EcoR1 is a type IIP restriction endonuclease which cleaves the palindromic

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1 Transfer of the Fungal cdna CIH-1 from the Plasmid Vector pbk CMV to the Plasmid Vector puc19 and sub- Cloning Mediated Recombinant puc19 Amplification INTRODUCTION Molecular cloning is a method used for amplifying a specific sequence and generating multiple copies of it. This procedure requires a series of steps. A restriction digestion must be undertaken by restriction endonucleas es on the DNA molecule in order to cut the target sequence. Also, the plasmid vector must be cut with the same enzymes. The next step is the ligation of the DNA sequence to the plasmid vector. This step is facilitated by a DNA ligase, which connects the sticky ends of the restricted DNA molecule to the complementary sticky ends of the plasmid. The completion of ligation is followed by the transformation of competent cells and screening for recombinants (Strachan and Read 2011). EcoR1 is a type IIP restriction endonuclease which cleaves the palindromic recognition sequence 5'- G/A A T T C -3' 3'- C T T A A /G -5', producing single stranded overhangs (sticky ends) on each cut sequence (Pingoud et al. 2005). Xba1 is a restriction endonuclease which cleaves the palindromic recognition sequence 5'- T/ C 3'-A G T A A G T C A -3' /T -5' thus, producing sticky ends (Sales et al. 2007). T4 DNA ligase is an enzyme which covalently joins the 3 hydroxyl end to the 5 phosphate end of DNA fragments to form a phosphodiester bond (Chow 2005). The plasmid puc19 includes the β-lactamase gene thus, conferring ampicillin resistance to the host cell thus, allowing only transformed cells to develop providing a convenient way for transformant screening.. A marker used for discrimination between cells which contain the recombinant plasmid and those who do not is based on the E.coli gene lacz. This gene codes for the enzyme β- galactosidase. The host E.coli (XLI-Blue) have a mutation on the lacz gene, which causes the production of a non-functional fragment of β-galactosidase. puc19 contains lacz, a portion of the lacz gene. Effective expression of lacz DNA produces

2 a fragment of β-galactosidase which binds to the host cell-derived protein fragment by α-complementation, to result in a functional enzyme (Strachan and Read 2011). IPTG acts as an artificial inducer of the lac operon and X-gal is an artificial substrate for β-galactosidase that produces a blue product when hydrolysed (Green 2013). In puc19 the polylinker is located inside the sequence coding for the β-galactosidase fragment. This detection system allows the production of blue colonies of cells with functional β-galactosidase. Recombinant plasmids which contain an insert in the polylinker can result in insertional inactivation thus, non-functional enzyme is produced and white colonies grow (Strachan and Read 2011). Transformation requires the successful introduction and replication of the plasmid by E.coli. The cells must undergo a process to become competent. Treatment with divalent cations and heat shock are procedures that allow a small amount of E.coli to acquire competence and incorporate the plasmid (Green 2013). The aim of this experiment was to transfer the fungal gene CIH-1 from the plasmid pbk CMV to the plasmid puc19. This was approached by restriction digestion of the DNA-donor and DNA-recipient plasmids and a T4 ligation reaction to bind CIH-1 to puc19. Subsequently, puc19 was introduced to competent E.coli. Ampicillin resistance was used as a transformant marker and white/blue marking method was employed for identification of recombinant and non-recombinant plasmids in transformed cells. METHODS (Green 2013)

3 RESULTS The restriction digest yielded DNA fragments of three different sizes. The restricted pbk CMV plasmid yielded a band near the 5000bp mark and a second band near the 500bp mark. The restricted puc19 plasmid yielded a band near the 3000bp mark. With respect to the marker bands, the two large bands showed a high intensity of staining with EtBr in contrast to the small band which showed moderate intensity (Figure 1.). From the ligation reaction colonies, 60% of recombinants contained the recombinant plasmid (Table 1.). The absorption spectra showed high concentration of plasmid DNA in the B1 (90.7ng/uL) and a clearly prominent peak in absorbance (A=1.815) was produced at 260nm. For W1 the concentration was 28.8ng/uL and an absorbance peak was observed (A=0.576). The absorption spectrum of W2 demonstrated low concentration (10.9ng/uL). The restriction digest of the puc19 plasmids isolated from colonies of transformed E.coli resulted in one band near the 3000bp mark for each of the three. Additionally, the white 1 plasmid produced a second band near the 500bp mark. Compared to the marker bands, the large bands showed high, moderate and low staining intensity for blue, white 1 and white 2, respectively. The small band showed a relatively low intensity (Figure 2.). The staining intensity showed correlation with the absorption spectra concentrations (Figures 3,4,5).

