Lab partners
The transformation of bacterial cells using Puc18 plasmid as a vector
Abstract
In molecular cloning experiments, the DNA to be cloned is obtained from an organism and then broken into smaller fragments through digestion with restriction enzymes. The fragments of restriction enzyme digestion are then ligated /combined with some vector DNA to produce recombinant DNA molecules. These recombinant DNA molecules are then introduced into a host organism. When the host organism replicates, the recombinant molecule is replicated together with it. With time, large populations of transformed cells or clones are generated expressing the genotype and phenotype of the inserted gene fragment.
In these experiments, some basic principles of DNA cloning and bacterial transformation were investigated. Some restriction enzymes were used to a fragment of genomic and the plasmid vector Puc18. The restriction digests were then analyzed using southern blotting technique. The vector DNA was then ligated with the DNA fragments of DNA molecule using DNA ligase enzyme. The recombinant molecule was then used to transform E coli cells in culture conferring the property of the ligated DNA fragment on the bacterial cells.
Purpose of the experiments
These experiments were performed to learn the basic principles of genetic engineering. The main objectives included
1) Learning the process of restriction enzyme digestion
2) Understanding the process of gel electrophoresis
3) Learning how to ligase two fragments of DNA using DNA ligase enzyme
4) Transforming e coli bacteria using puc18 plasmid as the vector
Materials, Equipments and reagents
1) Centrifuge tubes
2) Loop wash
3) Inoculating loops
4) 100ML OF TE buffer
5)100 ML OF Tris Hcl buffer
6)1vial plasmid DNA
7) Ethidium bromide
8) Autoclave
9) Shaking incubator
10) Fume cupboard
11) Thermometer
12) Sterile water
13) Water bath
14)0.5 m Tris Hcl
15) 0.5m Naoh
16)0.5m Nacl.
17 Selective media
18) Electrophoresis apparatus
19 Probe DNA, one vial.
20) Restriction enzymes Hind 1, sal1
21) Culture tubes
22) Microfuge tubes
23) Petri dishes
24) Beaker
24 Ampicilin stock
25 X- gal solution
Results
Restriction enzyme digestion
Preliminary calculations
The initial fragment of the gene before restriction digestion was as shown in the gene map below with all the restriction sites for various restriction Endonucleases.
When the fragment was subjected to restriction digestion various fragments of differing length were produced as in the table below
Enzyme name
Fragment size
When these fragments were run on agarose gel, varied banding patterns formed on the electrophoresis gel as in the picture of gels shown below.
A2 Gel one
A2 gel2 A2 AI gel 1
When the DNA fragments were subjected to southern blotting technique, the banding patterns produced on the nitrocellulose membrane by probing DNA on electrophoresis gel with probe L were as in the picture below.
The size of fragment detected by probe L were as follows
Enzyme name
EcoRI/ HindIII
Bacterial transformation results
The Transformation of E coli colonies with the recombinant DNA molecules produced too many colonies on the agar plates with some colonies having a blue color while other colonies had a white color. The control plates did not record any growth of bacterial colonies.
Control minus DNA
Vector only white
Vector only blue
Vector plus insert
White in color
Vector plus insert blue in color
Many blue colonies
2 white colonies
Many blue colonies
Discussion
Restriction digestion the linear DNA fragments yielded many fragments that produced different banding patterns when run on electrophoresis gel. The length that each fragment moved on the electrophoresis plate was determined by size and charge and of the fragments. The fragments that moved farthest in the electrophoresis gel were the fragments that had the smallest size.
The restriction enzyme Hind iii cuts out the Puc 18 plasmid at a single location within the lac Z gene. Since the plasmid was cut at only one location by Hind III enzyme, the plasmid became linear. The linear plasmids and a fragment of DNA can then joined together by DNA ligase to form a recombinant DNA molecule (Nichol, 2008).
Sal1 digestion of the linear DNA fragment yields a sticky end. Sal 1 restriction enzyme cuts the DNA fragment on base pair position 355 and 473 respectively. Hind III digestion also yields a sticky end (Clover, 1995). A southern blot technique was used to identify the fragments of DNA digestion to be used for DNA ligation. The probe used (probe l), had a complementary sequence to the portion of the linear DNA fragment used in the experiment.
The fragment digests of DNA that arose from the restriction digestion had sticky ends that made them qualify for ligation by DNA ligase to form a recombinant DNA with DNA from puc18 vector. DNA ligase joins two molecules of DNA through forming phosphodiester bonds between the 3’ and 5’ phosphodiester bonds in two nucleotides (Nichol, 2008).
When the recombinant DNA molecule was created, it was incubated with E coli cells. Incubation of recombinant DNA molecule with E coli cells resulted in some of the competent E coli cells taking some of the recombinant DNA molecules. The cells that took up the recombinant DNA molecules are the ones that were transformed.
The plasmid puc18 is an artificial plasmid that infects bacteria. The plasmid includes an antibiotic resistance gene ampR, lac z gene and many unique restriction sites for restriction enzyme digestion like hind III. Digestion of the plasmid with the restriction enzyme Hind iii allows the plasmid to become linear. Digestion of the Puc 18 Plasmid with a restriction enzyme also allows the plasmid to join with alien DNA that has been cut with another restriction endonuclease that makes a complementary end.
