Molecular cloning is the experimental method used to assemble the recombinant DNA and, this involves replication of the DNA in the living organism. This is a method to produce exact replicas. Cloning was performed in a cell or a DNA. The recombinant DNA is inserted into a cell which has the ability to accept the foreign DNA which is known as competency. If the cell naturally does not have the ability to accept the foreign DNA then these cells are forced to be competent by using calcium chloride or by electroporation method. The process of inserting or projecting a recombinant DNA into a host cell is called cloning.
Molecular cloning produces with an unlimited quantity of the DNA segments derived from a genome and these segments can be used in a range of biological sciences such as gene expression, gene therapy and so on. By this methods many chemical reagents such as acrylic acid, ethylene glycol, methanol, ethylene oxide and salicylic acid can be produced. Recombinant DNA molecules are molecules formed by laboratory methods of genetic recombination to bring together genetic material from various sources. In general it is the combination of at least two strands of DNA. These molecules are possible because in all organisms DNA share a same chemical structure having a variation in the nucleotide sequence within the structure.
Cloning of a recombinant DNA involves the process of making copies of recombinant DNA by inserting into a vector DNA. Normally used vector is pUC19 which has a high copy number. Molecular cloning involves four steps namely restriction, ligation, transformation and screening. Enzymes used for restriction are BamH1, Pst1, ECORI, and Xho1 which has unique restriction site and produce different sized fragments. By the use of recombinant DNA in cloning a specific combination of genes can be put into a vector DNA and then can be proliferated and expressed in a recipient cell.
Recombinant DNA technology is used in biotechnology, medicine and research. Some of the practical applications are found in agriculture, industry, food production, human and veterinary medicine such as recombinant insulin is synthesized by inserting the human insulin gene into E.coli or yeast which then produces insulin used in the treatment of diabetes.
Blue white screening is based on the method called alpha-complementation. It works by first cleaving the lacZ gene into two parts : one part coding the ?-peptide and the other part coding the ?-peptide. The ?-peptide remains in the genome, the E.coli cell. When the alpha peptide is placed inside a plasmid vector, when the plasmid is transformed into the corresponding cell and expressed with IPTG it creates the lacZ ?-peptide and lacZ ?-peptide. When these two peptide are induced together they create one functional ?-galactosidase(?-gal) enzyme. In the presence of X-gal, the ?-gal will cleave the X-gal and forming blue precipitate thus turning the colonies blue. (Ausubel, 1994)
When inserting a recombinant DNA sequence into the multiple cloning site of the lacZ? gene (i.e.) when DNA insert is put into the lacZ? gene and expressed with IPGT, it will not create lacZ ?-peptide, thus when the bacterial genome creates the lacZ ?- peptide, a non-functional beta gal enzyme is created. Thus in the presence of X-gal there is no cleavage, so in the plate there will be only white colonies.
?-galactosidase enzyme is used in genetics and molecular biology as a detection marker in gene expressions. This enzyme can be split into peptides lacZ? and lacZ?. These peptides are inactive by themselves but when both are present together they act as a functional enzyme. When the DNA fragments are inserted into the vector the production of the lacZ? is disrupted and therefore the cells show no activity of ?-galactosidase.
The vectors and the host cells does not have the b-galactidase enzyme activity. LacZ gene has a multiple cloning site within itself which can be cut by different restriction enzymes. Therefore when a gene fragment is inserted into the vector, the lacZ gene is disrupted and cannot form beta-gatatosidase enzyme. LacZ gene encodes for beta-galactidose which helps in metabolising Xgal to form 5-bromo-4chloro indole 3-acetic acid which is blue in colour.
The beta-galactidase enzyme is used in the blue white screening of the colonies. The presence or the absence of the b-galactosidase can be detected by X-gal. when X-gal is cleaved by the enzyme b-galactosidase it produces a characteristic blue dye, which make it easy for the identification of the cloned products in a plasmid. When a foreign DNA is inserted at the lacz? gene, the production of beta galactisodase is inhibited thereby lead to the formation of the white colonies. The colonies will be White if the DNA insert is present and has no enzyme function. Blue colonies are DNA which does not have the insert gene.
In this experiment we use unknown DNA samples, pMA and pMB will be digested initially and the restriction map has to be created And to subclone the fragment from the pMB into pUC19. This restricted insert DNA pMB is ligated with restricted pUC19 vector with the same enzyme and is transformed into the competent cell (E.coli XL1 blue) and analysed by electrophoresis.
Strains Ampicillin Tetracycline Kanamycin
DH5? – – –
pUC19 Resistant – –
pMA Resistant Resistant –
pMB – Resistant Resistant
XL1-blue – Resistant –
The strains of DH5?, PUC19, pMA, pMB, and XL1-blue are tested for antibiotic resistance in Luria-Broth agar plates and the results noted. pMA showed antibiotic resistance to ampicillin and tetracycline while pMB showed resistance to tetracycline and kanamycin. E.coli stain XL1-blue is resistant to tetracycline as well.
