (Figure 3.2) was added to the gene sequence of primer ECE1 forward after the XhoI sequence and before

ECE1 sequence to keep a proper translation for rECE1 protein. These restriction

enzymes were chosen because they both do not have recognition sites within the target gene but do have in pCR 2.1 and pRSET-B vectors. CCG and CATG nucleotides were added before XhoI sequence and after NcoI sequence respectively to increase the stability of the primers. Restriction enzyme reaction conditions were as specified by the manufacturer. In multi-enzyme reactions, the buffer was chosen based on maximum compatibility with both enzymes. About 1 µg of DNA was digested with 2 U of the enzyme in 1× buffer in a total volume of 20 µL at 37°C for 1-20 h. Reactions were incubated for over 4 h; acetylated BSA was added at a concentration of 100 µg/mL. Enzymes were heat inactivated at 80ºC for 20 min.

Figure 3.2 Sequence of ECE1 cloned to pRSET-B the (a) nucleotide in red colour was added to sequence to keep a proper translation of rECE1 protein

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Double digestion of Topo 2.1 and gel electrophoresis of DNA

To confirm that the ECE1 insert had been cloned into topo pCR 2.1 vector, a double digestion with both XhoI and NcoI restriction enzymes was performed before running on a gel. The components shown in Table 3.5 were mixed in an Eppendorf tube and then incubated in a 37°C water bath for 4 h, afterwhich the restriction products were viewed by gel electrophoresis (Section 3.3.5).

Table 3.5 Components of double digestion solution of ECE1 cloned into topo 2.1 vector

Component Topo 2.1 vector

Plasmid DNA 3.3 µl (300.3 ng/ µl) 10x Buffer H 5 µl XhoI enzyme 1 µl NcoI enzyme 1 µl BSA 5 µl N F W 34.7 µl Total volume 50 µl