Recombinant DNA technology is used for cloning of the genes of interest. The cloning involves isolation, restriction, ligation and insertion of recombinant DNA molecule into a host such as E. coli, yeast, an animal or a plant cell. In the following experiment phage λ and pUC19 were joined together by using DNA ligase followed by restriction digestion by EcoRI. The restriction and ligation products were analysed by agarose gel electrophoresis. The products of ligation were used to insert into bacterial cells and were identified by blue-white screening test. The results showed the size of pUC19 restricted with EcoRI was found to be 6.5 kb. The efficiency of transformation was 00 / µg mean while the percentage of recombinant containing inserts was also 00%. More probably the bacterial cells were died due to overheating. On the otherhand restriction of phage λ and pUC19 was achieved successfully. Recombinant DNA technology may becarried out accurately by following the standard methods carefully.
Introduction
Gene cloning has become a fast growing technique due to its wide ranging applications in life and health sciences. With the help of gene cloning large-scale production of therapeutic proteins and biobiopharmaceuticals has been made possible. Human insulin, human growth hormone, interferon, hepatitis B vaccine, tissue plasminogin activator, inter leukin 2 and erytheroproteins are some of the examples. Gene cloning has been proved very effective in improving crop plants and farm animals (Dominic et al., 1997). Gene cloning is a process of transforming a recombinant DNA molecule into a host cell such as, E. coli, yeast, an animal or a plant cell. Cloning involves isolation and restriction of plasmid DNA followed by its renaturation by DNA ligase under appropriate conditions. This is done by inserting the λDNA or gene of interest into a vector to produce a recombinant DNA molecule. Vector act as a vehicle to transport the gene of interest into the host cell. pUC19 is mostly used as a vector in gene cloning (Peter et al., 2001).
Various methods have been developed for plasmid isolation. Maxi-prep method is used for large scale whereas mini-prep is idael at small-scale because it is less labour intensive method. Alkaline lysis method (Binboim and Doly, 1979) and rapid boiling procedure (Holmes and Quingley, 1981) are widely used for mini-prep (Jaffery et al., 1990). After isolation of DNA restriction endonuclease enzyme is used to cut bacterial λDNA and vector DNA. Some enzymes cut the double stranded DNA at the same position to produce blunt-end fragments such as, HaeIII and SmaI. On the other hand many enzymes cut the DNA strands at restriction sites at different positions to produce cohesive or sticky ends such as, EcoRI and HindIII. The sticky ends can be easily joined by using DNA ligase to produce recombinant DNA molecule (Merz et al., 1972). The results of isolation, restriction digestion and ligation are analysed by using agarose gel electrophoresis.
Finally the recombinant DNA molecule is transferred into a host which is mostly a bacterial cell. Effeciency of transformation and the percentage of recombination is monitered by blue-white screening test. This test is based on the functional inactivation of E. coli Lac Z gene present in the vector. This gene encodes β-galactosidase required for the breakdown of Lactose into glucose and galactose. It is monitered by its ability to break 5-bromo-4-chloro-3-indolyl-β-D-galactoside (Xgal) into a blue coloured compound, 5-bromo-4-chloroindigo and galactose. Xgal is colourless so, if it is not brocken down by the enzyme β-galactosidase then white colonies may be observed. On the other hand if it is cleaved to give 5-bromo-4-chloroindigo and galactose then blue colonies may be observed. So if recombination occur between λDNA and pUC19 it causes the deactivation of β-galactosidase resulting white colonies instead of blue colonies (Snustad et al., 2001).
The purpose of present experiment was to isolate DNA from bacterial cell.Its restriction and ligation with a vector (pUC19) to form recominant DNA molecule. Finally its transformation into a bacterial cell to monitor the percentage of recombination and effeciency of transformation by using blue-white screening test.
Materials and methods
All the procedures including the micro isolation of plasmid DNA, restriction, ligation and transformation of the recombinant DNA molecules were followed as given in the practicl booklet.
Results
Results were analysed by using gel electrophoretic technique. Following table and figures show the data observed from experiment.
DH5α DH5α λ DNA pUC19 pUC19 pUC19 pUC19
pUC19 EcoRI EcoRI λDNA λDNA
-lig +lig
Figure 01. This figure shows the migration of DNA extracts of isolation, restriction and ligation in agarose gel.
