Cloning methods using Gibson Isothermal Assembly

2.2.2.1 Primer design for cloning

PCR primers for cloning were designed as outlined in Section 2.2.1, with the addition of a ≥ 16 bp tag sequence which was complementary to the sequence flanking the cut site in the backbone vector. In this case, cutting pGL3P with SmaI resulted in the following flanking sequences: AGCTCTTACGCGTGCTAG added to the 5’ end of all forward primers, and AGATCGCAGATCTCGAG added to the 5’ end of all reverse primers.

2.2.2.2 PCR amplification using a proof-reading enzyme

PCR amplification of regions for cloning was carried out using the proof-reading Phusion High-Fidelity DNA Polymerase (NEB), with the master mix as below.

Component Volume

Phusion HF Buffer (5x) 10 µl dNTPs (10 mM each nucleotide) 1 µl Forward primer (20 pmol/µl) 2.5 µl Reverse primer (20 pmol/µl) 2.5 µl

Betaine 2.5 µl

Phusion DNA polymerase (2 U/µl) 0.5 µl

DNA template 2.5 µl (10 ng/µl)

Nuclease free water 21 µl

Total volume 50 µl

2.2.2.3 Agarose gel electrophoresis

Both PCR reactions and diagnostic restriction digests were analysed by gel electrophoresis, typically using 1.5% agarose depending on the expected band sizes. DNA bands were visualised through the addition of 0.5 µl of ethidium bromide (Sigma 10 mg/ml) per 10 ml of gel, and band size estimated through comparison to either 100

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bp or 1 kb ladders (Promega), or a 2-log ladder (NEB). Gels were typically run at 100 volts for 1.5 hours, varying dependent on the gel percentage and size of the DNA fragments. Gels were visualised and imaged using the BioDoc-it Imagine System UV transilluminator.

2.2.2.4 Purification of DNA from agarose gels

After separation by gel electrophoresis, the appropriate bands were excised from the gel and column purified using the Wizard SV Gel and PCR Clean-Up System (Promega) following the manufacturer’s protocol. DNA was eluted in 30 µl of nuclease free water.

2.2.2.5 Restriction enzyme digests

Restriction enzyme digests were used both to cut a vector to create specific nucleotide overhangs in preparation for cloning, and later diagnostically to determine successful cloning through visualising the presence and/or orientation of an insert. Restriction enzymes were purchased either from Promega or NEB, and used in the reaction as follows: Component Volume Buffer (10x) 2 µl BSA (10 mg/ml) 0.2 µl Enzyme (10 U/µl) 0.5 µl DNA X µl

Nuclease free water Y µl Total volume 20 µl

Restriction enzyme digests were incubated at the appropriate temperature for the enzyme of use, typically for 1-2 hours. After digestion, the reaction mix was run on an

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agarose gel (Section 2.2.2.3) to confirm restriction and/or to confirm the presence and orientation of the insert.

2.2.2.6 Ligation using Gibson isothermal assembly

Before cloning, the amount of vector and insert for ligation using Gibson isothermal assembly was calculated. NEB guidelines recommended the use of 100 ng of cut vector, and an insert:vector ratio of 3:1 for the assembly of 2-3 fragments. The amount of insert for each fragment was calculated using the following equation:

𝐼𝑛𝑠𝑒𝑟𝑡 (𝑛𝑔) = 𝑣𝑒𝑐𝑡𝑜𝑟 (𝑛𝑔) 𝑥 𝑠𝑖𝑧𝑒 𝑜𝑓 𝑖𝑛𝑠𝑒𝑟𝑡 (𝑘𝑏)

𝑠𝑖𝑧𝑒 𝑜𝑓 𝑣𝑒𝑐𝑡𝑜𝑟 (𝑘𝑏) 𝑥 𝑟𝑎𝑡𝑖𝑜 (

𝑖𝑛𝑠𝑒𝑟𝑡 𝑣𝑒𝑐𝑡𝑜𝑟)

The ligation reaction was set up as follows:

Component Volume

Insert DNA X µl

Vector (50 ng/µl) 2 µl

Gibson assembly master mix (2x) 10 µl

Nuclease free water Y µl

Total volume 20 µl

The Gibson assembly master mix contains a 5’ exonuclease, a DNA polymerase, and a DNA ligase. All reactions were incubated at 50 ºC for 15 minutes. During the time taken for the thermocycler to reach 50 ºC, the exonuclease was able to chew back the 5’ ends of the cut vector and insert, producing complementary overhangs. Upon reaching 50 ºC, the exonuclease was inactivated, leaving the remaining time for the insert and vector to anneal at the complementary overhangs, the DNA polymerase to extend the 3’ ends, and the DNA ligase to repair the backbone (Figure 2.1).

