INTRODUCTION:

In the middle of nineteenth century Gregor Mendel formulated a set of rules to explain the inheritance of biological characteristics. The basic assumption of these rules is that, each heritable property of an organism is controlled by a factor called a gene, which is a physical particle present somewhere in the cell. The rediscovery of Mendel’s law in 1900 marks the birth of genetics, the science aimed at understanding what there genes are and how exactly they work.

The idea that gene reside in chromosome was proposed by W. Sutton in 1903 and received experimental backing from T.H. Morgan in 1910. Morgan and his colleagues then developed the techniques for gene mapping, and by 1922 had produced a comprehensive analysis of the relative position of over 2000 genes on the four chromosomes of the fruit fly, Drosophila melanogaster

CLONING:

Insertion of a fragment of DNA, carrying a gene, into a cloning vector, and subsequent propagation of recombinant DNA molecule in a host organism. Also used to describe those techniques that achieve the same result without the use of a cloning vector

OBJECTIVES:

Pseudomonas aeruginosa is a Gram- negative, rod –shaped bacterium that grows in soil, marshes and coastal marine habitats, as well on plants and animal tissues.

It is a very pathogenic organism responsible for much disease due to its resistance to antibiotics and disinfectants eliminates other environmental bacteria. Pseudomonas aeruginosa is now a significant source of bacteria in burn victims, urinary-tract infections and hospital-acquired pneumonia in patients.

Pseudomonas aeruginosa even though it is very pathogenic it’s DNA fragments (BL21) which contains the structural gene coding for the galactophilic PA-1L (pa-1L,369bp) is economically important as subunit vaccine . In genetics, a clone is a replica of all or part of a macromolecule (eg; DNA) . Cloning a gene means to extract a gene from one organism (for eg; by PCR) and insert it in to a second organism (usually via a vector), when it can be used and studied.

Selective cloning of the desired fragment of the organism can be done to avoid contamination with the other toxic products of this bacterium. This can be done by using a cloned vector, which is a small DNA vehicle that carris a foreign DNA fragment.

REVIEW OF LITERATURE:

GENE:

Genes are the units of heredity in living organism. They are encoded in the organisms genetic material (usually DNA or RNA), and control the physical development and behavior of the organism. During reproduction, the genetic material is passed on from the patients to the offsprings. Genetic material can also be passed between un-related individuals (eg; via transfection, or on viruses). Genes encode the information necessary to construct the chemicals ( proteins ) needed for the organism to function. The word ‘gene’ was coined in 1909 by Danish botanist William Johannes for the fundamental physical and functional unit of heredity.

LECTIN:

Lectins are a type of of receptor protein of non-immune origin that specifically interacts with sugar molecules (carbohydrates) without modifying them. These proteins recognize and bind specifically to monosaccharides and are classified by which sugar they recognize. most lectins recognize either N-acetyl neuraminic acid , N-acetyl glucosamine , N-acetyl galactosamine, galactose , manose or fucose lectins are found in a variety of species from plants to inserts to man.

GENE CLONING VECTORS:

Now cloning vectors were originally derived from naturally occurring extra chromosomal elements such as bacteria phages and plasmids. Most DNA fragments are incapable of self replication in E. coli or any other host cell, an additional segment of DNA , capable of autonomous replication , must be linked to the fragment to be cloned. This autonomously replicating fragment is the molecular cloning vectors and by definition plays a central role in recombinant DNA technology.

Stanley Cohen and co workers first reported the use of bacterial plasmid as molecular cloning vector, since that motel report thousands of cloning vectors have been constructed and their versatility in terms of cloning sites, host range, and function appears to be limited only by the ingenuity and imagination of the inventor. Whereas insertion of foreign DNA fragment into Psc101 was limited to a single restriction enzyme cleavage site and E. coli as a host, most vectors currently in use, carry multiple restriction enzyme cleavage sites and have been modified for use with most common laboratory microorganisms. Most vectors now include dual origin of replication that enables them to be shuttled between hosts as dissimilar as E. coli and human cells.

Specialized vectors are now available that enable the investigator to

1. Identify and isolate regulatory DNA sequences such as promoters and terminators.

2. Identify open translational reading frames.

3. Over produce useful RNA’s and proteins and

4. Determine the nucleotide sequence of genes and segment of DNA.

COPYNUMBERS:

The number molecules of a plasmid found in a single bacterial cell is termed as copy number. It ranges from 1 to more than 50 per cell but this number is specific for a given plasmid residing in bacterial cells.

AMPLIFICATION OF PLASMIDS:

The copy number of a plasmid can be increased manifold. This process is called amplification of the plasmid DNA. This property is exploited to increase the plasmid copy number. When the bacterial culture is at its exponential phase chloromphenical is added to the medium .

