FINAL YEAR PROJECT

submitted in part fulfilment for the degree of

BSc (Hons) Biology

Name : Božena Pálinkášová

Title : Investigations into the Lambda / HindIII Restriction Fragment Insertion Frequencies Using pUC18 as a Cloning Vector

Supervisor: Mr H N Hughes


Year : 2006 / 2007

SCHOOL OF BIOLOGY, CHEMISTRY AND HEALTH SCIENCE

FACULTY OF SCIENCE AND ENGINEERING


Abstract

Plasmids are essential tools in the molecular biology due to having a number of advantageous properties. They are easy to manipulate and their covalent closed circular form makes them easy to differentiate and purify from the linear genomic DNA. They are extremely useful in molecular cloning, as they replicate independently and produce up to 500 copies per cell.

In this project, frequencies of the lambda DNA / HindIII restriction fragments cloned into pUC18 were investigated. Eight lambda DNA restriction fragments produced by HindIII vary in size, ranging from 125bp – 23,130bp; therefore comparison of these fragments of different sizes can be made. The plasmid was cut with HindIII creating a complementary ‘sticky ends’, ligated at random with a mix containing eight lambda fragments and transformed to Escherichia coli. Cells were allowed to replicate and produce clones of the recombinant plasmid. After incubation for 24 hours, the recombinant plasmid DNA was extracted and analysed.

Results showed that half of the lambda fragments failed to be inserted and cloned into the pUC18, which was probably due to being considerably larger in comparison with the size of the vector. The remaining fragments that were successfully cloned were statistically analysed and the frequencies of the occurance were highly significantly different.

During the project, the actual number of lambda restriction fragments produced by HindIII was investigated due to contradictory literature statements. It was revealed that wild-type lambda DNA has six restriction sites for HindIII and a standard lambda laboratory strain (cIind 1 ts857Sam7), which used for this experiment, has seven HindIII restriction sites due to a single base substitution.

Index

1. Introduction………………………………………………………………….....5-10

2. Aims and Objectives……………………………………………………………...11

3. Materials and Methods……………………………………………………….12-20

3.1. Preparation of the Vector and Ligation……………………………..….12-13 3.1.1. pUC18………………………………………………………………………....12

3.1.2. pMAL –c2X…………………………………………………………….....12-13

3.2. Transformation…………………………………………………….……….14-16

3.2.1. Selective Media………………………………………………………...……..14

3.2.2. Transformation Reaction……………………………………………...…15-16

3.3. Purification of Recombinant Plasmid…………………………………...... 16-18

3.4. Plasmid DNA Digestion and Analysis by Agarose Gel Electrophoresis...18-19

3.4.1. Plasmid DNA digestion with HindIII………………………………………..18

3.4.2. Analysis by Agarose Gel Electrophoresis……………………………….18-19

3.5. Lambda DNA / HindIII Restriction Fragments………………………...... 19-20

4. Results…………………………………………………………………………21-24

4.1. pUC18………………………………………………………………………..21-23

4.2. pMAL – c2X…………………………………………………………………….23

4.3. Lambda DNA / HindIII Restriction Fragments…………………..………….24

5. Discussion………………………………………………………..…………….25-29

6. Acknowledgements…………………………………………………………….....30

7. References……………………………………………………………………..31-35

Appendix I…………………………………………………………………….....36-49

Appendix II……………………………………………………………………...50-54

1. Introduction

In vivo cloning using living bacterial cells was one of the first methods used for DNA amplification. The discovery of the existence of extrachromosomal circular DNA molecules, plasmids, has contributed greatly to the development of recombinant technology. One of the advantages of plasmids is that they can replicate independently and produce a significant number of copies, ranging from a single copy to several hundreds, within a single bacterial cell. The number of copies per cell depends on the “plasmid-encoded control elements that regulate the initiation”, and the rate of the replication of a plasmid (Del Solar et al. 1998). These naturally occurring double stranded DNA molecules usually give a specific advantage to the host cell. Cells may benefit from an antibiotic resistance gene carried by the plasmid or the ability to metabolise substances other than glucose for a carbon source, but some plasmids may also encode genes for virulence. Studies of plasmids have also led to the construction of synthetic plasmids that can be designed and used for a number of specific purposes (Viera and Messing 1991). Nowadays plasmids tend to be used as cloning vehicles to amplify DNA segments of interest, to express genes coding for a protein product, to sequence or even mutate genes. Synthetically derived plasmids are advantageous as they can be designed to carry a selective marker that would, under the specific conditions, differentiate cells that carry recombinant plasmid from cells that do not carry it.

