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Chapter 20: Problem 4

The human insulin gene contains a number of sequences that are removed in the processing of the mRNA transcript. In spite of the fact that bacterial cells cannot excise these sequences from mRNA transcripts, explain how a gene like this can be cloned into a bacterial cell and produce insulin.

Short Answer

Expert verified
Question: Explain how a human insulin gene containing intron sequences can be cloned into a bacterial cell and produce functional insulin, considering bacteria lack the machinery to properly process eukaryotic mRNA transcripts.

Step by step solution

01

Isolate and understand the human insulin gene

First, isolate the human insulin gene from the genomic DNA. To accomplish this, perform PCR or use restriction enzymes to cut the DNA at specific sites surrounding the insulin gene. Then, analyze the DNA sequence of the insulin gene to identify the exons (protein-coding regions) and introns (non-coding regions that must be removed).
02

Remove introns and create cDNA

As bacterial cells lack intron-splicing machinery, use a technique called reverse transcription to generate a complementary DNA (cDNA) copy of the mature mRNA, which contains only exon sequences. To do this, isolate mRNA by creating an mRNA transcript of the insulin gene in a eukaryotic cell and using poly-dT beads or columns to separate mRNA from other RNA species. Next, use a reverse transcriptase enzyme to generate the cDNA.
03

Clone the cDNA into a bacterial expression vector

With the cDNA of the human insulin gene in hand, clone it into a bacterial expression vector, such as a plasmid. This vector must include regulatory elements to control gene expression, like a promoter and a ribosome-binding site compatible with bacterial cells. This ensures the correct expression of the insulin gene in bacterial cells.
04

Introduce the recombinant plasmid into bacterial cells

Transform bacterial cells (typically, E. coli) with the recombinant plasmid containing the insulin cDNA. Use a laboratory technique such as electroporation or heat-shock to introduce the foreign DNA into the bacterial cells, allowing them to take up the recombinant plasmid.
05

Select for successfully transformed bacterial colonies

To find the bacterial colonies that successfully took up the recombinant plasmid, use a selectable marker, such as antibiotic resistance, included in the expression vector. Plate transformed bacteria on agar containing the appropriate antibiotic and only the transformed cells with the recombinant plasmid will survive and form colonies.
06

Expression of recombinant insulin protein

Grow the selected bacterial colonies that have the recombinant insulin plasmid and induce gene expression under optimized conditions. The bacterial cells express the insulin cDNA from the recombinant plasmid, producing the functional human insulin protein, which can then be isolated and purified for further use.

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Most popular questions from this chapter

How do next-generation sequencing (NGS) and third. generation sequencing (TGS) differ from Sanger sequencing?

Assume you have conducted a DNA sequencing reaction using the chain- termination (Sanger) method. You performed all the steps correctly and electrophoresced the resulting DNA fragments correctly, but when you looked at the sequencing gel, many of the bands were duplicated (in terms of length) in other lanes. What might have happened?

In this chapter we focused on how specific DNA sequences can be copied, identified, characterized, and sequenced. At the same time, we found many opportunities to consider the methods and reasoning underlying these techniques. From the explanations given in the chapter, what answers would you propose to the following fundamental questions? (a) In a recombinant DNA cloning experiment, how can we determine whether DNA fragments of interest have been incorporated into plasmids and, once host cells are transformed, which cells contain recombinant DNA? (b) When using DNA libraries to clone genes, what combination of techniques are used to identify a particular gene of interest? (c) What steps make \(P C R\) a chain reaction that can produce millions of copies of a specific DNA molecule in a matter of hours without using host cells? (d) How has DNA sequencing technology evolved in response to the emerging needs of genome scientists?

What is quantitative real-time PCR (qPCR)? Describe what happens during a qPCR reaction and how it is quantified.

The introduction of genes into plants is a common practice that has generated not only a host of genetically modified foodstuffs, but also significant worldwide controversy. Interestingly, a tumor-inducing plasmid is often used to produce genetically modified plants. Is the use of a tumor-inducing plasmid the source of such controversy?
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