This arrest chromosomal DNA replication and cell division. The culture is then incubated for another 12 hours for the plasmid molecules to replicate. This increases the plasmid per cell. Sometime the plasmid copy number may react to several thousand.

TYPES OF PLASMID:

Plasmids can be categorized on the basis of number of copies per cell as

1. Relaxed plasmids , which are normally maintained at multiple copies per cell and

2. Stringent plasmids [which have a limited number of copies per cell] Plasmid with larger copy number is more useful for gene cloning experiments.

3. Plasmids can also be classified as conjugative and non conjugative plasmids, depending on whether or not they carry a set of transfer gene called the tra gene.

4. These tra genes promote bacterial conjugation. Generally conjugative plasmids are of high molecular weights and are present as 1-3 copies per cell, where as non – conjugative plasmids have low molecular weight and are present in multiple copies are 20- 28 copies per cell.

PET 28(a) VECTOR:

In the pET system, protein-coding sequence of interest is cloned downstream of the T7 promoter and gene 10 ribosome binding site, and then transformed into E. coli. Protein expression is achieved either by IPTG induction of a chromosomally integrated cassette in which the T7 RNA polymerase is expressed from the lacUV5 promoter (BL21(DE3) cells), or by infection with the polymerase-expressing bacteriophage lambda CE6 (BL21 cells).³ Due to the specificity of the T7 promoter, basal expression of cloned target genes is extremely low in strains lacking a source of T7 RNA polymerase. However, upon induction, the highly active polymerase produces an abundant number of transcripts. This phenomenon together with high-efficiency translation result in such high expression levels that, after only a few hours, the target protein may constitute the majority of the cellular protein.

pET System has become recognized as one of the most powerful approaches available for producing recombinant proteins. Based on the T7 promoter-driven system originally developed by Studier and colleagues (1–3),the pET System has been greatly expanded and now includes over 23 vector types, 11differentE. coli host strains and many other companion products designed for efficient detection and purification of target proteins. The various vectors and hosts provide important options for maximizing the yield of different types of proteins. In addition, several vector-encoded affinity tags, including11aa and 260aa T7•Tag™, N-terminal and C-terminal His Tag®, 15aa S•Tag™, and 11aa HSV•Tag™ sequences are available to assay expression levels and purify proteins by several independent strategies Whereas systems available from other sources tend to offer few options for cloning and expression, Novagen has made the commitment to offer a wide selection of strategies for taking advantage of the power of the T7 promoter. These options are necessary since no one strategy or condition is suitable for every target protein. This article highlights some of the unique advantages of the pET System and the reasons for

“Tuning” the vector/host combination to optimize the expression of specific target proteins. Control Over Basal Expression Levels .A surprising percentage of cloned sequences slow the growth of E. coli when expressed as protein. Genes whose products severely affect the host cell’s growth rate at low concentrations are considered to be toxic and can be difficult to maintain as stable plasmids if even only weakly transcribed by the host. In these cases, cells carrying non-expressing

Proteins:

Proteins, on the other hand, can have different charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulfate/sodium dodecyl phosphate (SDS/SDP) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like they were long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape. Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.

APPLICATIONS:

Gel electrophoresis research often takes advantage of software-based image analysis tools, such as those used in two-dimensional gel electrophoresis, or 2-DE. In proteomics research, these tools primarily analyze biological markers by quantifying individual markers, and showing the separation between one or more protein "spots" on a scanned image of a 2-DE product. These tools may also be used to match spots between gels of similar samples to show, for example, proteomic differences between early and advanced stages of an illness. However, though some tools tend to agree on the quantification and analysis of well-defined, well-separated protein spots, they deliver different results and tendencies with less-defined, less-separated spots.

Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry. The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software.

Primers:

The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial DNA strands —often not more than 50 and usually only 18 to 25 base pairs long — that are complementary to the beginning or the end of the DNA fragment to be amplified. They anneal by adhering to the DNA template at these starting and ending points, where the DNA polymerase binds and begins the synthesis of the new DNA strand.

The choice of the length of the primers and their melting temperature (Tm) depends on a number of considerations. The melting temperature of a primer is defined as the temperature at which half of the primer binding sites are occupied. Primers that are too short would anneal at several positions on a long DNA template, which would result in non-specific copies. On the other hand, the length of a primer is limited by the maximum temperature allowed to be applied in order to melt it, as melting temperature increases with the length of the primer. Melting temperatures that are too high, i.e., above 80°C, can cause problems since the DNA polymerase is less active at such temperatures. The optimum length of a primer is generally from 15 to 40 nucleotides with a melting temperature between 55°C and 65°C.

