Cloning of PMB Insert Inside the Competant Cell
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Cloning is the process of making the exact replicas. Cloning can be done in cell, DNA. This recombinant DNA gets inserted to the cell which should have the ability to accept the foreign DNA called competency. Some cells naturally have the ability of accepting the foreign DNA otherwise these cells are forces to be competent by using CaCl2/Electroporation method. This process of projecting recombinant DNA into the new host is called cloning.
Recombinant DNA cloning involves the process of making many copies of the recombinant DNA by introducing it to the host that is normally used is E.coli which can produce number of copies of DNA. These recombinant DNA is used for blotting, microarray, sequencing, Transcript mapping, Band shift assaying and footprinting. Insulin is the best example of a recombinant product. And hence, the main application of recombinant DNA technology is that, it is used to produce the desired protein for medical use and to read the sequence as easy as possible and also to read the regulatory pathways of the cells.
Recombinant DNA technology is inserted DNA into the vector DNA. Most prominently used vector is PUC19.Molecular cloning involves 4 steps: Restriction, Ligation, Transformation and Screening. Restriction is done with 4 enzymes namely Bam HI, EcoR1, PstI and XhoI. Each enzyme has unique restriction sites and produce different sized fragments.
In this experiment we use unknown DNA samples, pMA and pMB will be digested initially (both single and double digest) and the restriction map has to be created. pMB is restricted by PstI. This gives a small and large fragment. The small fragment is known as pMB insert fragment. This insert is ligated with restricted PUC19 vector restricted with the same enzyme and is transformed into the competent cell (E.coli XL1 blue).
BLUE WHITE SELECTION:
The concept of alpha-complementation is a quick, easy and single step process of determining the transformation efficiency of a competent cell. The competent cell is the E.coli cell that can accept the DNA that we need to insert into them. The key to alpha complementation is the fact that the lac-Z gene product (β- galactosidase) is a tetramer, and each monomer is made of parts - lacZ-alpha and lacZ-omega.
Researchers found that α fragment encodes for the protein and thus when its deleted renders the galactosidase inactive. However, alpha fragment functionality can be restored in trans via plasmid (ultimers.utp.edu). We need an E.coli strain that has the deletion of the lac Z alpha, and a plasmid with the lacZ-alpha fragment as the scorable marker (Arana et al., 2007). If plain plasmid is success fully transformed into a cell, then the cell will express functional β-galactosidase.
We have made a new plasmid containing the insert DNA from the pMB which is ligated to PUC19 vector DNA. This is having the Pst fragment of pMB ligated to the PUC19. This is transformed into the competent cell XL1 blue (E.coli). The efficiency of transformation is calculated by growing the strain in the selection medium containing a specialised compound call IPTG which is compound having similar structure to lactose (Chen and Dubnav et al., 2004).
If the recombinant plasmid are transformed successfully into the cell, then it will express the non functional β-galactosidase (Copeland et al., 2001). Plate the cells out onto selection media based on the selectable marker, IPTG and X-gal and the white colonies are the ones with the recombinant DNA and the blue ones are having the plain plasmids without insert thus, expressing the β-galactosidase because, the lacZ is not disrupted.
AMP RESISTANCE SELECTION:
The Ampicillin resistance in the E.coli species is used to screen many genes in ampicillin selection medium (containing 50 - 100 mg/ml of ampicillin). Here, a gene named bla (TEM-1) is inserted into the artificial plasmid and made to transformed into normal competent cell namely DH5α. Thus, the ordinary bacterium which lacks the insert gene along with the resistance gene cannot withstand the increase in the concentration of the antibiotic in the medium. Thus, the bacterium which contains the gene which may also contain the necessary gene of interest ought to survive in our selection medium. The major mechanism of resistance to β-lactum antibiotics in the gram negative bacteria results from the production of lactamases (Bibbal et al., 2007). Most of these are coded by the plasmid mediated blaTEM-1 gene. The continuous introduction of new lactum antibiotics with different activity spectra in human medicine has led to the selection od lactamase mutations, which confer resistance to newly developed β-lactum antibiotics with different activity spectra in human medicine has led to the selection of lactamase mutations, which confer resistance to the newly developed β-lactum antibiotics and so, this can make the bacteria to become less sensitive to ampicillin and other types of β-lactum antibiotics.