Table1. This table indicates the data about distance travelled by the λ DNA fragments restricted with EcoRI against their size expressed in kb and log kb
Migrated distance in mm
Size of cut λ DNA fragment (kb)
Log size of cut λ DNA fragment (log kb)
19
21.80
1.338
23
7.52
0.876
25
5.93
0.773
25
5.54
0.744
27
4.80
0.681
30
3.41
0.533
On the basis of above results a garph was plotted with standard size of λ DNA on x-axis and distance covered by the fragments of λ DNA restricted with EcoRI on y-axis.
Figure 2. The graph plotted with standard size of λ DNA on x-axis against distance covered by the fragments of λ DNA restricted with EcoRI on y-axis.
Results were analysed by using gel electrophoretic technique. In lane 01 and lane 02 no band was observed. In well three five bands were observed. Again no band was observed in lane 04. Where as lane 05 shows one band of cut pUC19. Lane 06 appears with 06 bands indiacating the cut fragments of λ DNA and pUC19. Finally no band was observed in lane 07.
Table2: This table shows substances added on agar plates containingLB broth withampicillin an X-gal.
Plate
Substance Added
1
DH5α
2
DH5α +pUC19
3
λ DNA +pUC19
4
λ DNA +pUC19+DNA ligase
Table3: This table gives information about the number of white and blue colonies observed on each plate, after incubating at 37 degree C
Colour of the colonies
Plate1
Plate2 diluted
Plate2 undiluted
Plate3 diluted
Plate3 diluted
Plate4undiluted
Blue
No Growth
No Growth
No Growth
No Growth
No Growth
No Growth
White
No Growth
No Growth
No Growth
No Growth
No Growth
No Growth
In blue-white screening test no colonies were observed.
Calculation:-
No of white colonies X100
% of recombinant = Total colonies
% of recombinant = 00X100
00
% of recombinant = 00% of recombinant
Efficiency of transformation = No of blue colonies Xdilution factor
Amount of µg of DNA added
Efficiency of transformation = 00 / µg of cfu.
Discussion
According to gel electrophoresis figure the lane 1 showed no dands because it contains E.coli DH5α so no band is observed. Where as a band was expected in lane 02 indicating the isolated super coiled plasmid DNA (Old et al. 1989). But from the results no band is obseved, because of the reason that the concentration of stopping solution was very low and samples were also no loaded into the wells properly. By careful and proper loadind the results may be obtained.
In well three five bands were observed. indicating the restriction digestion of λ DNA. These 05 bands reflect that restrition digestion occurred by EcoRI and λ DNA is restricted into six fragments. But results show that only five light bands wereobserved because the distance between bad 3 and 4 is very small so may be they are merged together representing one band.
In lane 4 there should appear a band to represent supercoiled, un-cut λ DNA(Old et al. 1989). But results show no band may be some pipetting error was made. In lane 5 one band indicates that pUC19 is restricted with EcoRI. In lane 6 bands were observed they indicate the presence of cut fragments of λ DNA andpUC19.
In lane 7 also no bad was observed may be the the well was not properly loaded with samples. In screening test no colonies were obseved because the sample were over heated durin heat shok(instead of 2 minutes samples were kept in water bath for 20 minutes) Most probably the cells were died due to extra heating reulting oo% recombination and 00 cfu per µg of DNA. The molecular mass of pUC19 was foun from the standard curve and was found to be 2.5 kb. But in actual practice the mass of pUc19 is 2.686 kb (Russell et al. 2001).
Conclusion:
The restriction was carried out but ligation and transformation was not obseved due to im proper handlig.
Refrences:
Dominic, W. S. Wing. (1997). The ABC of gene cloning. (pp.13-14). London: Chapman and Hall.
Jaffery M. Becker, Guy A. Caldwell, Eve Ann Zachgo. (1990). Biotechnology: A Laboratory course. (pp.47-48). London: Academic Press Limited.
Merz, J. E. and Davis, R. W. (1972). Leavage of DNA by R1 restriction endonuclease gnerates cohesive ends. Proc. Natn Acad. Sci. USA, 69, 3370-4.
Old, R. W. and Primrose, S.B. (1989). Principles of Gene manipulation: An Introduction to Genetic Engineering. (4th ed). (pp. 06). London: Blackwell Scientific Publication.
Peter J. Russell. (2001). iGenetics. (pp.165-169). London: Pearson Education.
Snustad and Simons. (2001). Principles of Genetics. (3rd ed). (pp.493-497). USA: John Wiley and Sons, Inc.
