Cloning of Gene Coding Glyceraldehyde 3-Phosphate Dehydrogenase

Niharika Mishra¹, Riyazul Hasan Khan², Bhavna Dwivedi¹, Skand Kumar Mishra¹

¹Department of Botany, Govt. New Science College, Rewa (M.P)

²Genentech Laboratories, Biotech Park, Lucknow

*Corresponding Author E-mail:

ABSTRACT:

Glyceraldehyde-3-phosphate dehydrogenase (GPD, E.C. 1.2.1.12) is one of the key enzymes in the Embden Meyerhof Parnas or glocolysis pathway. It catalyzes phosphorylation of glyceraldehyde-3-phosphate to pro-duce 1, 3-diphosphoglycerate. GPD is a tetrameric enzyme composed of four identical subunits. It is an essential enzyme used to maintain life activities through contributing in thisway to the formation of ATP and providing additional energy to the cell by reducing NADH to NAD+ and H+ upon its action. Moreover, GPD protein also has many other important functions, such as abiotic stress tolerance (Liu and Yang, 2005).The pUC18 vector was isolated from E.coli Top10 strain. It is a prokaryotic vector carrying a multiple cloning site with 13 unique restriction sites. This multiple cloning sites are located within the lacZ’ gene resulting in the disruption of β -galactosidase activity by cloned inserts allowing blue/white selection. The vector pUC18 was restricted using the restriction enzyme Eco.RI. Further it was thymidized to provide sticky ends so that desired gene can be inserted in it. The GAPDH gene was amplified using PCR. Then it was inserted/ ligated into cloning vector pUC18.It was then transformed into the suitable competent cells which were freshly prepared using JM107 strain of E.coli which is devoid of plasmid. Inside the host cell the recombinant DNA had undergone replication; thus, a bacterial host gave rise to a colony of cells containing the cloned target gene i.e. GAPDH. Various screening methods could be used to identify such colonies, enabling them to be selected and cultured.

KEYWORDS: β –galactosidase, Glyceraldehyde 3-phosphate dehydrogenase, PCR.

MATERIALS AND METHOD:

Preparation of Pure Bacterial Culture

In order to get pure culture, it is necessary to achieve an increase in relative number of species, preferably to the point where the species become numerically dominant component of the population. This can be achieved by using the growth of desired species, while killing or inhibiting others. A culture of bacteria (E. coli) is normally achieved as a broth culture on a Petri dish or as a freeze-dried culture. Agar plates were prepared

Single Colony Isolation

The principle of this technique is to streak a suspension of bacteria until single cells are separated on the plate. Each individual cell then grows in isolation to produce a clone of identical cells known as a “colony”. The majority of these cells are genetically identical. However, during growth, mutation of even a single colony can give rise to low levels of mutant cells. Repeated single colony isolation

Isolation of Plasmid DNA from Bacteria ( E.coli)

Plasmid isolation is achieved by the disruption of cell wall by alkali lysis method. Bacteria are suspended in an isotonic solution of sucrose, after addition of EDTA the cells are exposed to detergent and lysed by treatment with alkali. This treatment disrupts base pairing and cause the linear chromosomal DNA of the host to denature. RNA/SDS/Protein/membrane complexes, which can be removed by centrifugation. Plasmid DNA is purified and concentrated with phenol/chloroform/iso-amyl alcohol.

Quantification of DNA

We have used Nanodrop spectrophotometer for the quantification of DNA samples. Quantitative analysis may be performed in the UV/Visible regions to identify certain classes of compounds both in the pure form and biological mixtures. The approximate total amount and purity of DNA isolated was estimated by taking absorbance reading at 260 nm and 280 nm.

Amplification of Gene on PCR and Purification of Amplified Gene

Amplification of the gene were done by the PCR. The Gene JET purification kit is designed for the rapid purification of PCR products. In a high salt buffer, DNA is bound tomembrane in a spin column. Following a wash step, DNA is eluted in low-salt buffer or water without alcohol precipitation or desalting. This kit removes DNA polymerase, dNTPs and salts.