4 Table 1.* Colony counts of competent, transformed and control E.coli agar plates Sample Dilution No. blue colonies No. white colonies % recombinants Plate 1 Plate Mean Plate 1 Plate 2 Mean 2 Competent cells Transformation negative control (tube3) Transformation positive control (tube2) Transformation ligation (tube1) None None None Table 2. Nanodrop absorption spectra for measuring concentration of puc19 in 3 solutions B1 W1 W2 conc. (ng/ul) Abs. at 260nm

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7 Absorbance at 260 nm Figure 6. Absorbance at 260nm versus concentration of puc19 in B1, W1 and W2, using the Nanodrop system 2,4 2,2 2 1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0, concentration (ng/ul)

8 DISCUSSION The migration pattern of fragments observed in the agarose gel after the restriction digest, was expected to occur. The 4518bp band was pbk CMV, the 2686bp band was puc19 and the 600bp band was the fungal cdna CIH-1. This evidence demonstrates a successful restriction digest. Their electophoretic mobility was related only to their base pair size because the fragments generated by restriction digest were linear. Figure 6. verified that the relationship between absorbance and concentration was linear for Nanodrop system readings. After the transformation, the CIH-1 was expected to be present within the plasmid in the white colonies. Analysis with gel electrophoresis confirmed this hypothesis, as from W1 a 600bp band was observed (Figure 2.). However the W2 lane did not show to contain a second band apart from the restricted plasmid. This, combined with the fact that W2 yielded a considerably low concentration and the low staining intensity in the gel, could mean that the 600bp band was present, but not detectable. As expected the B1 lane yielded a single high intensity band, which is the restricted puc19 without the insert. The results of the blue/white screening provide evidence that the CIH-1 was successfully transferred in the polylinker of puc19. The slightest interference in the junction between the pipette and the pipette tip could allow air to enter with every use. This could result in pipetting wrong volumes in the solutions. Regarding the high dependence of the restriction digests on optimal conditions, wrong volumes could alter the conditions enough to promote cleavage at star sites. Star sites are sequences that differ from the recognition sequence by only one base pair. Non-optimal conditions compromise the specificity of restriction nucleases (Pingoud and Jeltsch 1997). More specifically, from the 18 possible star

9 sites for each endonuclease, puc19 contains 11 for Xba1 and 4 for EcoR1 (NCBI 2008). Consequently, non-optimal conditions could promote non-specific cleavage and as a result, DNA fragments of undesired size. Regarding the antibiotic choice, the hydrolysis of β-lactam by secreted β-lactamase and the acidic environment surrounding high-density cultures, could degrade ampicillin on agar plates (Sorensen and Mortensen 2005). This could result in formation of satellite colonies, which consist of non-transformed cells (Bitesize Bio 2011). Carbenicillin is an alternative antibiotic which could be used as it is more resistant to degradation than ampicillin (Sorensen and Mortensen 2005). An automated system could be employed for agarose gel electrophoresis for enhanced reproducibility. The device can add and remove reagents from the staining tray by the movement of pumps, regulated by a microcontroller (Raymer and Smith 2007). A method which involves photochemical regulation could be implemented to enable more precise control on restriction endonuclease activity. EcoR1 has been shown to become blocked by a caging group applied on the restriction site. Removal of the caging group, triggered by UV irradiation, allows the endonuclease to proceed to cleavage (Young et al. 2009). An additional method for providing solid evidence for the existence of CIH-1 in the white colonies could be undertaken. CIH-1 is a gene of eukaryotic origin. To assay for the CIH-1 protein directly from the white E.coli colonies could be inefficient, due to the difference in post-translational processing between prokaryotic and eukaryotic cells. The CIH-1 coding cdna could be isolated from the white colonies and transfected into insect cell lines via the autographa californica nuclear polyhedrosis virus (AcMNPV). Substitution of the polyhedrin-coding sequence by CIH-1 cdna in the viral vector could enable considerably high expression rates, due to the polyhedrin promoter. This expression system results in higher quantities of protein production than mammalian cell expression systems (Strachan and Read 2011). The insect cells then could be lysed and prepared for western blot analysis. After separation of proteins by molecular weight by polyacrylamide gel electrophoresis,

10 the proteins can be stained with UB25, a monoclonal antibody which recognizes the CIH-1 protein (Perfect et al. 1998). The protein can be detected by adding a secondary antibody bound to a reporter enzyme in order to produce colour change when the appropriate substrate is added. In conclusion, this experiment had the purpose of transferring CIH-1 from pbk CMV to puc19. This was effectively undertaken by restriction digest, ligation, transformation, transformant and recombinant screening, plasmid isolation and analysis by agarose gel electrophoresis. Some aspects of this experiment produced limitations and suggestions have been made for future improvements. Word count: 1497 *Table 1. Data was obtained from Moodle

11 REFERENCES Bitesize Bio (2011) What s The Problem With Ampicillin Selection? [online] available from < [2 December 2013] Chow, P. (2005) Cloning of λ DNA fragments into puc19 vector to study the ligation efficiency of NdeI-digested puc19 and HindIII-digested puc19 by T4 DNA ligase. Journal of Experimental Microbiology and Immunology [online] 8, Available from < [1 December 2013] Green, E. (2013) 216BMS Molecular Genetics DNA Cloning Labs. Coventry: Coventry University NCBI (2008) Cloning vector puc19, complete sequence [online] available from < [8 December 2013] Perfect, S.E., O Connell, R.J., Green, E.F., Doering-Saad, C., Green, J.R. (1998) Expression cloning of a fungal proline-rich glycoprotein specific to the biotrophic interface formed in the Colletotrichum-bean interaction. The Plant Journal [online] 15 (2), Available from < [10 December 2013] Pingoud, A., Fuxreiter, M., Pingoud, V., Wende, W. (2005) Type II restriction endonucleases: structure and mechanism. Cellular and Molecular Life Sciences [online] 62, Available from < > [1 December 2013]

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