Question

You wish to express a particular peptide in \(E\). coli using a high-copy-number plasmid. You have the amino acid sequence for the peptide. a. Explain the experimental process for generating and selecting the genetically engineered \(E\). coli using restriction enzymes, ligase, \(E\) coli, plasmid with neomycin resistance, and the known amino acid sequence. b. What control elements would you place on the plasmid to regulate expression and to prevent read-through?

Short answer

Introduce a DNA sequence into a plasmid, transform E. coli, select with neomycin, and use promoter and terminator for expression control.

Step-by-step solution

Digest the Plasmid and DNA Sequence

Use restriction enzymes to cut the plasmid and the DNA sequence encoding the peptide. Select restriction enzymes that create compatible ends to ensure that the plasmid and DNA sequence can be ligated together.

Ligate the DNA Fragment into the Plasmid

Use DNA ligase to join the DNA sequence encoding the peptide with the plasmid. This will form a recombinant plasmid containing the peptide sequence.

Transform the Recombinant Plasmid into E. coli

Introduce the recombinant plasmid into E. coli cells via a process called transformation. This can be done using heat shock or electroporation.

Select for Successfully Transformed E. coli Cells

Plate the transformed E. coli cells on agar plates containing neomycin. Only the cells that have successfully taken up the recombinant plasmid will survive, as the plasmid contains a neomycin resistance gene.

Control Elements for Regulating Expression

To regulate expression of the peptide, place a promoter upstream of the peptide coding sequence. Common choices include the T7 promoter or the lac promoter. Additionally, include a terminator sequence downstream of the coding sequence to prevent read-through and ensure proper termination of transcription.

Verification of Clone

Verify the presence and correct orientation of the inserted peptide sequence in the plasmid through techniques such as colony PCR or restriction digestion followed by gel electrophoresis.

Key concepts

Restriction Enzymes

To successfully express a peptide in E. coli, the first step involves using restriction enzymes. These enzymes act like molecular scissors, cutting DNA at specific sequences. You start by finding restriction sites on both your high-copy-number plasmid and the DNA sequence encoding your peptide. Common restriction enzymes include EcoRI, HindIII, and BamHI. The goal is to create 'sticky ends,' which are single-stranded overhangs that allow the cut DNA pieces to join more easily. Always ensure the restriction enzymes you select create compatible ends on both your plasmid and DNA sequence for efficient ligation.

Plasmid Ligation

After using restriction enzymes, the next step is to insert the cut DNA sequence into the plasmid. This is done through a process called ligation. DNA ligase is the enzyme that 'glues' the DNA fragments together by forming phosphodiester bonds between the sticky ends of the cut fragments. This results in a recombinant plasmid, a circular DNA that now includes your peptide sequence. Successful ligation is essential for the plasmid to carry and express the desired sequence when introduced into E. coli.

Transformation

Transformation is the process of introducing your recombinant plasmid into E. coli cells. Two common methods are used: heat shock and electroporation. In the heat shock method, cells are treated with calcium chloride to make their membranes permeable. Then, a sudden increase in temperature allows the plasmid to enter the cells. Electroporation involves applying an electrical field to the cells, which creates temporary pores in their membranes, allowing the plasmid to slip through. This step is crucial for the E. coli to acquire the ability to produce your peptide.

Neomycin Resistance

Selecting successfully transformed E. coli cells is done using neomycin resistance. The plasmid carrying your peptide sequence also includes a gene that provides resistance to the antibiotic neomycin. After transformation, E. coli cells are plated on agar containing neomycin. Only the cells that have taken up the plasmid will survive and grow. This allows you to selectively amplify the bacteria that contain your recombinant DNA.

Promoter Regulation

To control the expression of your peptide, a promoter sequence is required upstream of the peptide coding region. The promoter acts as an 'on' switch for transcription in the cell. Commonly used promoters include the T7 promoter, which is induced by T7 RNA polymerase, and the lac promoter, which is regulated by lactose or IPTG (a lactose analog). Carefully choose the promoter based on the desired level and control of expression. This step is vital for ensuring that the peptide is produced in adequate amounts at the right time.

Terminator Sequence

Finally, include a terminator sequence downstream of your peptide coding region to ensure proper termination of transcription. The terminator signals the RNA polymerase to stop transcription and release the newly synthesized mRNA. This prevents 'read-through,' where transcription would continue unabated, potentially producing unintended and disruptive sequences. Two common terminator sequences in bacterial systems are the rho-independent and rho-dependent terminators. Including a strong terminator makes sure your peptide is expressed correctly and efficiently.