RESULT AND DISCUSSION:
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. pMA and pMB DNA samples were digested with BamH1, EcoRI, Pst1 and Xho1 in both single double digest to map their restriction sites. The correct results of double digest samples were got from group 3 since our samples had failed. λ HindIII and Ï•X174 markers are used in the restriction digestion experiments.
pMA has restriction sites of EcoRI, BamH1 and Pst1 when pMB has a restriction site for Xho1 as well. Based on restriction fragment size got from this samples that we got from the 1kb marker that is used in double digest gel we can categorise the fragments that are formed by the digest using the four enzymes EcoRI, BamHI, Pst1 and XhoI. When pMB was digested with Pst1 it produced fragments of 3.8kb and 1.2kb. pMB and PUC19 were digested with Pst1 and phosphate from 5' end of PUC19 were removed enzymatically for transformation process. While restricted fragment of pMA was isolated and estimated for vector and insert concentration (pMB was found at 0.0128pmoles/µl and PUC19 was found at 0.011pmoles/µl).
Ligations were carried out in 3 different ratios. These ligated fragments were transformed into competent cells and later identified by blue white selection. Blue white screening depends on LacZ protein with multiple cloning sites. When our gene of interest is inserted into lacZ gene, its functionality gets inactive. LacZ encodes for β-galactosidase which helps in metabolising Xgal to form 5-bromo-4chloro indole 3-acetic acid which is blue in colour.
If lacZ gene gets disrupted, β-galactosidase is not formed and hence white colonies are formed. Ampicillin is added in medium since PUC vector is resistance to it and IPTG is added to induce Lac operon. This is the basis for Blue/white selection. Usually expression of LacZ gene in PUC19 will not produce functional β-galactosidase. It has to be complemented with Host lacZ gene to produce a functional β-galactosidase. This whole process is known as alpha complementation. However in rare occasions white colonies may be formed in control plates which may be due to mutation or some bacteria with ampicillin resistance may also grow and produce negative results. On the other hand, also some blue colonies may also have insert which is hidden due to repair mechanism of cells.
There was no contamination in transformation. The transformation was successful to 100%. But one of the sample seemed to be repeated and so we used the gel picture of group 3 to manipulate the sizes of the restriction fragments. White colonies from the transformed samples were selected and restricted with Pst1 and analysed by gel electrophoresis for ligation confirmation. This ligated DNA was then sequenced using known forward and reverse primers of PUC19 by thermo cycler. pMB also has kanamycin resistance. Also this shows that Pst1 restricts the kanamycin resistance gene. This pMB can be used to transfer kanamycin resistance to organism of our interest.
Results of double digests of pMA and pMB:
λ/HindIII and Ï•X HaeIII where used as markers and the pMB and pMA were restricted by EcoRI, BamH1, Xho1, Pst1 enzymes and the undigested pMA and pMB were also loaded into the gel. A standard graph has been plotted to find the molecular weight of the digested DNA. It is inferred that XhoI has no restriction site in pMA and PstI has two restriction sites in pMB.
The table shows the fragment length obtained by restriction enzymes. These details obtained about the fragments caused about various enzymes is analysed by keeping the standard graph which is plotted with the markers but later on we have inferred that there is some problem in the marker λ/HindIII and the total data expected to be wrong but however number of restriction sites we have noted for the restriction enzymes are correct and the number of bands also seems to be correct. The standard and the expected molecular weight has been mentioned in the table.
The Restriction maps of pMA and pMB:
The restriction enzyme PstI have been used to restrict both pMB and PUC19 it is identified that the molecular weight of the PUC19 which is restricted by PstI and produce a linear fragment of weight 3.6kb which is inserted with the pMB of 1.2kb with the help of DNA ligase these recombinant DNA got inserted to E.coli XL-1 Blue strain whose competency and transformation efficiency has been checked by previous experiments and these recombinant cells were allowed to grow in LB Ampicillin plates containing IPTG and incubated overnight. These cells were screened for the transformation results by colony colour. Blue colour colony suggests that the cell is non recombinant as the lacZ that is supposed to be cleaved when the insert is combined to the vector.
Thus, the blue colonies are selected in the samples plates and the transformation was successful which can be inferred such that the control plate has no white colonies. So the colonies are isolated and the DNA is taken and digested using PstI and run on agarose gel for testing whether the fragments that are supposed to be there are ther. i.e., the vector and the insert fragment. Which we can see in the below photograph which represents that the transformation was successful. Competent cells efficiency was found to be 1.448-106
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