Restriction enzymes BAMH1 ECORI,
Fragment length Band1 Cm 1.4 1.4 1.5-1.6 1.4-1.5 1.4 1.6-1.7 1.4
Bp 4000 4000 3200 3600 4000 2800 4000
Band2 Cm – – 2.8 3.3 – 2.4-2.5 –
Bp – – 800 400 – 1200 –
Restriction enzymes BAMH1 ECORI,
Fragment length Band1 Cm 1.3 1.5-1.6 1.6-1.7 1.3-1.4 1.6-1.7 1.7 1.4-1.5
Bp 5000 3100 2900 4600 2700 2500 3700
Band2 Cm – 1.9-2 2.3-2.4 3.3 1.8 2.4 2.5
Bp – 1900 1300 400 2300 1300 1100
Band3 Cm – – 2.8 – – 2.4-2.5 –
Bp – – 800 – – 1200 –
The table shows the fragment length obtained by the restriction enzymes. These details obtained about the fragment caused about various enzymes is analysed by keeping the standard graph which is plotted with the markers.
pma and pmb were restricted by ecori(E),bamh1(B), pst1(P) and xho1(X) enzymes. The fragment length obtained by the restriction enzymes were obtained with the 1kb marker used in the double digest gel.
Pma has restriction sites of ecori, bamh1, pst1 and no restriction for xho1.pmb has restriction two restriction for pst1 and also has a restriction for xho1
Restriction maps were drawn based on the double digest (figure()). From the the restriction maps, the fragments in pma and pmb, restricted by ecori and bamh1, have the fragment size 0.4kb (400bp). The fragment cutting by bamh1 and pst1 in pma has the same fragment size 1.2kb (1200bp) with one of the fragments restricted by ecori and pst1 in pmb. The fragment restricted by ecor1 and pst1 in pma has the sam fragment size 0.8kb (800bp) with one of the fragments restricted by ecor1 and pst1 in pmb.
The plasmids pMA and pMB was digested (single and double digest)with ther estriction enzymes BamH1, ECORI, pst1 and xhol. The results of the experiments showed that the xho1 had no restriction site in pMA and pst1 had two restriction sites in pMB. The plasmid pMB was found to be similar to the pMA with the 1.2kb, 0.8kb and 0.4kb fragments
The double digest with the
Restriction enzymes BamHI and ECORI in the plasmids pMA and pMB had the same weight of 0.4kb
Restriction enzymes ECORI and pst1, in pMA has the same weight (0.8kb) with one of the fragment restricted ECORI and pst in pMB
Restriction enzymes BamH1 and pst1, in pMA has the same weight(1.2kb) with one of the fragment restricted by ECORI and pst1 in pMB.
The size of the restriction fragments of the plasmids pMA and pMB when digested with the enzymes was roughly obtained with the help of 1kb marker and these values were not accurate. As the pst1 has one restriction site in the pMA and two restriction sites in the pMB, we can assume that pMA is part of pMB. If the longer fragment of the pst1 site in the pMB when re-circled will have the same restriction enzymes sites as the pMA(BamH1,ECORI,pst1). The xhol restriction site in the pMB is not in the longer fragment and so this xhol site will not digest the longer fragment which is consistent with pMA.
The strains of DH5?, PUC19, pMA, pMB, and XL1-blue are tested for antibiotic resistance in Luria-Broth agar plates and the results noted. The bacterial host, DH5? has no resistance to any of the three bacterial strains, pUC19 is resistant to ampicillin, pMA is resistant to ampicillin and tetracycline, pMB is resistant to tetracycline and Kanamycin, and XL1-Blue is resistant to tetracycline. Therefore in the presence of an antibiotic cells containing recombinant dna and different plasmids could be selected by the growing host cells. For example, kanamycin can be used to select cells containing only pMB from a mixture of cells containing pMB and pUC19. As the results noted, pMA to be both ampicillin and tetracycline resistant whereas pMB to be resistant to tetracycline and kanamycin.
This may be because the tetracycline resistant gene is in pMA which is a part of the pMB, and the kanamycin resistant gene is in the Pst1 fragment which is not in the pMA that is it is in the Xho1 fragment which is not present in the pMA. The ampicillin resistant gene in the pMA which might be located in the pst1 restriction site which is insertion inactive that is when insert the pst1 fragment to pMA to become pMB therefore, pMB does not have ampicillin resistant gene
We have a new plasmid containing the pst1 fragment of pmb ligated to the puc19. This plasmid is transformed into the component cell XL1 blue and the transformation efficiency is calculated by growing the strain in the medium containing IPGT (compound having similar structure to the lactose) . IPGT inactivates lac repressor and thus derepresses peptide synthesis, and posses white that is when dna insert is present. Xgal, which is turned blue by the enzymatic activity of beta-galactosidase. On this medium, these vector-containing cells posses beta-galatosidase activity and turn blue.
Verification of dna insertion by restriction enzyme digestion and agarose gel electrophoresis is a step which provides evedience that the insert dna is cloned at the correct side of the vector. The pmb fragment of size 1.2kb is cloned between the pst1 sites at the multiple cloning sites of vector puc19 assuming that pst1 are unique in vector and insert. As shown in figure(), after ligation and transformation, plasmid DNA is isolated from selected colonies. After digestion of the plasmid DNA with the same restriction enzyme pst1, electrophoresis will be carried out. The results showed pattern of the DNA bands as expected. That is, the two bands are the same sizes as the insert and vector dna used for ligation as shown in the figure().
Apart from the blue white screening there are several methods that can recover only bacterial clones harbouring recombinant pUC19 containing the PstI fragment from pMB. The other methods include positive selection vector, diagnostic restriction digest, colony PCR and sequencing. Eventhough the blue white screening could verify the recombinant colonies the most accurate method to verify the recombinant coloniesis by sequencind. The puc19 plasmid vector dna is first isolated from an overnight bacterial culture, and the pmb insert dna can be identified by sanger sequencing usind forward and reverse primers for the puc19 vector dna. The result will be an exact sequence of insert. pMB is resistant to kanamycin and pst1 also restricts kanamycin resistant gene. Thus pMB can be used to transfer kanamycin resistance to organism of interest.