Question

You wish to develop a genetically engineered \(E\). coli producing a peptide hormone. You know the amino acid sequence of the peptide. Describe the sequence of steps you would use to obtain a culture expressing the gene as a peptide hormone.

Short answer

Translate the amino acid sequence into DNA, design and synthesize the gene, clone it into a plasmid, transform E. coli, select transformants, and confirm expression.

Step-by-step solution

Determine the DNA sequence

Translate the known amino acid sequence of the peptide hormone into its corresponding DNA sequence using the genetic code.

Design the gene

Include additional sequences needed for expression in bacteria such as a promoter, ribosome binding site, and a terminator. Consider codon optimization for expression in E. coli.

Synthesize the gene

Use a gene synthesis service or PCR to construct the gene according to the designed sequence.

Clone the gene into a plasmid

Insert the synthesized gene into an appropriate plasmid vector that can replicate within E. coli and includes a selection marker.

Transform E. coli

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

Select successful transformants

Grow the transformed E. coli on selective media containing an antibiotic to ensure that only cells containing the plasmid vector survive.

Confirm gene expression

Verify that the E. coli is expressing the peptide hormone using techniques such as PCR, RT-PCR, or protein assays.

Key concepts

DNA sequence translation

The first step in genetically engineering E. coli to produce a peptide hormone is to translate the amino acid sequence of the peptide into its corresponding DNA sequence. This is done by referencing the genetic code, which maps each amino acid to a set of codons (sequences of three DNA bases). For example, the amino acid Methionine is translated to the codon ATG. This process is crucial because DNA is the form of genetic material that can be manipulated, synthesized, and ultimately expressed in a bacterial system like E. coli.

The DNA sequence determines the arrangement of nucleotides that will be later synthesized and inserted into a plasmid vector. This sequence must be accurate to ensure the correct peptide is produced during expression.

Remember:

• Each amino acid corresponds to specific DNA codons.
• A correct translation of the amino acid sequence is essential.

Gene synthesis

After translating the peptide sequence into DNA, the next step involves designing and synthesizing the gene. This is more than just the coding sequence for the peptide; additional sequences are needed for successful expression in E. coli.

Key elements include:

• **Promoter**: A DNA sequence at which RNA polymerase can bind and start transcription.
• **Ribosome Binding Site (RBS)**: Ensures the mRNA can be efficiently translated by ribosomes.
• **Terminator**: Signals the end of transcription.

Codon optimization ensures the gene uses codons preferred by E. coli, enhancing expression efficiency. Once the gene is designed, it can be synthesized using a gene synthesis service or PCR. The synthesized gene contains all the necessary sequences for its insertion into E. coli.

E. coli transformation

With the gene synthesized and cloned into a plasmid, the next step is to introduce this plasmid into E. coli. This process is called transformation. There are commonly used methods to achieve this:

• **Heat shock:** Briefly exposing E. coli cells to a sudden increase in temperature helps them take up the DNA.
• **Electroporation:** An electric pulse creates temporary pores in the cell membrane, allowing the plasmid to enter.

Both methods increase the permeability of the bacterial cell membrane, enabling the plasmid DNA to enter the cell. Successfully transformed cells can then start utilizing the inserted gene for peptide hormone production.

Plasmid cloning

Plasmid cloning involves inserting the synthesized gene into a plasmid vector. A plasmid is a small, circular piece of DNA that can replicate independently within the bacterial cell.

Important elements on a plasmid include:

• **Origin of Replication (ORI):** Allows the plasmid to replicate within the bacterial cell.
• **Selection Marker:** Typically an antibiotic resistance gene to identify and select successful transformants.

During cloning, restriction enzymes are used to cut both the plasmid and the synthesized gene, creating compatible ends for ligation. DNA ligase is then used to join these ends, forming a recombinant plasmid. This recombinant plasmid contains the gene necessary for peptide hormone production and can be introduced into E. coli during transformation.

Gene expression verification

After transformation, it is essential to verify that E. coli has successfully taken up the plasmid and is expressing the gene for the peptide hormone. Verification can be done through several methods:

• **PCR** (Polymerase Chain Reaction): Amplifies specific DNA sequences to confirm the presence of the gene.
• **RT-PCR** (Reverse Transcription PCR): Converts RNA transcripts back into DNA to check the gene's transcription.
• **Protein Assays:** Detects the presence and amount of the peptide hormone produced.

These techniques confirm whether the genetically engineered E. coli is expressing the desired peptide hormone. This step is critical to ensure the success of the genetic engineering process and can help identify areas for improvement in the synthesis, cloning, or expression stages if necessary.