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The previously described methodology was immediately successful for the production of the pcDNATM5/FRT/TO/AURKAIP1-FLAG construct (Figure 3.3B), but despite several attempts, these methods could not produce successful ligation of the truncated AURKAIP1 construct in pGEX-6P-1. Further attempts at ligation using an increased insert:vector molar ratio of 8:1 were still unsuccessful at producing the desired construct.

To address this problem it was first important to ensure that the restriction endonucleases were digesting the vector and insert correctly. Digestion of the pGEX- 6P-1 vector was confirmed by subjecting double digested vector to agarose gel electrophoresis and visualising a single band of approximately 5kb on the gel (Figure 3.4). This showed that the vector had been successfully linearised. However, linearisation could have occurred if only one of the restriction endonucleases were active. Since XhoI was also used in the successful generation of the pcDNATM5/FRT/TO/AURKAIP1-FLAG construct, it was only necessary to confirm the activity of the BamHI enzyme. A restriction digest reaction using only the BamHI Figure 3.3: Successful cloning of pcDNATM5/FRT/TO/AURKAIP1-FLAG. A) An aliquot of

digested pcDNATM_{5/FRT/TO was subjected to 0.8% agaroge gel electrophoresis B) Vector from A and}

gel extracted insert (Figure 3.2C) were ligated and used to transform α-select cells. Several clones were selected for cells transformed with either the FLAG tagged (lanes 2 and 3) or non-FLAG-tagged (lanes 4 and 5) construct. Clones were cultured at 37°C in LBamp and plasmid DNA was extracted by miniprep. Plasmid DNA from the clones were digested with HindIII and XhoI and an aliquot was subjected to 0.8% agarose gel electrophoresis.

enzyme still resulted in successful linearisation of the vector. This proved that the

BamHI enzyme used was active.

Figure 3.4: Confirmation of pGEX-6P-1 vector linearisation. An aliquot of isolated pGEX-6P-1 vector

(lane 2) or pGEX-6P-1 vector digested with BamHI and

XhoI (lane 3) were subjected to separation through a 0.8%

agaroge gel.

Since each of the restriction enzymes were confirmed to linearise the vector, it was more probable that the problem was with the insert. Though there were nucleotides added to the primers downstream of the restriction sites in order to allow endonuclease activity, it was possible that they were insufficient to allow for effective digestion of the inserts. As described earlier, linearisation of the vector was easy to confirm via electrophoresis, but it was impossible to confirm digestion of the insert in the same manner. The few nucleotides that should have been removed from the ends of the insert would have been undetectable by electrophoresis. This reasoning led to the use of the PCR-Script Amp cloning kit (Agilent technologies), which allows any blunt ended PCR product to be cloned into the PCR-Script Amp SK(+) vector. Cloning the truncated AURKAIP1 insert into this vector may improve endonuclease activity and would allow visual confirmation of insert digestion by electrophoresis.

The truncated AURKAIP1 insert was cloned into the PCR-Script Amp SK(+) vector by adding a 100ng aliquot of the PCR product to a 10µl ligation reaction containing 1x PCR Script reaction buffer, 0.5mM rATP, 10ng pre-digested PCR-Script Amp SK(+) vector, 0.5U/µl SrfI and 0.4U/µl T4 DNA ligase. The reaction was incubated for 1 hour at room temperature before heating at 65°C for 10 minutes. A 2µl aliquot of the reaction was then used to transform 40µl of Bioline α-select chemically competent cells (2.2.2). Colonies that were transformed by recombinant plasmid were selected by blue/white colour screening after overnight incubation at 37°C on LB-ampicillin agar plates spread

with 100µl of 10mM IPTG and 100µl of 2% X-gal 30 minutes prior to plating the transformation reaction.

Blue/white colony screening is possible due to the presence of a lacZ’ gene in the PCR- Script Amp SK(+) vector which encodes the α-fragment of β-galactosidase. β- galactosidase is an enzyme that can catalyse X-GAL to 5-bromo-4-chloro-indoxyl, which then spontaneously dimerises and oxidises to form 5,5'-dibromo-4,4'-dichloro- indigo, which is a blue insoluble pigment. Bacterial colonies with functional β- galactosidase will therefore appear blue when grown on agar plates spread with X-GAL. Several E.coli strains (including α-select cells) express a mutated form of β- galactosidase, which has an N-terminal deletion and is inactive. Colonies of these bacteria will appear white when grown on agar plates spread with X-GAL due to a lack of β galactosidase activity. The mutated β-galactosidase can have its function restored with expression of the α-fragment, therefore α-select cells transformed with the PCR- Script Amp SK(+) vector would have active β-galactosidase activity. However, since the multiple cloning site of the PCR-Script Amp SK(+) vector is situated within the

LacZ’ gene, ligation of an insert would prevent the expression of the α-fragment of β-

galactosidase.

