Second cloning step

3.2.4.1 Preparing electrocompetent DH5α E. coli

Competent E. coli DH5α were prepared for electrotransformation according to the Gene Pulser apparatus user’s manual (BioRad) with minor alterations. All steps were performed aseptically. An overnight E. coli DH5α culture was used to inoculate 200 ml of LB broth at a 1/100 volume. The cells were incubated with shaking at 37°C until an OD 600 of about 0.6 (early to mid-log phase) was obtained. The culture was then placed on ice for 30 min before the bacteria were pelleted by centrifugation at 5,000 x g for 15 min at 4ºC. The supernatant was removed, and the pellet resuspended in 200 mL of ice-cold sterile Milli-Q water. The resuspension was placed on a flat shaker at slow speed until the pellet disappeared, then centrifuged at 3500 x g for 15 min at 4°C. This

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centrifugation process was repeated twice, with the cells firstly resuspended in 100 mL of ice-cold sterile Milli-Q water followed by resuspension in 10 mL of ice-cold 10% glycerol. The cells were then finally pelleted by centrifugation at 4,000 x g for 15 min and resuspended in 400 µL of ice-cold 10% glycerol and immediately aliquoted into 40 µL amounts in tubes and stored at -80ºC.

3.2.4.2 Assessing the efficacy of the electrocompetent cells

To measure the preparation effectiveness of electrocompetent cells, 0.5 µl of PUC19 plasmid (0.1 ng/µl), which has been previously shown to be efficiently transformed into electrocompetent cells, was mixed with 50 µl of the prepared electrocompetent cells and then transferred to a cuvette tube. The transformation was done by placing the cuvette in a chamber attached to the BIORAD electroporation system set at 2.5 volts, 25 µF, 200 ohms and then giving the cells an electrical shock. Following the addition of 1 ml of LB broth to the cells, they were then transferred to a microcentrifuge tube and incubated at 37°C on a shaker at 225 rpm for one hour. The culture (100 µl) was plated on LB agar containing 50 µg/ml of ampicillin and incubated overnight at 37°C and afterwards the colony forming units (CFU) were counted and used in the transformation efficiency calculation. The calculation was done as follows:

1) Total DNA transformed (ng) = plasmid concentration x the added volume

= 0.1 ng/µl x 0.5 µl =0.0001 ng.

2) Transformation efficacy (CFU/µg) = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐶𝐹𝑈

𝑇𝑜𝑡𝑎𝑙 𝐷𝑁𝐴 𝑡𝑟𝑎𝑛𝑠𝑓𝑜𝑟𝑚𝑒𝑑 (𝑛𝑔) x 10 3 _x 𝑇𝑜𝑡𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 𝑡𝑟𝑎𝑛𝑠𝑓𝑜𝑟𝑚𝑒𝑑 𝑇ℎ𝑒 𝑝𝑙𝑎𝑡𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 = 540 𝐶𝐹𝑈 0.0001 𝑛𝑔 x 10 3 _x (250+50)µ𝑙 100 µ𝑙 = 3.24 x 1010 _CFU/µg

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From this result of 3 x 1010 _{it was concluded that the prepared E. coli DH5α cells were} suitable for further electroporation as the number of viable cells in a preparation for a transformation reaction may range from 2×108 to 1011 and most common methods of E.

coli preparation yield around 1010 viable cells per reaction.

3.2.4.3 Isolation of pRSET-B vector

The vector pRSET-B was purified from DH5α E. coli using the Plasmid Mini Kit (Bioline) using the high copy plasmid DNA isolation protocol according to the manufacturer’s instructions. Briefly, the E. coli DH5α that included the pRSET-B plasmid was grown overnight in 5 mL LB broth containing 50 µg/mL of ampicillin. Cells were pelleted for 30 s at 11,000 x g and the supernatant was discarded. Cells were then lysed through resuspension in 250 µl of buffer P1 and vortexed until no cell clumps remained. 250 µl of buffer P2 was then added to the mixture, and the cells were mixed gently by inverting the tube 6-8 times. 300 µl of neutralisation buffer P3 was then added to the mixture and mixed by inverting the tube 6-8 times, then centrifuged 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 then washed with 500 µl of buffer PW1 followed by 600 µl of buffer PW2 and centrifuged for 1 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 and the column was then transferred to a new 1.5 Eppendorf tube. The resulting DNA was eluted into 50 µl of elution buffer after incubation at room temperature for 1 min and then centrifuged for 1 min at 11,000 x g. The elution step was repeated to increase the DNA yield, and the DNA was stored at -20°C.

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Figure 3.3 pRSET-B 2.9 kbp PCR cloning plasmid. A) shows the sequence

sites of the start codon and the polyhistidine and restriction enzyme sites used in this study. B) shows the complete design of pRSET-B.

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3.2.4.4 Digestion of pRSET-B vector DNA and ECE1 cloned into topo pCR 2.1 vector DNA by restriction enzymes

To clone ECE1 digested from the Topo pCR 2.1 vector into the pRSET-B vector, both the insert and the dedicated cloning site of the pRSET-B vector were digested with both

XhoI and NcoI restriction enzymes before running on a gel. The components shown in

Table 3.6 were mixed in a microfuge and then incubated in a 37°C water bath for 4 h. The restriction products were then viewed after separation in 1.5% gel electrophoresis.