In this project an example of the synthetically derived plasmid, pUC18 (Figure 1), has been used for in vivo cloning of lambda phage DNA fragments. The pUC18 vector was originally derived from the plasmid pBR322, containing an ampicillin resistance gene, which was fused with the E. coli lacZ’ gene containing an introduced multiple cloning site (Norrander et al. 1983 and Yanish-Perron et al. 1985). The lacZ’ gene consists of the promoter, operator and the first 146 codons that code for the part of the amino acid sequence of the product β-galactosidase. The rest of the sequence is complemented (α - complementation) by the E. coli host that has a partly deleted version of the gene, lacZ ∆M15 (Brown, 2000). This small plasmid vector has 2686 base pairs (bp) and a high copy number, with 500-700 copies per cell. The small size and high copy number makes it extremely advantageous for cloning as it is easily taken up by the host cell and produces a high number of target DNA (Parke, 1990). Also, a functional lacZ’ gene within the pUC18 enables scientists to distinguish recombinant colonies from non-recombinants by simple colour selection on medium containing the lactose analogue, 5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside (X-Gal). The enzyme product of the lacZ gene, β-galactosidase, hydrolyses X-Gal producing a coloured substance that stains colonies that carry the plasmid blue (Messing, 1991). However, if a DNA fragment is cloned into the polylinker site located within the lacZ, it disrupts the gene resulting in white colonies (ibid). The ampicillin resistance gene in pUC18 also aids to selection of recombinants as only the colonies carrying the plasmid are selected from untransformed colonies that would also appear to be white. The resistance gene encodes β-lactamase that hydrolyses ampicillin in the selective medium; hence the phenotype of the recombinants is ampr lacZ-. However, occasionally small “satellite colonies” could appear in the close proximity of recombinant colonies due to the degradation of ampicillin by β-lactamase, produced by recombinants, in the nutrient agar. Subculturing the colonies into liquid media containing ampicillin enables the selection of purely recombinant colonies (Sambrook and Russel 2001a).

Other essential tools in recombinant DNA technology include restriction endonucleases. Restriction endonucleases are enzymes isolated from microorganisms, and their primary function is to protect the organism’s DNA from foreign viral DNA that can invade the cell. Restriction endonucleases function by cleaving the phospho-diester bonds at specific recognition sites, hence degrading the foreign DNA (Takasaki, 1994). Restriction enzymes fall into three main groups, Type I, II and III that are further subdivided. Type I comprises a multisubunit protein complexes that require ATP to function. They nick the DNA sequences at inconsistent sites (Roberts et al. 2003). Type II restriction enzymes recognise DNA at specific sequences and require metal ions (Mg2+) as co-factors to function (Cowan, 2004). They cleave at or adjacent to the palindromic sequences, cleaving both DNA strands and producing free 5’ phosphate and 3’ hydroxyl groups (Smith and Wilcox 1970). Type III, composed of two subunits, has an absolute requirement for ATP, as Type I, but cleaves at a specific distance from their recognition sequence. In addition, recently a new category of restriction enzymes was recognised as Type IV that cleaves methylated DNA sequences (ibid). In this project a type II restriction endonuclease HindIII (isolated from Haemophilus influenzae, serotype d) was used. This enzyme recognises a specific palindromic sequence and cleaves the DNA of both complementary strands between the two adenines of 5’ A↓AGCTT 3’ nucleotide sequence (Roberts, 1979). Cleavage by the enzyme produces complementary sticky ends and therefore any DNA fragments cleaved with HindIII can be ligated together to form a recombinant molecule.

In this project, E. coli bacteriophage lambda was cut with HindIII to produce restriction fragments for in vivo cloning into pUC18. Lambda phage DNA is 48,502bp double stranded linear molecule with 12 single stranded complementary bases extending in 5’ direction from the ends of a molecule (Chauthaiwale et al. 1992) and seven restriction sites for HindIII (Fermentas, 2006a). Therefore, lambda DNA is cleaved into eight restriction fragments: 23,130bp; 2,027bp; 2,322bp; 9,416bp; 560bp; 125bp; 6,557bp and 4,361bp. However, it was noted during the literature search that a number of sources (Roberts, 1979; Roberts and Macelis 2007; Sambrook and Russel 2001a) state that lambda DNA had only six HindIII restriction sites resulting in seven fragments (with no 125bp and 6,557bp fragments, but a single 6,682bp fragment). Therefore, the above aspect was also investigated as a part of this project. The pUC18 was cut with HindIII at a single restriction site, which is located in the multiple cloning site (MCS), disrupting the lacZ’ gene. Both, digested lambda DNA and pUC18 were ligated together using DNA ligase by restructuring the phosphodiester bond to produce recombinant molecules (Higgins and Cozzarelli 1979). The ligated mix was then transferred into competent E. coli cells via transformation. Transformation was chemically induced with CaCl2 and heat shocking the cells (Chung and Miller 1993). The transformed mix was then grown on nutrient agar plates containing X-Gal, lac operon inducer isopropyl-β-D-thiogalactopyranoside (IPTG) and ampicillin. Plasmids carrying the lambda phage insert (white) were harvested from the recombinants via an alkaline lysis method. Isolates were digested with HindIII and the restriction fragments (plasmid and lambda DNA insert) were separated using agarose gel electrophoresis to confirm the transformants and determine the size of the lambda insert.