White colonies from the transformation were selected on the basis that they must have been transformed with the PCR-Script Amp SK(+) vector, as this has conferred ampicillin resistance, and that the PCR-Script Amp SK(+) vector must have the insert in the multiple cloning site because there was no β-galactosidase activity. These white colonies were cultured in 5ml LBamp overnight at 37°C in order to amplify the plasmid copy number. Plasmid DNA was then isolated from bacterial lysate (2.3.1) and digested with BamHI and XhoI. A 2µg aliquot of the digested construct was subjected to electrophoresis through a 1% agarose gel with 0.2µg/ml ethidium bromide. This technique confirmed that both restriction enzymes had correctly digested the insert because two bands were clearly visible corresponding to the size of the vector and the size of the insert respectively. Only one band with a size consistent with the sum of the sizes of vector and insert would have been seen if only one restriction enzyme had successfully cut the plasmid DNA.

Figure 3.5: Generating Correctly Digested Truncated AURKAIP1 Insert via Cloning into PCR- Script Amp SK(+). A) Two bacterial colonies that were identified as being transformed with PCR-Script

Amp SK(+)/truncated AURKAIP1 by blue/white colour colony screening were cultured in LB amp overnight at 37°C. Plasmid DNA was isolated from the bacterial lysate and subjected to double restriction digest with BamHI and XhoI. An aliquot of the digestion reactions from two clones were each loaded on a 1.5% GTG low-melt agarose gel (lanes 2 and 3 respectively). B) the bands corresponding to the truncated AURKAIP1 insert were excised from the gel for use in subsequent ligation reactions.

Correctly digested pGEX-6P-1 vector and truncated AURKAIP1 insert, as confirmed by electrophoresis were excised and extracted from their respective gels using the QIAquick® Gel Extraction Kit. Further attempts to ligate the vector and insert and subsequently transform α-select cells were carried out. Though some colonies grew on the experimental LB-ampicillin agar plates, plasmid DNA isolation and double digests of all colonies from several ligation and transformation attempts failed to show any evidence of successful construct production.

To overcome this problem, an in-gel ligation reaction was used. The two different clones of the truncated AURKAIP1-PCR-Script construct were digested with BamHI and XhoI before a 2µg aliquot was subjected to electrophoresis through a 1.5% GTG low melt agarose gel (Figure 3.5A). The insert was then excised from the gel (Figure 3.5B) and re-melted by placing at 68°C for 10 minutes. The re-melted gel was then placed at 37°C and an extra 10µl of dH2O was added to prevent re-formation of the gel.

The ligation reaction was prepared in a final volume of 25µl with 9µl 10mM Tris-HCL pH 7.5, 2.5µl 10x T4 DNA Ligation buffer, 0.5µl vector, 0.5µl T4 DNA Ligase and 12.5µl of re-melted agarose gel containing insert. The reaction was mixed and incubated

at room temperature for 3 hours. The ligation reaction was then re-melted at 68°C for 10 minutes and a 2µl aliquot was used to transform α-select cells as described previously. After overnight culture at 37°C on LB-ampicillin plates there were zero colonies on the control plate (from ligation reaction lacking insert), three colonies from the transformation with the truncated AURKAIP1 clone 1 ligation and 15 colonies from the transformation with clone 2. Each of the 3 colonies from the clone 1 plate and 5 colonies from the clone 2 plate were cultured in 5ml LBamp overnight at 37°C and had plasmid DNA isolated by miniprep (2.3.1). Plasmid DNA from each colony was then double digested before an aliquot of each digested plasmid was subjected to electrophoresis through a 1% agarose gel with 0.2µg/ml ethidium bromide (Figure 3.6). The results show that two truncated AURKAIP1 pGEX-6P-1 clones (clones 3 and 4 from ligation with the insert from truncated AURKAIP1-PCR Script clone 2) were successfully produced (Figure 3.6, lanes 7 and 8).

Figure 3.6: Successful Generation of pGEX-6P-1/truncated AURKAIP1 constructs.

Transformed colonies from ligation reactions performed with digested pGEX-6P-1 vector and either digested PCR-Script/AURKAIP1 clone 1 (Figure 3.5B lane 2) or clone 2 (Figure 3.5B lane 3) insert were selected and each cultured at 37°C in LBamp overnight. Plasmid DNA was isolated from each clone and subjected to 1% agarose gel electrophoresis (lower panel). An aliquot of plasmid DNA from each clone was subjected to double restriction digest with BamHI and

XhoI before separation through a 1%

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