18. Molecular Genetic Tools

Genetic Cloning

18. Molecular Genetic Tools

Genetic Cloning

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Problem 31b

Textbook Question

You have cloned a gene for an enzyme that degrades lipids in a bacterium that normally lives in cold temperatures. You wish to transfer this gene into E. coli to produce industrial amounts of enzyme for use in laundry detergent.
You have managed to produce transgenic E. coli expressing mRNA of your gene, but only a low level of protein is produced. Why might this be so? How could you overcome this problem?

Verified step by step guidance

Step 1: Understand the problem. The gene for the lipid-degrading enzyme originates from a bacterium adapted to cold temperatures. When transferred to E. coli, the gene is expressed at the mRNA level, but protein production is low. This suggests a problem with translation or protein stability in the new host organism.

Step 2: Consider codon usage. Different organisms have varying preferences for codons that encode the same amino acid. If the gene from the cold-adapted bacterium uses codons that are rare in E. coli, the ribosome may struggle to efficiently translate the mRNA into protein. To address this, you could optimize the codon usage of the gene to match E. coli's preferred codons.

Step 3: Evaluate protein folding and stability. Proteins from cold-adapted organisms may require specific conditions to fold properly. E. coli, which grows at higher temperatures, might not provide the optimal environment for the enzyme to fold and function. You could try expressing the protein in E. coli grown at lower temperatures or co-expressing chaperone proteins to assist in proper folding.

Step 4: Assess promoter strength and regulatory elements. The promoter driving the expression of the gene might not be strong enough in E. coli, leading to low levels of mRNA and protein. You could replace the existing promoter with a stronger, E. coli-compatible promoter to enhance transcription and subsequent protein production.

Step 5: Investigate post-translational modifications. If the enzyme requires specific modifications (e.g., glycosylation) that E. coli cannot perform, the protein may be non-functional or unstable. In this case, you could consider using a different host organism, such as yeast, that can perform the necessary modifications.

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Key Concepts

Here are the essential concepts you must grasp in order to answer the question correctly.

Gene Expression Regulation

Gene expression regulation refers to the mechanisms that control the timing and amount of gene product (protein) produced in a cell. In bacteria like E. coli, factors such as promoter strength, ribosome binding sites, and transcription factors can significantly influence how efficiently a gene is expressed. If the cloned gene's regulatory elements are not optimal for E. coli, it may lead to low protein production.

Codon Optimization

Codon optimization involves modifying the DNA sequence of a gene to use codons that are more frequently used by the host organism, in this case, E. coli. Different organisms have preferences for certain codons, and using less common codons can result in inefficient translation and low protein yield. By optimizing the gene sequence for E. coli, one can enhance the translation efficiency and increase protein production.

Post-Translational Modifications

Post-translational modifications (PTMs) are chemical changes that occur to a protein after its synthesis, affecting its activity, stability, and localization. E. coli may not perform the same PTMs as the original organism from which the gene was cloned, potentially leading to an inactive or unstable enzyme. Understanding and engineering the necessary PTMs can help improve the functionality and yield of the expressed protein.

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Related Practice

Textbook Question

The gel presented here shows the pattern of bands of fragments produced with several restriction enzymes. The enzymes used are identified above the lanes of the gel, and six possible restriction maps are shown in the column to the right.

One of the six restriction maps shown is consistent with the pattern of bands shown in the gel.

The highlighted bands (magenta) in the gel were hybridized with a probe for the gene pep during a Southern blot. Where in the gel is the pep gene located?

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Textbook Question

One of the six restriction maps shown is consistent with the pattern of bands shown in the gel.

From your analysis of the pattern of bands on the gel, select the correct restriction map and explain your reasoning.

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Textbook Question

The RAS gene encodes a signaling protein that hydrolyzes GTP to GDP. When bound by GDP, the RAS protein is inactive, whereas when bound by GTP, RAS protein activates a target protein, resulting in stimulation of cells to actively grow and divide. As shown in the accompanying sequence, a single base-pair mutation results in a mutant protein that is constitutively active, leading to continual promotion of cell proliferation. Such mutations play a role in the formation of cancer. You have cloned the wild-type version of the mouse RAS gene and wish to create a mutant form to study its biological activity in vitro and in transgenic mice. Outline how you would proceed.

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Textbook Question

A widely used method for calculating the annealing temperature for a primer used in PCR is 5 degrees below the melting temperature, Tₘ(°C), which is computed by the equation 81.5+0.41×(%GC)−(675/N), where %GC is the percentage of GC nucleotides in the oligonucleotide and N is the length of the oligonucleotide. Notice from the formula that both the GC content and the length of the oligonucleotide are variables. Assuming you have the following oligonucleotide as a primer,

5′-TTGAAAATATTTCCCATTGCC-3′

Compute the annealing temperature for PCR. What is the relationship between %GC and? Why? (Note: In reality, this computation provides only a starting point for empirical determination of the most useful annealing temperature.)

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Textbook Question

You have cloned a gene for an enzyme that degrades lipids in a bacterium that normally lives in cold temperatures. You wish to transfer this gene into E. coli to produce industrial amounts of enzyme for use in laundry detergent.

How would you accomplish this?

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Textbook Question

Most of the techniques described in this chapter (blotting, cloning, PCR, etc.) are dependent on hybridization (annealing) between different populations of nucleic acids. The length of the strands, temperature, and percentage of GC nucleotides weigh considerably on hybridization. Two other components commonly used in hybridization protocols are monovalent ions and formamide. A formula that takes monovalent Na⁺ ions (M[Na⁺]) and formamide concentrations into consideration to compute a Tₘ (temperature of melting) is as follows:

Tₘ=81.5+16.6(log M[Na+])+0.41(%GC)−0.72(%formamide)

Given that formamide competes for hydrogen bond locations on nucleic acid bases and monovalent cations are attracted to the negative charges on nucleic acids, explain why the Tₘ varies as described in part (a).

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Textbook Question

Most of the techniques (blotting, cloning, PCR, etc.) are dependent on hybridization (annealing) between different populations of nucleic acids. The length of the strands, temperature, and percentage of GC nucleotides weigh considerably on hybridization. Two other components commonly used in hybridization protocols are monovalent ions and formamide. A formula that takes monovalent Na⁺ ions (M[Na⁺]) and formamide concentrations into consideration to compute a Tₘ (temperature of melting) is as follows:

Tₘ=81.5+16.6(log M[Na+])+0.41(%GC)−0.72(%formamide)

For the following concentrations of Na⁺ and formamide, calculate the Tₘ. Assume 45% GC content.

[Na⁺] % Formamide

0.825 20

0.825 40

0.165 20

0.165 40

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Textbook Question

About 1% of occurrences of nonautoimmune type 1 diabetes are due to loss-of-function alleles in the insulin gene. Individuals heterozygous for such mutations develop diabetes as infants or in the first few years of their lives. Outline how you might approach gene therapy for such a disease and what difficulties you might encounter.

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