Pre-lab Introduction

Outline of Practical

Practical Title: Using Bacteriophages to Make Mutations in Bacterial SpeciesStructure of Practical:

• Introduction: Overview of the practical, including objectives and significance in microbial genetics.

• Background to the Hybrid Practical (Week 31): Contextualize the experiment's purpose within the field of bacteriophage research and its implications for genetic engineering.

• Section 1: Identify the Cultures

• Section 2: Regrowth Assay

• Section 3: Plaque Assay: Procedures used to identify bacteriophage activity and select optimal strains for experimentation.

• Pre-practical Lecture (Week 33): A session designed to prepare students for the practical by discussing methodologies and expected outcomes.

• Section 4: Transduction (Week 34): Introduces the mechanism of gene transfer between bacteria via bacteriophages.

• Section 5: Mutagenesis: Focus on creating specific mutations and the tools utilized in evaluating their success.

Guidelines:

• Comprehensive guidelines on the practical write-up and research ethics.

• Weight: The practical accounts for 40% of the final mark, emphasizing its importance in the curriculum.

• Deadline: 12 noon Tuesday 6th May 2025

Aims & Learning Outcomes

Aim: To create 6 gene knockout mutations in Escherichia coli using the bacteriophage P1vir to facilitate studies on gene function and microbial resistance.Learning Outcomes:

• Develop in-depth knowledge in bacterial and bacteriophage genetics, understanding their interactions and implications in biotechnology.

• Acquire essential techniques in microbiology and virology, including culture practices and genetic manipulation.

• Gain skills in molecular biology techniques, such as PCR, cloning, and sequencing.

• Engage in experimental planning and design, including hypothesis formulation and data analysis.

• Enhance intellectual development, decision-making, and logical reasoning through practical problem-solving abilities.

Note: The practical includes significant problem-solving elements and requires comprehensive reasoning and external reading to achieve complete understanding and execution.

Introduction to Viruses

Virus Host Specificity:

• Different viruses exhibit a high degree of specificity for certain host cells, which typically belong to closely related strains or species within a given kingdom.

• Understanding host specificity is crucial for the application of bacteriophages in targeted therapies.

Bacteriophage Definition:

• A bacteriophage is a virus that specifically infects bacteria, playing a pivotal role in regulating bacterial populations in various ecosystems.

General Structure:

• Detailed structural characteristics of bacteriophages, including the capsid, tail fibers, and genome type.

• References to transduction processes previously discussed in Dr. Alessia Buscaino’s Week 28 lecture.

Bacteriophage Attachment

Attachment Process:

• The attachment of a bacteriophage to a bacterial cell is mediated by specific cell-surface receptors—proteins that are integral to bacterial host functions, making them targets for infection.

• Understanding this interaction is essential for manipulating phage applications in biocontrol and therapeutics.

Phage Genome Entry:

• Most bacteriophages inject only their genetic material into the host cell, leaving the empty capsid outside—a process often referred to as the 'ghost' phase.

Bacteriophage Life Cycle

Life Cycle Types:

• Lytic Cycle: Involves the destruction of the host bacterial cell, leading to the release of new phage particles.

• Lysogenic Cycle: The phage DNA integrates into the host genome, allowing it to replicate along with the host without killing it.

Cycle Decision Factors:

• Environmental factors significantly influence whether a phage will enter the lytic or lysogenic cycle; for instance, a lytic response occurs when the host is under environmental stress or threat.

The Lytic Cycle

Process Steps:

1. Host Recognition and Attachment: Initial contact and binding to the specific receptors on the bacterial surface.

2. Genome Entry: The phage injects its genome, initiating the infection process.

3. Assembly of Phages: New phage components are synthesized and assembled.

4. Exit and Transmission: The release of progeny phages occurs as the host cell lyses, leading to the infection of new bacterial cells.

Progeny Definition:

• Offspring produced during the replication cycle of phages, capable of initiating new infections.

The Lysogeny Cycle

Temperate Phages:

• Example: Lambda phage, which can either cause lysis or reside as a prophage integrated into the host genome.

Quiescent State:

• In this state, the bacteriophage's genetic material remains dormant within the host, with the potential to reactivate under favorable conditions to resume the lytic cycle.

Types of Bacteriophages

Structural Features:

• Tail fibers, head (capsid), and the presence of dsDNA genomes, highlighting the complexity of the structural proteins involved (e.g., gp10A, gp10B).

Genomic Variability:

• Mutation rates and structural variations present across different bacteriophage types, which can influence their effectiveness in targeting specific bacterial strains.

Flow of the Practical

Section Overview:

1. Identify E. coli Cultures for practical usage through selective methods.

2. Regrowth Assay: Applied methodologies to ensure the viability of the chosen E. coli culture for subsequent sections.

3. Plaque Assay: Techniques used to determine the appropriate bacteriophage for experiments and measure their efficacy in lysis.

4. Transduction: Hands-on application using P1vir bacteriophage for genetic modification.

5. Mutagenesis Analysis: Rigorous evaluation of the defined guidelines to measure success in the practical write-up.

Identify Cultures

Aim:

• To determine which cultures are pure Escherichia coli strains suitable for manipulation in experiments.Method:

• Evaluate 10 sets of microscope slides, agar, and antimicrobial plates with cultured inoculations to ascertain purity and identity.

Identify the E. coli Strain

Aim:

• Choose suitable E. coli culture(s) (A, B, C) for transduction experiments based on defined characteristics.Data Provided:

• Raw OD600 (optical density 600) cell density data for comparative analysis, accessible via Moodle to support informed decision-making.

Identify the Bacteriophage

Aim:

• Select appropriate bacteriophage (A, B, or C) through analysis of results from plaque assays.Task:

• Calculate plaque-forming units (PFUs) to assist in decision-making regarding phage selection based on their effectiveness.

Transduction

Aim:

• Conduct 6 gene knockout mutations in Escherichia coli with P1vir bacteriophage for insights into gene function.Pre-practical Lecture:

• Scheduled for Thursday in Week 33, covering essential theoretical knowledge related to transduction processes.

Mutagenesis Analysis

Aim:

• Evaluate the success of six transduction experiments via quantitative and qualitative measures.Evidence Collection:

• Use laboratory data (e.g., colony counts on plates), PCR results, and online sequencing data for in-depth analysis of outcomes.Discussion:

• Engage in comprehensive results interpretation and context-based analysis of findings.

Guidelines

Practical Guidelines:

• Promote adherence to comprehensive guidelines during practice to clarify objectives and ethical considerations.Pre-practical Session:

• Attendance is highly recommended to enhance understanding and performance during practical lab sessions.

Summary

Preparation:

• Ensure readiness for practical session with all necessary materials and understanding of protocols.Additional Resource:

• Watch the "Bacteriophage Lysis and Lysogen" video on Moodle for an animated understanding of the cycles, enhancing theoretical knowledge with practical visualization.Next Step:

• Proceed to Section 1 - Identify the culture to initiate practical work.