Question

You wish to produce a small protein using E. coli. You know the amino acid sequence of the protein. The protein converts a colorless substrate into a blue product. You have access to a high-copy-number plasmid with a penicillin- resistant gene and normal reagents for genetic engineering. Describe how you would engineer \(E\). coli to produce this protein. Consider: source of donor DNA; regulatory elements that need to be included; how the donor and vector DNA are combined; how the vector DNA is inserted; and how you would select for a genetically engineered cell to use in production.

Short answer

Amplify donor DNA, ligate into plasmid, transform E. coli, and screen for blue product on penicillin agar.

Step-by-step solution

- Identify the Source of Donor DNA

Obtain the DNA that encodes the amino acid sequence of the desired protein. This could be from a genomic library, cDNA library, or synthesized directly if the amino acid sequence is known.

- Isolate and Amplify the Donor DNA

Use PCR (Polymerase Chain Reaction) to amplify the donor DNA fragment that encodes the protein. Confirm the sequence through sequencing.

- Prepare the Plasmid Vector

Select a high-copy-number plasmid vector with a penicillin-resistant gene. Cut the plasmid vector with restriction enzymes to create compatible ends for the insertion of the donor DNA.

- Incorporate Regulatory Elements

Ensure the plasmid contains necessary regulatory elements such as a promoter, ribosome binding site, and terminator specific for efficient gene expression in E. coli.

- Insert Donor DNA into Vector

Ligate the amplified donor DNA into the prepared plasmid vector using DNA ligase, creating a recombinant plasmid.

- Transform E. coli

Introduce the recombinant plasmid into competent E. coli cells through a transformation method such as heat shock or electroporation.

- Select for Transformed Cells

Plate the transformed E. coli on agar containing penicillin. Only cells that have taken up the plasmid with the penicillin-resistant gene will grow.

- Screen for Protein Production

Cultivate the colonies on a medium containing the colorless substrate. Identify colonies that produce the blue product, indicating successful expression of the protein.

Key concepts

plasmid vector

A plasmid vector is a circular DNA molecule that is used to introduce foreign genes into a host cell, like E. coli. Plasmids can replicate independently of the chromosomal DNA, which makes them useful tools for genetic engineering. They usually contain several key elements:

• Origin of replication (ori): This allows the plasmid to duplicate itself inside the host cell.
• Selectable marker gene: Example is the penicillin-resistant gene. It allows for the selection of cells that have successfully taken up the plasmid.
• Multiple cloning site (MCS): A region containing various restriction enzyme sites that make it easier to insert the donor DNA.

For your engineering task, you would use a high-copy-number plasmid to ensure that the foreign gene is present in many copies within the bacterial cells, thereby increasing the yield of the desired protein.

polymerase chain reaction (PCR)

Polymerase Chain Reaction (PCR) is a technique used to amplify specific DNA sequences. Here are the vital steps involved in PCR:

• Denaturation: The double-stranded DNA is heated to around 95°C to separate it into two single strands.
• Annealing: The temperature is lowered to about 50-65°C to allow primers to bind to their complementary sequences on the single-stranded DNA.
• Extension: The temperature is raised to around 72°C, enabling the DNA polymerase enzyme to synthesize a new strand of DNA by adding nucleotides to the primers.

Using PCR, you can isolate and amplify the donor DNA fragment encoding the protein. This amplified DNA can then be inserted into the prepared plasmid vector. This step is crucial for ensuring enough copies of the gene are present for subsequent steps.

protein expression

Protein expression refers to the process by which proteins are synthesized from the genetic code. In genetic engineering, it's essential to include proper regulatory elements to ensure efficient protein production. These regulatory elements include:

• Promoter: A DNA sequence where RNA polymerase attaches and begins transcription of the gene. The promoter must be recognized by bacterial transcription machinery.
• Ribosome Binding Site (RBS): A sequence on the mRNA that helps the ribosome bind and initiate translation.
• Terminator: A sequence that signals the end of transcription.

After inserting the gene into the plasmid and transforming the E. coli cells, the presence of these regulatory elements allows the bacterium to produce the target protein. Screening for colonies that express the protein can be done by observing the conversion of the colorless substrate into a blue product, signifying successful protein expression.