Restriction Digestion of cloning vector

The cloning vector p^uc18 was cleaved with the help of EcoRI restriction enzyme.

Thymidization of the restricted vector

With the advent of PCR, one of the most common cloning reactions in the current molecular biology laboratory is the ligation of PCR-amplified DNA fragments to plasmid vectors and subsequent introduction into E. coli cells. Although several methods exist for sub cloning PCR products the T/A procedure is the most popular choice. TA cloning is brought about by the terminal transferase activity of certain type of DNA polymerase such as the Taq polymerase. This enzyme adds a single, 3'-A overhang to each end of the PCR product. As a result, the PCR product can be directly cloned into a linearized cloning vector that have single base 3'-T overhangs on each end. Such vectors are called T- vectors. The plasmid vector can be prepared by digestion with a restriction enzyme which produces a 3’-T overhang (e.g.BspCl) or by incubating blunt-end plasmid DNA molecules with Taq polymerase in the presence of excess dTTP. Linear vector DNA with 3’-T overhang are also available commercially.

Ligation of Gene into p^uc18 Vector

Construction of any recombinant molecule of DNA is dependent on ligation of 5’ phosphate and 3’ hydroxyl terminus. The ligase enzyme catalyses the formation of phosphodiester bonds between 5’ phosphate and 3’ hydroxyl terminus of dsDNA. The T4 DNA ligase has unique ability to join sticky and blunt ends of DNA fragments.

After that agarose gel electrophoresis were performed then comprtent cells preparation and tranformation done.

Screening and Selection of Recombinant Clones

A typical vector of this type is pUC18. pUC18 also carries an ampicillin resistance gene, so transformants are plated on to ampicillin agar, on which all cells containing a vector molecule are able to grow to produce colonies. The cloned DNA is inserted into a restriction site within lacZ’, which means that recombinants are amp^r lacZ^-and non-recombinants are amp^r lacZ⁺. The two types of colony can be distinguished by including X-gal in the agar, as recombinants will be white and non-recombinants blue. Unlike pBR322, this system therefore allows recombinants to be identified during the first plating-out.IPTG is a non-metabolizable inducer of the lac operon and is therefore needed to switch on expression of lacZ’. If we have used a phosphates vector, and therefore expect no non-recombinants, then you will need an additional control to check that the color reaction is working. Transform an aliquot of cells with 1 ng unrestricted pUC18 vector and treat in the same way as one of the test dilutions.

RESULTS :

The plasmid DNA was isolated from a freshly prepared culture of E coli Top 10. It was then run on Agarose gel electrophoresis for its Qualitative analysis and gave the result as shown here.

Fig- Bands of Plasmid DNA on Gel

Amplification and Purification of the gene (GAPDH)

Amplification of the GAPDH gene was performed. It was then run on agarose gel electrophoresis for its purification. Then band was cut and purified.

Fig- Bands of Purified Gene on Gel

Restriction of the plasmid DNA

The Plasmid vector (pUC18) was restricted by an enzyme EcoRI so that the gene of interest (GAPDH) can be inserted resulting in the formation of recombinant vector can be formed. After restriction the sample was run on Agarose gel electrophoresis which gave the following result.
The GAPDH gene was ligated into the vector (pUC18).The sample was then run on Agarose gel electrophoresis which gave the following result.

Transformation of E.coli, Screening and Selection

The LB Agar plate containing IPTG, X-gal and Amp was inoculated with E.coli a day before. The cultured plates were examined next day for colony morphology and the presence of possible contaminants. Blue colonies of uniform size and appearance were clearly visible on the plate and hence it was confirmed to have obtained pure culture. This plate was stored at 4^oC for future use. The recombinant DNA molecules were transformed into freshly prepared competent cells E.coli. Immediately after transformation the competent cells of E.coli were spread cultured on LB Agar plate containing IPTG, X-gal and Ampicillin and incubated overnight at 37oC. The plate was examined next day for the growth of blue and white colonies.