The above mentioned considerations make primer design a very exacting process, upon which product yield depends:

RESULTS AND DISCUSSION:

Our objective was to clone PA-1L gene (Lectin) present in Pseudomonas aeruginosa. pET is used as a vehicle or vector to carry this gene and transformed in the (BL-21). Once transformation is done successfully the plasmid of cloned colonies isolated and screened by PCR. pET plasmid DNA was isolated using alkali lysis method. Spinning down the cells separates pieces of cell wall released from the bacteria and floating around in the supernatant. These cell wall pieces can inhibit enzyme action so it is important to get rid of these. Cells were resuspended in resuspention buffer, solution 1, where glucose helps in maintaining osmotic pressure in and out of the cell, EDTA chelates divalent Mg2+ ions and Tris HCl maintains pH of the solytion. Highly alkaline solution gives rise to the name of this technique. Lysis buffer, Solution II, SDS is an anionic detergent helps in the rupruting the cell wall by breaking lipid membrane and solubilising cellular proteins. NaOH denatures the chromosomal DNA in to single stranded DNA and also increases pH of the solution. Solution III potassium acetate, glacial acetic acid and distilled water together called as neutralizing buffer. The plasmid DNA is allowed to renature and the chromosomal DNA remains as a single stranded DNA as it takes more time to renature. Adding potassium aceytate to SDS farms KDS. This will allow easy removal of SDS from plasmid DNA. The beutralising solution also neutralizes basicity and decreases pH. Ethanol helps in the precipitation of DNA and 70% ethanol will wash salts and 30% water will dissolve salt completely and lead to rehydration of plasmid. Plasmid then air dried and dissolved in TE buffer that maintains pH and prevent any DNase action. When isolated plasmid was electrophoresed on 0.8% Agarose gel three bright bands were observed. These three bands separate according to their molecular weight. The one which is heaviest is near to the well and is a linear form; Nicked form is slightly lighter than linear and hence ahead; Supercoiled forms molecular weight is less compared to other two forms and is seen as smiling form ahead from both forms. For gene cloning super coiled form of DNA is required. As considerable amount of this form was obtained it was further processed for restriction digestion.

The objective is to isolate genomic DNA of Pseudomonas aerations in pure form. This involves different steps like bacterial growth and harvesting, lyses of the cell, the cell extract is then treated to remove all components except the DNA and the resulting DNA is then concentrated and purified. Overnight grown culture is harvested and centrifuged at Farley low speeds to pellet the bacteria at the bottom of the centrifugation tube. Techniques for breaking open bacterial cell can be physical or chemical. Chemical lyses are brought about by EDTA. EDTA removes Mg2+ ions that are essential for preserving the overall structure of the cell envelope and also inhibits cellular enzymes that could degrade DNA. Anionic detergent like SDS is used along with EDTA, which further denatures the cell wall by removing lipids, and also disrupts interaction between nucleic acids and proteins. The polysaccharide degraded by SDS is precipitin by sodium chloride. A mixture of acetyl tri methyl ammonium bromide (CTAB) protein extraction was performed with phenol and is amyl alcohol mixture. Chloroform denatures protein surfaces and facilitates the action of phenol, which precipitates proteins. Is amyl alcohol acts as anti foaming agent. Ethanol is used for precipitation. Treatment with 70% ethanol removes salts and helps in dehydration of DNA. Pellet is then air dried and dissolved in TE buffer that maintains pH and prevent any Danes action. When isolated DNA was electrophoreses on 0.8% Agars gel a thick bright band was observed near the well because of its high molecular weight. Its large size restricts the movement through the gel matrix. The DNA thus obtained was further used for specific gene amplification by PCR. Once the insert is ligated with vector, it is important to introduce the ligated product in to a system that will amplify it, such as bacterium. Under normal conditions bacteria will not take up these free plasmids and have to become “competent” to dissolve, this process is known as transformation.

REFERENCES:

1. Old and Primrose. Principles of gene manipulation, 5^th Edition, 2001.

2. Christophee Howe. Gene cloning and manipulation, 1^st Edition, 1995.

3. Thomas M Delvin. Text book of biochemistry, 5^th Edition, 2002.

4. Robert H. Lavirin. Principles of genetics, 7^th Edition, 2003.

5. Fox TA. Brown. Molecular biology lab, 2^nd Edition, 2007.Helen Abuzer. Recombinant DNA and Biotechnology-A lab manual, 2^nd Edition, 2001.

Received on 29.08.2013 Accepted on 01.09.2013

Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 187-190