Lecture 13

Introduction to Plasmids

• Plasmids Overview

  • Natural DNA structures found mainly in bacteria.

  • Serve as extrachromosomal DNA that can carry additional genes.

  • Function: Transfer genes, confer virulence, and provide antibiotic resistance.

• Structure of Plasmids

  • Typically divided into two halves:

    • Bacterial Side: Contains elements necessary for bacterial replication and survival.

      • Origin of Replication: Critical for plasmid DNA replication.

      • Incentive Mechanism: Bacteria earn antibiotic resistance by harboring the plasmid.

    • Researcher Side: Contains elements for gene manipulation and experiments.

      • LacZ Gene: Produces enzyme that splits lactose; disruption indicates successful DNA insertion (blue/white screening).

      • Inserts disrupt LacZ activity, resulting in white colonies, signifying the presence of introduced DNA.

Chapter 2: Synthesizing RNA

• Key Genes in Plasmids

  • Antibiotic resistance marker: Ensures bacteria maintain the plasmid.

  • LacZ gene disruption verifies successful DNA insertion during cloning (ectopic expression leading to blue/white screening).

• Plasmid Applications

  • Sequence DNA (less common now due to next-gen sequencing).

  • RNA synthesis for proteins and genetic engineering.

• Sequencing with Plasmids

  • Historical Context: Previously utilized for sequencing prior to the advent of more efficient next-gen technologies.

  • Primer Walking: Technique used for sequencing that has been largely replaced due to limitations compared to newer methods.

Chapter 3: Viral Messenger RNA

• Bacterial Viruses:

  • T7 and SP6: Viral polymerases that drive transcription from their respective promoters.

• Restriction Sites: Regions where endonucleases cut; critical for termination of transcription during RNA synthesis.

• Transcription Process

  • The ribosomal binding sites and terminators influence RNA production.

  • Antisense probes created for targeting mRNA during detection experiments (like in situ hybridization).

  • Example Uses: Monitoring viral RNA presence and transcriptional regulation.

Chapter 4: Detecting Messenger RNA

• Detection Techniques:

  • Probes and PCR: Alternatives to antibody-based methods such as Western blots for detecting mRNA expression.

  • Antisense Probes: Used to bind specifically to target mRNA; detection through fluorescence or radioactivity.

  • Sense Control: Ensures the specificity of the detection system; sense mRNA lacks a complementary binding partner.

• Protein Synthesis Implications:

  • RNA synthesized for probes can also lead to direct protein production.

  • Expression Systems: Considerations for producing proteins in prokaryotes vs. eukaryotes require different mRNA modifications (5' cap for eukaryotes, ribosomal binding site for prokaryotes).

Chapter 5: Conclusion

• Plasmids in Experimental Research:

  • Overview of plasmid components:

    • Similarities in structure between prokaryotic and eukaryotic systems but with distinct functional elements (promoters, terminators, and operators).

  • Inducible Systems:

    • Lac operon system for transcription regulation through controlled expression.

• Applications in Protein Research:

  • Plasmids serve as vehicles for studying proteins, gene modifications, and transient gene expression in eukaryotic systems.

  • Techniques for modifying DNA, including adding tags to proteins or making specific mutations.