The colonies which were creamy white or egg shell blue in the center were recombinant plasmid and do not contained active β-galactosidase.

The colonies which were pale blue in center and dense blue at their periphery, were non-recombinant colonies and carried wild type plasmid containing active β-galactosidase.

Fig: Blue-White Colony Selection

BLUE = NON - TRANSFORMED.

WHITE = TRANSFORMED.

Colony PCR

Colony PCR was used further to confirmed the amplification of cloned product. Two to three white colonies were picked from LB agar plate used for blue white screening. A master mix was prepared containing forward an reverse primers of GAPDH gene. The PCR product was checked on 1% agarose gel and viewed under UV Transilluminator. A sharp band was visible. The band was compared with amplified GAPDH gene band both bands covered the same distance from the well. This confirmed that the white colonies contained GAPDH gene and was amplified by primer in colony PCR.

Fig- White colonies contained GAPDH gene and amplified GAPDH gene in well no. 1, 3respectively.

DISCUSSION:

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis. Despite lacking the secretory signal sequence, this cytosolic enzyme has been found localized at the surface of several bacteria and fungi. As a surface protein, GAPDH exhibits various adhesive functions, thereby facilitating colonization and invasion of host tissues (Nagarajan and Ponnuraj , 2014). Although increased GAPDH gene expression and enzymatic function is associated with cell proliferation and tumourigenesis, conditions such as oxidative stress impair GAPDH catalytic activity and lead to cellular aging and apoptosis(Nicholls et al., 2012).In the years 1971-1973 genetic researches was thrown back into gear by what at the time was described as a revolution in experimental biology. A whole new methodology was developed, enabling previously impossible experiments to be planned and carried out, if not with ease, then at least with success. This suggested that this plasmid did not have any contamination. The purified plasmid (pUC 18) was digested with sticky end producing restriction endonuclease that has single restriction site within the MCS of the vector. For this purpose EcoRI (GAATTC) was employed which converted circular plasmid to sticky ended linear plasmid DNA. The restricted plasmid was visualized on 0.8% Agarose gel with the control (non – restricted plasmid) plasmid. It was observed that the band of non – restricted plasmid was slightly below the band of restricted plasmid. This suggested that the restriction had been done because the molecular weight of restricted plasmid DNA and non restricted plasmid DNA are same but the restricted plasmid DNA was linear and non restricted plasmid DNA was circular. Hence the band of restricted plasmid DNA was slightly above non restricted plasmid DNA. Hence the plasmid was restricted so that our gene of interest i.e. GAPDH could be inserted into it. Amplification of the GAPDH gene was performed. It was then run on Agarose gel electrophoresis for its Purification. This amplified product was purified using Fermentas PCR product purification kit to remove contaminants like primer dNTPs, Taq DNA Pal, buffer etc. The purified product was visualized on 1.0% Agarose gel. The sharp band of purified gene was visible. Hence Fermentas can be considered good for purification of gene. The amplified gene was ligated into the pUC18 vector using T/A cloning because the amplified gene product by PCR contained Adenine overhang at 3’ end. So, to ligate the gene into vector it must have Thymidized overhang at 5’ end. Adenine overhang and Thymidine overhang was complimentary to each other so they were easily ligated into the presence of T₄ DNA ligase enzyme. The recombinant DNA was visualized on 0.8% Agarose gel with a marker and control (non recombinant plasmid) with the help of UV Transilluminator it was observed that band of ligated DNA was slightly above the band of restricted plasmid because the molecular weight of ligated plasmid was more as compared to restricted plasmid . In Agarose gel electrophoresis separation was done on the basis of molecular weight. Hence it was confirmed that the gene was ligated into vector and give rise to new recombinant DNA. For uptake of recombinant DNA or foreign DNA competent cell are required. Competent cell was prepared by JM107 strain of E.coli. The competent cells were maintained in LB Broth.The selection of recombinant colonies was done on the basis of blue /white colony screening. When the colonies of transformed cells were placed on the LB Agar media contained IPTG, X-gal and Ampicillin. The β- galactosidase enzyme present into the LacZ+ gene of non-recombinant pUC 18 vector acts on X-gal substrate and produced blue colonies while the inactive β-galactosidase enzyme present in the LacZ- gene of recombinant pUC 18 vector did not act on X-gal substrate and produce white colonies. The LB-Agar plate contains some blue and some white colonies. Therefore the blue colonies were non- recombinant and white colonies were recombinant.