The two genes present in puc18 form the basis of the use of the plasmid in bacterial transformation. The ampicilin resistance gene allows the bacteria transformed with this vector to grow in media incorporated with the antibiotic penicillin.
Bacteria without this gene cannot grow in ampicilin enriched media. The lac z gene produces the enzyme b galactosidase that digests lactose. When the bacteria with the lac z gene are grown in media containing an analogue of lactose called x gal. The b galactosidase gene breaks down xgal into a blue compound. The bacteria with a functioning lac z gene produces bacteria a blue color when they are inoculated in media enriched with x-gal the bacteria with a non functional lac z gene cannot metabolize x gal and remain white in color when inoculated in media with x-gal.(Garcia & Durand, 2010).
X gal is routinely used in cloning experiments as a visual indicator of whether a cell does express a functional lac z gene that produces the b galactosidase enzyme. The presence of bacterial cells that express active b lactosidase gene; lac z gene is detected through growing cell in plates that contain x-gal. Blue colored colonies are unsuccessful transformation colonies because successful ligation of DNA molecules makes lac z gene non-functional. This process is called blue white screening (Garcia & Durand, 2010).
The control plate without DNA did not record any colony growth because the selective media used in the selection process hindered the growth of bacterial cells that have not taken the recombinant DNA from the plasmid Puc18. The plates containing the vector and the insert do record growth of colonies because they are able to grow on selective media that hinders their replication. This means they had been transformed to express the trait of the recombinant DNA molecule, which is the ampicilin antibiotic resistance gene
Some bacteria take the vector only without the insert. These bacteria could not grow on selective media because they do not have the recombinant DNA from the recombinant molecule. The presence of ampicilin in the selective media hinders their growth because they do have the ampicilin resistance gene.
There were some significant differences in the color of the transformed colonies. There were many blue and few white colonies. The presences of x gal in the media that produce blue colonies allow the bacteria to hydrolyze the antibiotic ampicilin to a blue product that make the bacterial colonies to appear blue.
Transformation was observed in only two plates of the four plates that were inoculated. The control plate which had no DNA from the plasmid cannot survive the inoculation medium because it had ampicilin antibiotic incorporated in it that killed all the bacteria. The ampicilin resistance gene is in the plasmid for which the bacteria do not take. In the two plates, which had media broth and the plasmid, bacteria grew because there was no ampicilin that can kill the bacteria.
The plate with bacteria that had taken the recombinant DNA from the vector, and cultured in media with ampicilin are the ones that provide the chance in the experiment of observing the transformation patterns the bacteria. In the plates that had the antibiotic, and the bacteria still grew, the growth could be attributed to obtaining of the ampicilin resistance gene from the plasmid .This conferred immunity to the antibiotic. These resistant bacteria could therefore, grow in the presence of media with ampicilin antibiotic.
The plate with media broth ampicilin and x-gal -plasmid combination supported the growth of colonies because the bacteria had taken the recombinant DNA from the plasmid. This plate with media containing ampicilin and plasmid combination includes both blue and white colonies. The plate with media containing ampiciln x-gal and the plasmid ampicilin and plasmid has white and blue colonies.
The presences of x-gal in the media mixture allow the bacteria to hydrolyze x-gal into blue substances that gives their colonies a blue color. The bacteria that do not take the recombinant DNA have a disrupted lac z gene that makes unable to hydrolyze x-gal into blue compounds and are therefore; remain white in color (Nichol, 2008).
The evidence of transformation in this practical was the ability of the bacteria to grow in the media that has the antibiotic ampicilin and the few white colonies. This means if the bacteria grew in this media containing ampicilin they had obtained the ampicilin resistance gene from the plasmid vector. The phenotype of the transformed colony expressed by the plasmid was an indication of the type of recombinant DNA molecule the bacteria took.
If the recombinant plasmid was blue in color, then the transformed bacteria had taken a recombinant plasmid with an intact lac z gene because the bacteria were able break x-gal into a blue product that gave the colonies their color. If the bacteria colony was white, then the bacteria has taken a recombinant plasmid with a deformed lac z gene hence unable to break down the x gal into a blue substance making the colonies have a white color.
A non-functional Lac Z gene cannot produce b galactosidse gene for breaking x-gal to blue colored compounds. Successful colonies transformations are therefore, white in color (Nichol, 2008). The experiment recorded only a few successful transformations because most of the bacteria colonies were blue in color meaning they were not successful transformations.
Conclusion
The experiments were not very successful and some of the intended results were obtained. It was possible to digest the DNA fragments with restriction enzymes and obtain the desired banding patterns in the southern blot experiment. The transformation efficiency of the bacteria in the second experiment was very low because only a few successful transformations were obtained. The possible cause for the low transformation efficiency was contamination in the experiment. The experiment can be improved through careful consideration of each stage in the experiment to minimize errors to obtain better results in transformation efficiency
Clover D. (1995) DNA Cloning: A Practical Approach. Oxford: Oxford University Press.
Garcia A. & Durand C. (2010) Bioengineering principles, methodologies and applications.
London: Cengage learning
Nichol D. (2008) an introduction to genetic engineering. Cambridge: Cambridge University
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