Concept explainers
Describe how the process of gene doning results in a cell clone contaning a recombinant plasmid.
To explain: The gene cloning process that produces a cell clone having a recombinant plasmid.
Concept introduction:
To study a particular protein, multiple copies of the gene fragment encoding that protein are synthesized. These gene fragments are then inserted into the expression vector to synthesize a large quantity of protein that is encoded by the gene. This is known as gene cloning.
Explanation of Solution
For cloning a eukaryotic gene, the bacterial plasmid vector and the gene segment to be cloned are cleaved with the same restriction sites. Fig. 1 shows the cloning procedure using EcoRI restriction enzyme as both the sequences have their restriction sites. The arrows between the bases in Fig. 1 represent the restriction sites. The restriction digestion of the linear gene sequence and the plasmid sequence produces sticky ends. The digested gene sequence is ligated into bacterial plasmid and the recombinant plasmid is synthesized. This recombinant plasmid is reintroduced into the host cells (bacteria), where the eukaryotic gene expresses and forms proteins. The bacterial cells containing the recombinant plasmids will divide to form cell clones.
Pictorial representation:
Fig. 1 shows the cloning procedure using a gene sequence and bacterial plasmid.
Fig. 1 Cloning procedure
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Study Guide for Campbell Biology
- With the use of well-illustrated diagrams, reconstruct the entire cloning process by explaining different stages of the cloning process that involves the following:a. Isolation of target DNA fragments (often referred to as inserts)b. Ligation of inserts into the plasmid, creating recombinant molecules c. Transformation of recombinant plasmids into bacteria or other suitable host for propagationd. Screening/selection of hosts containing the intended recombinant plasmid. For this stage(d), discuss the importance of a second marker that can be used for screening of genomic DNA for colonies containing the pka-1 under the principle of insertional inactivation. This should be properly explained using all the attributes of the plasmid described above.arrow_forwardexplain how Plasmids Carry Genes in Addition toThose in the Bacterial Chromosomearrow_forwardExplain how you would use the blue-white colony screening technique to identify bacteria containing the recombinant plasmidarrow_forward
- Explain how to identify mutant genes molecularly bytransformation with recombinant plasmids.arrow_forwardExplain what restriction enzymes are, how they function and how they can be used to make recombinant DNAarrow_forwardDescribe how restriction enzymes like EcoR1 are used to create recombinant plasmids and what the process is for using these plasmids to replicate a piece of target DNA. Include information about how to create sticky ends, the makeup of the bacterial plasmid and how to tell if the gene was successfully inserted in the plasmid and if the plasmid has been transformed by the bacteria. You may use a drawing to enhance your description.arrow_forward
- Explain why recipient cells do not successfully takeup plasmids during natural transformation.arrow_forwardExplain the Cleavage of DNA by the restriction enzyme EcoRI?arrow_forwardWhen E. coli cells are mixed with recombinant vector DNA and subject to a stress such as heat shock, a small fraction of the cells will take up the plasmid DNA, a process known as : A. Ligation. B. Transformation. C. Transfection. D. Digestion.arrow_forward
- a. Using nucleotide letters, show the kind of cut that could be made on a DNA molecule to circularize it into a plasmid. b. What are restriction length polymorphisms, and how are they used?arrow_forwardAfter cloning, they transformed and plate bacterial cells using their cloned plasmid. Onto what type of growth medium will they plate their cells in order to distinguish between bacterial cells that obtained the plasmid and those that did not?arrow_forwardUsing nucleotide letters, show the kind of cut that could be made on a DNA molecule to circularize it into a plasmid.What are restriction length polymorphisms, and how are they used?arrow_forward
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