CONCLUSION:

Gene cloning, or molecular cloning, has several different meanings to a molecular biologist. In the literal sense, cloning a gene means to make many exact copies of a segment of a DNA molecule that encodes a gene. Cloned genes also make it easier to study the proteins they encode. Because the genetic code of bacteria is identical to that of eukaryotes, a cloned animal or plant gene that has been introduced into a bacterium can often direct the bacterium to produce its protein product, which can then be purified and used for biochemical experimentation. Cloned genes can also be used for DNA sequencing, which is the determination of the precise order of all the base pairs in the gene. All of these applications require many copies of the DNA molecule that is being studied. Gene cloning also enables scientists to manipulate and study genes in isolation from the organism they came from. This allows researchers to conduct many experiments that would be impossible without cloned genes. For research on humans, this is clearly a major advantage, as direct experimentation on humans has many technical, financial, and ethical limitations. Cloning genes is now a technically straightforward process. Recombinant DNA methods make it feasible to clone specific DNA fragments from any source into vectors that can be studied in well-characterized bacteria, in eukaryotic cells, or in vitro. Applications of DNA cloning are expanding rapidly in all fields of biology and medicine. In medical genetics such applications range from the prenatal diagnosis of inherited human diseases to the characterization of oncogenes and their roles in carcinogenesis. Pharmaceutical applications include large-scale production from cloned human genes of biologic products with therapeutic value, such as polypeptide hormones, interleukins, and enzymes. Applications in public health and laboratory medicine include development of vaccines to prevent specific infections and probes to diagnose specific infections by nucleic acid hybridization or polymerase chain reaction (PCR). The latter process uses oligonucleotide primers and DNA polymerase to amplify specific target DNA sequences during multiple cycles of synthesis in vitro, making it possible to detect rare target DNA sequences in clinical specimens with great sensitivity.

REFERENCES:

1. Liu ZH, Yang Q (2005). Cloning and characterization of glyceraldehyde- 3-phosphate dehydrogenase gene from Chaeto-mium globosum. Acta Microbiol. Sin. 45 (6): 885-889.

2. Mullis KB, Faloona FA (1987), Specific synthesis of DNA in vitro via a polymerase catalyzed reaction.Meth. Enzymol, 255, 335-350.

3. Nagarajan R, Ponnuraj K (2014). Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis ofglyceraldehyde-3-phosphate dehydrogenase from Streptococcus agalactiae NEM316 Acta Crystallogr F Struct Biol Commun. Jul;70(Pt 7):938-41.

4. Nakajima H, Amano W, Kubo T, Fukuhara A, Ihara H, Azuma YT, Tajima H, Inui T, Sawa A, Takeuchi T: Glyceraldehyde-3-phosphate dehydrogenase aggregate formation participates in oxidative stress-induced cell death. J Biol Chem 2009, 284:3 4331-34341.

5. Nicholls C¹, Li H, Liu JP (2012). GAPDH: a common enzyme with uncommon functions. Clin Exp Pharmacol Physiol. Aug; 39(8):674-9

6. Sirover MA: New nuclear functions of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in mammalian cells. J Cell Biochem 2005, 95:45-52.

7. Sojar HT, Genco RJ (2005), Specific enzymatic amplification of DNA in vitro: the PCR, 25-30.

Received on 02.12.2014 Accepted on 16.12.2014

Asian J. Pharm. Tech. 2014; Vol. 4: Issue 4, Oct.-Dec., Pg 184-188