In this project, 121 isolated plasmids from the transformed E. coli colonies were analysed. The insertion of lambda phage fragments cut with HindIII was studied to determine the size and the frequency of the all possible inserts. In addition, the intention of this study was to identify the maximum size of lambda phage fragment that can be inserted into the pUC18. This plasmid vector is relatively small in size in comparison with half of the cut lambda fragments, which are considerably larger. Therefore, in this study it was investigated whether any of the larger fragments would insert into this comparatively smaller plasmid vector.

For comparison with pUC18, the larger plasmid pMAL – c2x that has 6,648bp was used (Brown, 1998). This vector is usually used for protein expression and has a structural gene for maltose-binding protein (MBP) that fuses with the target protein product of the inserted gene and is produced in the cytoplasm of the host cell (Di Guan et al. 1988). The MBP is then used for the recombinant protein purification (Maina et al. 1988). The vector is a synthetic derivative of pUC18 and has lacZ’ gene with MCS and gene for ampicillin resistance (New England BioLabs, 2006). Therefore, the recombinants can be easily screened for on X-Gal, IPTG and ampicillin containing selective agar plates similarly to the pUC18 recombinants. Another property of this vector is that it has a single HindIII restriction site within the MCS, which allows a direct comparison of the frequencies of integrated HindIII cut lambda DNA fragments with the frequencies of those that integrated into the pUC18.

2. Aims and Objectives:

The aim of this project was to clone lambda phage fragments in Escherichia coli using pUC18 plasmid vector and compare cloned fragment frequencies.

The objective was to test the frequency of insertion of lambda phage DNA fragments and test the following hypotheses.

Hypothesis: There will be a difference between the frequencies of the eight lambda phage fragments that have been inserted into the plasmid and taken up by the E. coli competent cells.

Null hypothesis: There will not be a difference between the frequencies of the eight lambda phage fragments that have been inserted into the plasmid and taken up by the E. coli competent cells.

3. Materials and Methods

3.1. Preparation of the Vector and Ligation

3.1.1. pUC18

A ligation mix containing 500ng of pUC18 plasmid (Sigma) and 1μg Lambda DNA HindIII fragments (Invitrogen) in total volume of 50μl was provided for this study (provided by MMU laboratory technician Mrs Noshina Shaheen).

3.1.2. pMAL - c2x

Phagemid vector pMAL - c2x was purchased from New England BioLabs. Vector was digested with FastDigestTM HindIII (Fermentas) in 1.5ml sterile Eppendorf tubes, so that 2μg of pMAL - c2x was digested with 20Units of enzyme. The enzyme buffer 10x FastDigestTM buffer (Fermentas) made 10 per cent of 50μl total volume reaction mixture in double deionised water. The vector was digested at 37oC (water bath) for one hour, centifuging the tubes in the bench top micro-centrifuge at 13,000rpm for 30seconds after every 30minutes to overcome any effects of condensation.

The vector was dephosphorylated with calf intestinal alkaline phosphatase (CIAP, New England BioLabs). In this process the removal of 5’ phosphate groups from the vector was catalysed by the alkaline phosphatase. The treatment usually helps to avoid the self ligation of the vector as the two ends cannot be joined with DNA ligase that has an absolute requirement for a free 5’ phosphate group (Powell and Gannon 2000). Two units of alkaline phosphatase in 1x NEB Buffer3 (New England BioLabs) were used to dephosphorylate the digested 2 μg of pMAL - c2x. Samples were incubated at 37oC (water bath) for 30 minutes. Samples were microcentrifuged at 13000rpm for 30 seconds at the end of the reaction. Both FastDigestTM HindIII and CIAP were inactivated by heat in a single step by incubation at 65oC (water bath) for one hour prior ligation reaction.

The ligation reaction of the digested and dephosphorylated pMAL - c2x with HindIII digested Lambda DNA fragments was performed using T4 DNA ligase in 1x T4 DNA ligase buffer (New England BioLabs). The T4 DNA ligase enzyme catalyses the formation of phosphodiester bonds between the complementary cohesive 5’ phosphate and 3’ hydroxyl termini (ibid). Various concentration ratios of the vector and insert were used and summarised in Table1. Ligation was performed at room temperature (22oC) for 20minutes and the ligation mix was incubated at 4oC for 24 hours prior to transformation. In addition, T4 DNA ligase was inactivated in 1:2 and 1:3 ligation mixtures with 1μl of 0.5M ethylene-diamine-tetra-acetate (EDTA) and the mixture was purified using Qiagen spin column (section 3.3.). In addition to the method using Qiagen spin column, the first step in vector purification was by addition 200μl of PB buffer to the ligation mixture which was applied to the spin column. The first step was due to having already isolated vector, followed by the remaining steps of the plasmid purification method.