Table 3.6 Conditions for double digestion of ECE1 and pRSET-A vector

Component ECE1 cloned into topo 2.1 pRSETB

Plasmid DNA 3.3 µl (300.3 ng/µl) 2 µl (500.3 ng/µl) 10x Buffer H 5 µl 5 µl XhoI enzyme 1 µl 1 µl NcoI enzyme 1 µl 1 µl BSA 5 µl 5 µl N F W 34.7 µl 36 µl Total volume 50 µl 50 µl

3.2.4.5 Gel extraction of DNA

Both the insert (ECE1 cloned into topo pCR 2.1 vector) and the digested pRSET-B vector were isolated from the gel to clone ECE1 into the pRSET-B vector. Consequently, restriction reactions were prepared for both the clone pCR 2.1 and the pRSET-B in 50 µl total volume separately. The digested products were loaded (20 µl) into 1.5% agarose gel (1.5 g of agarose dissolved into 100 ml TAE buffer) and electrophoresed for all the reactions. The DNA products were visualised with a UV illuminator and photographed using a Biorad Geldoc imaging system running Quantity One software. The bands corresponding to the ECE1 insert and the pRSET-B backbone were excised from the gel using a sharp scalpel and weighed in 1.5 ml Eppendorf tubes.

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The bands were purified using Isolate II PCR and Gel Kit (Bioline) according to the manufacturer’s instructions. Briefly, the excised gel slice was dissolved in 200 µl binding buffer and incubated at 50°C for 10 min. The sample was then vortexed briefly every 3 min until the gel slice was completely dissolved. To bind DNA, the dissolved mixture was loaded to a column provided in the kit and centrifuged for 30 s at 11,000 x

g and the flow through was discarding. The silica membrane was then washed with 700

µl CW buffer and centrifuged for 30 s at 11,000 x g. The centrifugation was repeated to dry the silica membrane and remove residual ethanol. The column was then transferred to a new 1.5 ml Eppendorf tube and the DNA eluted in 30 µl of elution buffer. To calculate the concentration of the purified DNA, 2 µl of each sample was tested using a NanoDrop spectrophotometer. The resulting DNA was stored at -20°C.

3.2.4.6 Ligation of ECE1 into pRSET-B vector

The ligation reaction was performed using 50 ng of pRSET-B vector and 40.34 ng of the purified ECE1 insert, by applying a 1:3 ratio of insert to vector and by using the calculations below. The ligation reactions were prepared according to the components listed in Table 3.7, and the tubes were incubated overnight at 14°C.

ng of DNA insert = ng vector x size of insert (bp) _{size of the vector (bp)} X molar ratio

ng of DNA insert = 50 ng x 781 (bp) _{2900 (bp)} X 3

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Table 3.7 pRSET-B vector and ECE1 ligation conditions

Components Volume

Gel extracted pRSET-B backbone 1.46 µl (50 ng)

Gel extracted ECE1 insert 2 µl (40.4 ng)

20 mM ATP 1 µl

T4 DNA Ligase 1 µl

10x T4 DNA Ligase Reaction Buffer 2 µl

N F W 12.54 µl

Total volume 20 µl

3.2.4.7 DH5α electroporation protocol

To confirm that ECE1 had been ligated into the pRSET-B vector, 2 µl of the ligation product was transformed into electrocompetent E. coli DH5-α using the method applied for the positive control plasmid (Section 3.2.4.2), and then the transformed cells were plated on LB agar containing 100µg/ml of ampicillin and incubated at 37°C.

3.2.4.8 Analysis of transformants

Conventional PCR and double digestion were performed as described in section 3.2.3.3, using the DNA of isolated pRSET-B vector from overnight E. coli DH5-α competent cells in order to confirm that ECE1 had been cloned into the pRSET-B vector.

Sequencing and alignment of rECE1

To confirm that ECE1 had been cloned into pRSET-B vector, the DNA of the pRSET- B vector was isolated and purified from DH5α E. coli cells using the Plasmid Mini Kit (Bioline) protocol described in section 3.2.3.3 and sequenced using the forward and reverse primers used for cloning. The purified plasmid was sequenced at the Australian Genome Research Facility LTD (AGRF, La Trobe University, Bundoora, Victoria, Australia), which recommends sending 12 µl containing 600 to 1500 ng of double-

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stranded plasmid and 0.8 pmol/1 µl of primers. The purified DNA was measured using a Nanodrop spectrophotometer which determined a final concentration of 626.6 ng/µl of DNA. The calculation below was performed to determine the volume of 10 µM

ECE1 primers to be added to the sample. This determined that 1 µl of the 10 µm ECE1

forward primer was to be added to 2 µl of the purified plasmid and made up to a total of 12 µl of nuclease free water. The same step was performed with the ECE1 reverse primer. The two 12 µl samples were then sent to AGRF for sequencing.

𝐶1 × 𝑉1 = 𝐶2 × 𝑉2

10 𝑝𝑚𝑜𝑙/µ𝑙 × 𝑉1 = 0.8 𝑝𝑚𝑜𝑙/µ𝑙 × 12 µ 𝑉1 = 1 µ𝑙