Question

The right template. Ovalbumin is the major protein of egg white. The chicken ovalbumin gene contains eight exons separated by seven introns. Should one use ovalbumin cDNA or ovalbumin genomic DNA to form the protein in \(E .\) coli? Why?

Short answer

Use ovalbumin cDNA to ensure correct protein synthesis in \( E. coli \).

Step-by-step solution

Understand the Problem

We need to determine whether ovalbumin cDNA or genomic DNA should be used to produce the ovalbumin protein in \( E. coli \). This involves understanding the differences between cDNA and genomic DNA, and how protein synthesis works in prokaryotes like \( E. coli \).

Concept of cDNA

cDNA, or complementary DNA, is synthesized from a mature mRNA template. It therefore contains only exons, which are the coding regions necessary for protein synthesis, with introns already removed.

Concept of Genomic DNA

Genomic DNA contains the entire gene sequence including both exons and introns. In eukaryotes, introns are non-coding sequences that are removed during mRNA processing, a process that doesn't naturally occur in prokaryotes like \( E. coli \).

Protein Synthesis in E. coli

\( E. coli \) is a prokaryote and lacks the machinery to splice out introns from eukaryotic genomic DNA. If genomic DNA is introduced into \( E. coli \), the presence of introns would prevent proper protein synthesis.

Conclusion

To form the ovalbumin protein in \( E. coli \), one should use ovalbumin cDNA because it only contains the necessary exons for protein synthesis, and \( E. coli \) cannot process introns.

Key concepts

Protein Synthesis

Protein synthesis is a fundamental biological process by which cells build proteins. This process involves two main stages: transcription and translation. - **Transcription**: The first stage, where DNA is used as a template to create messenger RNA (mRNA). The mRNA then carries genetic information from the DNA out of the nucleus and into the cytoplasm of eukaryotic cells. In prokaryotes like _E. coli_, this all occurs in the same cellular compartment. - **Translation**: During this stage, the mRNA is read by ribosomes to assemble amino acids in the correct order to produce a protein. Molecules called transfer RNAs (tRNAs) bring the amino acids, which are added one by one to a growing polypeptide chain. In _E. coli_, like many other bacteria, protein synthesis is a streamlined process as they lack a cell nucleus, allowing transcription and translation to be coupled directly. This simplicity is why prokaryotes are popular systems for studying protein synthesis and are often used to produce recombinant proteins. To produce ovalbumin efficiently in _E. coli_, an understanding of protein synthesis is crucial, particularly the necessity to overcome the differences between prokaryotic and eukaryotic systems.

cDNA vs Genomic DNA

cDNA and genomic DNA have distinct roles and characteristics, which are crucial when expressing proteins in another organism, like _E. coli_. - **Genomic DNA**: This contains the complete set of genetic information, including exons (coding regions) and introns (non-coding regions). In eukaryotic cells, introns are removed through a process called splicing during mRNA formation. - **cDNA**: Stands for complementary DNA. It is synthesized from mRNA and thus only contains the exons necessary for protein production. As it's derived from mRNA, introns are already removed, mimicking the processed mRNA form. When producing proteins in _E. coli_, using cDNA is necessary because _E. coli_ do not have the machinery to remove introns like eukaryotes do. Therefore, cDNA should be the preferred substrate for heterologous protein expression in _E. coli_. Understanding this distinction helps ensure accurate protein synthesis without additional modifications.

E. coli Expression System

The _E. coli_ expression system is a widely used method in scientific research for producing proteins. This prokaryotic system offers several advantages: - **Speed and Simplicity**: _E. coli_ grows rapidly and cloning vectors are well-developed, making it a straightforward system for expressing proteins. - **Cost-Effectiveness**: Due to its rapid growth and ease of use, _E. coli_ provides an economical solution for producing large quantities of protein. However, it also comes with limitations, especially when expressing eukaryotic proteins: - **Lack of Splicing Machinery**: _E. coli_ cannot process introns found in eukaryotic genes, which is why cDNA is preferred over genomic DNA. - **Post-Translational Modifications**: It lacks the ability to conduct complex post-translational modifications, which might be necessary for some proteins to become fully functional. When expressing ovalbumin in _E. coli_, choosing the correct form of DNA (cDNA) is essential to account for these limitations and to facilitate successful protein synthesis.