If I clone a complete eukaryotic gene, including the eukaryotic promoter region, ligate it into a plasmid, and transform it into E. coli, will I be able use the transformed E. coli to make the corresponding protein? Explain why, or why not? If you decide to do this, what would your cloning strategy be?

If I clone a complete eukaryotic gene, including the eukaryotic promoter region, ligate it into a plasmid, and transform it into E. coli, will I be able use the transformed E. coli to make the corresponding protein? Explain why, or why not? If you decide to do this, what would your cloning strategy be?

Human Heredity: Principles and Issues (MindTap Course List)

11th Edition

ISBN:9781305251052

Author:Michael Cummings

Publisher:Michael Cummings

Chapter13: An Introduction To Genetic Technology

Section: Chapter Questions

Problem 11QP: Cloning Genes Is a Multistep Process In cloning human DNA, why is it necessary to insert the DNA...

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You want to express the protein of a newly discovered human gene using E. coli, so you clone the human cDNA for the gene with an additional C-terminal purification tag into a plasmid. You then transform that plasmid into E. coli, grow up 1L of cells while inducing the promoter on the plasmid controlling the production of the protein from the gene. You then want to purify the protein using the C-terminal purification tag. What would be the best way of monitoring the purification process? Why did you choose this method? Please keep brief - 2 sentences/dot points max.
a）What two restriction sites are you going to use to clone your PCR product into the pL4440 plasmid? What are their DNA sequence? b) State the primer sequence you will use to amplify the F27C1.7 gene ready to be cloned into the pL4440 plasmid? c) How would you go about cloning this amplified DNA into pL4440? Using your knowledge of cloning list 5 important aspects of the method.
A molecular geneticist hopes to find a gene in human liver cells that codes for an important blood-clotting protein. He knows that the nucleotide sequence of a small part of the gene is CTCGACTCACA. Briefly explain how to obtain the desired gene. Briefly describe how to clone the desired gene into a cloning plasmid.
A) Outline the experimental procedure for cloning a eukaryotic gene and expressing it in E. coli. Focus on the essential steps starting with eukaryotic gene amplification to transformation of E. coli cells B) Explain how insertional inactivation can help you identify the colonies that carry the plasmid with your eukaryotic gene of interest C) Plasmids containing antibiotic resistance genes are widely used in gene cloning and other molecular biology techniques. What would happen if the eukaryotic gene was inserted into an antibiotic resistance gene on the plasmid?
Why do adult human cells (other than germ cells and stem cells) NOT express the enzyme telomerase? In other words what benefit does not having telomerase provide to these cells?
The human hexokinase enzyme has the same function as the bacterial hexokinase enzyme but is somewhat different in its amino acid sequence. You have obtained a mutant bacterial strain in which the gene for hexokinase is missing. If you introduce into your mutant strain a DNA plasmid engineered to contain the DNA coding sequence of the human hexokinase gene, what must you also include? a)The human hexokinase promoter b)The bacterial hexokinase promoter c)Both the human and bacterial promoters d)You cannot engineer a bacteria to produce a human enzyme
You were provided with the genetic sequence for one of these genes and asked to design a cloning strategy to generate a dsRNA expression construct to knock down that gene by RNAi . 1. What two restriction sites are you going to use to clone your PCR product into the pL4440 plasmid? What are their DNA sequence? 2. How would you go about cloning this amplified DNA into pL4440? Using your knowledge of cloning list 5 important aspects of the method.
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.
You want to clone a eukaryotic gene and express the corresponding protein in yeast. However, the protein typically localizes within mitochondria. How will you perform your gene cloning so that the protein is secreted from the cell, rather than localized within yeast mitochondria?
You’re working in a research lab, and your current task is to clone the gene that codes for tyrosinase from potatoes. You grind up some potato, extracts the DNA from it and digests the DNA with two different restriction enzymes (separately, not together): EcoRI and BamHI. You then obtain the cloning vector, pUC19, and digest it with the same two enzymes. You then run a gel which is shown here. You notice that the cloning vector made nice, tight bands on the gel, but the potato DNA just looks like a smear with no distinct bands. However, this is just what you expected. Explain why there are so many bands. Which enzyme would be the better choice to use for cloning the potato DNA, EcoRI, or BamHI? Explain why? Be specific.
As part of the steps necessary to clone a human gene with multiple introns (so it can be expressed in a bacterial cell, which does not have the enzyme for RNA splicing), the intron sequences have to be removed first. Explain how such an intronless human gene for cloning is made.
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