Transformation

Competent cells (E. coli DH5α, Top10, SoluBL21, BL21 (DE3) plysS) and plasmids were thawed on ice for 10 minutes. Plasmids (1 µL, 50~300 ng/µL) and 70 µL competent cells were added to a sterile 1.5 mL Eppendorf tube, and the mixture was placed on ice for 30 minutes. Then the tube was heated at 42o_{C for 45 seconds, and} immediately placed on ice for 2 minutes. LB broth (1mL) culture medium was then

added and the bacterial culture was incubated in a shaker (37o_{C, 250 rpm). After 60} minutes incubation, the cells were centrifuged and the supernatant was discarded. The cell pellet was resuspended in 100 µL LB culture. Twenty µL cell culture was spread onto a solid LB agar (MERCK) medium plate containing an appropriate antibiotic (50 µg/mL ampicillin sodium salt or 100 µg/mL kanamycin sulfate), and bacteria were incubated in a 37o_{C overnight. Finally, the plate was usually stored at} 4°C until required.

2.1.3 Plasmid amplification in DH5α cells and extraction from the cells

The desired plasmid was transformed into DH5α cells, as described in Section 2.1.2. A single colony containing the plasmid was picked from the agar plate and inoculated into 6~8 mL LB broth medium and the culture was incubated in a 37o_C shaker overnight. The bacteria were collected by centrifugation at 4000 g room temperature for 10 minutes and the supernatant was discarded. The plasmid was extracted from the bacteria pellet with QIAprep Spin Miniprep Kit (Qiagen, Manchester, UK) and stored at -20oC.

2.1.4 DNA fragment amplification by Polymerase chain reaction (PCR)

The PCR components, including polymerase (Merck), MgSO4, dNTP, primers, templates, buffer and water, were added to a PCR tube (STARLAB (UK), Ltd, Milton Keynes, UK) by following the protocol in Table 2.1 and mixed. Then, the samples were put into a PCR cycler (TC-PLUS, Bibby Scientific, Staffordshire, UK). The DNA fragment was amplified for 30 cycles with the conditions in Table 2.2. The PCR product was analysed by DNA agarose gel electrophoresis (Section 2.1.5) and stored at -20oC.

Table 2.1 PCR reaction (50 µL) components and volume:

PCR Reaction Components Volume/ µL

10 X buffer for KOD Hot Start DNA polymerase 5 25 mM MgSO4 3 dNTP (2 mM each) 5 Sense Primer (30 mM) 2 Anti-sense primer (30 mM) 2

Template DNA (200 ng/µL) 1

KOD Hot Start DNA Polymerase (1 U/µL) 1

PCR Grade water 31

Total reaction volume 50

Table 2.2 PCR cycling conditions:

Cycling conditions Temp./ oC Time Circles

Stage 1 Activation 98 5 min 1

Stage 2.1 Denature 95 15 s

30

Stage 2.2 Annealing Tm - 5 15 s

Stage 2.3 Extension 70 40 s

Stage 3 Complete Extension 70 10 min 1

Stage 4 Hold 4 +∞ 1

2.1.5 DNA agarose gel

TAE (Tris-acetate-EDTA) buffer (10 X, 1 L): 20 mL EDTA (0.5 M, pH 8.0), 48.4 g Tris-Cl, 11.42 mL acetic acid and H2O.

TAE buffer (1 X) was made by diluting 100 mL 10 X TAE buffer into 900 mL dH2O. Agarose gels (1.2% (w/v)) were made by dissolving 0.36 g agarose into 30 mL 1 X TAE buffer and heating in a microwave oven. SYBR® Safe DNA Gel Stain (2 µL, Life technologies, Paisley, UK) was added to the gel liquid when its temperature was about 40oC to 50oC. Then the warm gel liquid was added into a gel making kit and allowed to set.

DNA solutions were mixed with 6 X loading dye (Thermo Scientific, Leicestershire, UK) and loaded into the wells and 1 kb DNA ladder (Thermo Scientific) was used to identify the size of the DNA fragments. Agarose gels were run at 100 V in 1 X TAE buffer for 30 minutes.

2.1.6 Restriction enzyme digestion

The plasmid (vector) and PCR fragment (insert) were digested with two desired enzymes (NEB, Hitchin, UK) to open the plasmid and expose the restriction sites, such as NcoI and BamHI. The digestion reaction was set up, as described in Table 2.3 and the buffer was chosen according to that recommended by the manufacturer of the enzymes. The reaction proceeded at 37o_{C for 2 hours.}

Table 2.3 Restriction enzyme digestion setup:

Components Volume/ µL

DNA (plasmid or PCR product) 5

Enzyme 1 1

Enzyme 2 1

Buffer (10 X) 2

2.1.7 DNA extraction from DNA gel

The target DNA fragment was excised from the agarose gel with a scalpel, and the gel slice was weighed in a 1.5 mL Eppendorf tube. Then, the DNA fragment was extracted from the gel by using QIAquick Gel Extraction Kit (Qiagen).

2.1.8 T4 Ligation

The ligation components (Table 2.4) were mixed and the ligation reaction was carried out at room temperature overnight. The ligation product was transformed into Top 10 competent cells to amplify the ligated plasmid.

Table 2.4 T4 ligation setup

Ligation components Volume/ µL

Insert x

Vector y (50 ng)

T4 ligase Buffer (10 X) 2

T4 ligase 2

H2O (20-x-y)

Note: The volumes of insert (x μL) and vector (y μL) are based on different molar ratios (4:1 and 6:1).

2.1.9 In-Fusion Ligation

In-Fusion cloning is a ligation method, which is based on the overlap of base pairs at the ligation sites rather than the cleavage sites. The insert was prepared by PCR, and the vector was opened by enzyme digestion. The PCR product was designed to

possess around 15 bp complementary ends, which overlapped the respective ends of the opened vector. The in-fusion ligation (In-Fusion HD Cloning Kit, Clontech, Takara Bio Europe SAS, Saint-Germain-en-Laye, France) was prepared according to Table 2.5. The reaction was incubated for 15 minutes at 50°C, and then placed on ice for transformation.

Table 2.5 In-Fusion Ligation setup:

In-Fusion Ligation components Volume/ µL

5X In-Fusion HD Enzyme Premix 2

Linearized Vector x (50 ng)

Purified PCR Fragment y (100 ng)

H2O z (z=10-2-x-y)

Total volume 10

Note: The values of x and y are based on the concentration of the DNA fragment.

2.1.10 Colony screening for ligation with PCR and DNA sequencing

The colonies growing on the ligation plate were labelled and used as template in the PCR sample preparation. The PCR products of the predicted size were identified by DNA agarose gel electrophoresis and the corresponding colonies were transformed into 8 mL LB broth medium for plasmid extraction (Section 2.1.3). The extracted plasmid was sequenced across the insert in both directions by GATC Biotech (GATC Biotech Ltd., London, UK).