OSN-K 2024 - No 06 - Biselmol

Summary

This video script delves into the process of gene expression and cloning in *E. coli*, specifically focusing on the use of a plasmid vector to express the protein Tobisin. The presenter explains the essential components of the plasmid, such as the origin of replication, selectable markers, and multiple cloning sites, and how these elements enable successful gene insertion and expression. Key points include the importance of promoter and terminator sequences, codon optimization, and the role of *E. coli* as a host organism for protein production. The script also addresses potential challenges in gene expression and highlights important concepts such as transcription, translation, and plasmid replication in different host cells.

Takeaways

• 😀 The script discusses a 2024 biology problem from the Indonesian Biology Olympiad (OSN) focusing on protein expression using plasmids in bacteria.
• 😀 The plasmid used for protein expression contains crucial components like an origin of replication, selectable markers, and multiple cloning sites (MCS).
• 😀 The process begins by using restriction enzymes to cut the plasmid and insert the gene of interest (TBC), followed by ligation to incorporate the gene into the plasmid.
• 😀 Promoter sequences are essential to initiate transcription in bacterial cells, allowing proper expression of the inserted gene.
• 😀 A terminator sequence is necessary to signal the end of transcription, ensuring proper gene regulation.
• 😀 The gene inserted into the plasmid should have an intact coding sequence, including start (AUG) and stop codons, for proper protein translation.
• 😀 The script emphasizes that bacterial cells express proteins more efficiently using cDNA (reverse transcribed mRNA), as it lacks introns present in the genomic DNA.
• 😀 Codon usage bias refers to the preference for specific codons by different species, and adjusting codon sequences can help optimize protein expression in the host organism.
• 😀 The selectable marker, often a resistance gene like ampicillin resistance, allows for easy identification of transformed bacterial colonies.
• 😀 The script highlights that plasmids optimized for bacterial expression cannot replicate in human cells due to differences in the origin of replication, preventing protein expression in human cells using these plasmids.
• 😀 The overall process outlined in the script involves cloning, transcription, and translation steps that result in the production of large quantities of the desired protein in bacteria.

Q & A

What is the primary purpose of the plasmid discussed in the transcript?

The plasmid is used as a vector for gene expression, specifically for expressing the Tobisin protein in E. coli cells.

Why is a high-copy-number plasmid important in this process?

A high-copy-number plasmid ensures that multiple copies of the plasmid are present in each bacterial cell, which leads to high levels of gene expression and the ability to isolate large amounts of the protein.

What role do restriction enzymes play in this genetic process?

Restriction enzymes cut the plasmid DNA at specific sites, creating openings where the target gene (in this case, Tobisin) can be inserted, allowing for the cloning of the gene into the plasmid.

What is the function of the 'selectable marker' in the plasmid?

The selectable marker, such as a gene for antibiotic resistance (e.g., ampicillin resistance), helps identify and select bacterial cells that have successfully incorporated the plasmid, as these cells can survive in media containing the antibiotic.

What is the significance of the promoter sequence in the plasmid?

The promoter sequence is crucial for initiating transcription, as it is the binding site for RNA polymerase, enabling the transcription of the inserted gene (Tobisin) into mRNA.

How does the 'terminator sequence' function in gene expression?

The terminator sequence signals the end of transcription, telling RNA polymerase to stop transcribing the gene, ensuring proper regulation of gene expression.

Why must the gene inserted into the plasmid be free of introns?

Bacterial cells lack the machinery to remove introns, so the inserted gene must be in the form of cDNA, which is synthesized from mature mRNA that already lacks introns.

What is 'codon usage bias,' and why is it relevant in this process?

Codon usage bias refers to the preference of certain codons in different species to encode the same amino acid. It's relevant because the plasmid must be optimized for the host species (E. coli) to ensure efficient protein expression.

Why can't the plasmid used for E. coli expression work in human cells?

The origin of replication (ori) of the plasmid is specific to E. coli, and human cells have a different ori, meaning the plasmid cannot replicate in human cells and cannot be used to express proteins in them.

What would happen if a bacterial colony containing the plasmid is grown in ampicillin media?

Bacterial colonies that contain the plasmid with the ampicillin resistance gene will survive and grow, while those without the plasmid will be killed by the ampicillin, allowing easy selection of the transformed bacteria.