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Figure 2.1 Gibson isothermal assembly reaction

Before amplifying the desired region of DNA which will form the insert for the construct, PCR primers are designed to add ≥ 16 bp overhangs to the insert which will be complementary to the sequence on either side of the restriction enzyme site used to linearise the vector. Once amplified, a 3:1 ratio of insert to vector is added to the Gibson isothermal assembly mix, which contains a 5’ exonuclease, a DNA ligase, and a DNA polymerase. The reaction mixture is heated to 50 ºC. During the time it takes for the reaction to reach 50 ºC, the 5’ exonuclease chews back the 5’ strands of the insert and vector revealing complementary overhangs. Once the reaction reaches 50 ºC, the exonuclease is inactivated. The complementary overhangs anneal, and the polymerase fills in any gaps using the complementary strand as a template, with the ligase ligating the backbone of the vector and insert to form a complete vector with insert.

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2.2.2.7 Transformation of ligation mix into competent E. coli cells

Gibson assembly ligation mix was transformed into XL Gold ultracompetent cells (Agilent) as detailed in the manufacturer’s protocol. Briefly, 100 µl aliquots of cells were thawed on ice and placed in pre-chilled 14 ml falcon tubes. 4 µl β- mercaptoethanol was added to each aliquot and incubated on ice for 10 minutes, swirling gently every two minutes. 2 µl of ligation mixture was added to an aliquot of cells, swirled gently to mix, and then incubated on ice for 30 minutes.

After incubation, cells were heat shocked in a water bath at 42 ºC for 30 seconds and incubated on ice for 2 minutes. Next, 0.9 ml NZY+ _{broth (pre-heated to 42 ºC) was}

added to each, and tubes were incubated at 37 ºC for one hour, shaking at 225 rpm.

Following one hour incubation, 200 µl of each transformation mix was plated on LB agar plates supplemented with 100 µg/ml ampicillin and incubated overnight at 37 ºC.

2.2.2.8 Purification of plasmid DNA from transformed E.coli 2.2.2.8.1 Mini-prep of plasmid DNA

Individual colonies were picked from an overnight incubation of transformed E.coli culture on LB agar plates (100 µg/ml ampicillin) (Section 2.2.2.7) and grown overnight in 3 ml LB broth with 100 µg/ml ampicillin at 37 ºC, shaking at 225 rpm. Plasmid DNA was extracted and purified from 1 ml of the 3 ml overnight culture using the QIAprep spin miniprep kit (Qiagen) or the Wizard plus SV miniprep DNA purification system (Promega) according to the manufacturer’s protocols. DNA was eluted in 30 µl nuclease free water and either stored at – 20 ºC or directly used for restriction enzyme digest (Section 2.2.2.5) to determine the presence or absence of an insert.

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2.2.2.8.2 Maxi-prep of plasmid DNA

In order to extract plasmids with greater purity and yield for use in reporter gene assays, 100 ml bacterial cultures were grown overnight by adding 100 µl of 3 ml mini- prep culture (Section 2.2.2.8.1) to 100 ml LB broth with 100 µg/ml ampicillin, grown overnight at 37 ºC and shaking at 225 rpm. Plasmid DNA was extracted and purified using the Qiagen plasmid maxi kit according to manufacturer’s protocols for purifying high copy plasmids. DNA pellets were resuspended in 150 µl nuclease free water and the concentration determined using a Nanodrop 8000 before storing for later use at – 20 ºC.

2.2.2.9 Sequencing

Sequencing verification of plasmids with successfully cloned inserts was carried out externally by Source Bioscience, with samples of 5 µl at 100 ng/µl, and 5 µl of each sequencing primer at 3.2 pmol/µl. Sequencing primers for the pGL3P vector are outlined in Section 8.