Development of a reverse split-ubiquitin system to characterize protein-protein interactions

Table of Contents

1. Introduction

1.1 Autophagy

1.1.1 The lysosomal pathway

1.2 The Rab GTPase family

1.2.1 The glo-1 gene

1.2.2 The Rab cycle

1.3 Protein-protein interactions

1.4 Yeast Two Hybrid System

1.4.1 The Split Ubiquitin System

1.4.2 The rUra3p based split-ubiquitin system

1.4.3 Screening with the reverse split-ubiquitin system

1.5 Aim

2. Material and Methods:

2.1 Yeast strains:

2.2 Yeast transformation

2.3 Polymerase Chain reaction:

2.4 Ligation into the pGEMT vector

2.5 Cloning into target vectors:

2.6 Bacterial Transformation

2.7 DNA Mini-Preparation

2.8 Control digestion

2.9 Sequencing

3. Results

3.1 Creating vectors with TEF1 promoter

3.1.1 GLO-1 and C35B1.2a

3.1.2 C35B1.2a (Aa95-301)

3.2 Creating vectors with SSA1 and SSB1 promoter

3.3 Yeast Two Hybrid Assay

4. Discussion

Research Objectives & Topics

The primary aim of this bachelor's thesis is to establish and optimize a variant of the split-ubiquitin system to characterize protein-protein interactions (PPI) between the Rab GTPase GLO-1 and its effector protein C35B1.2a in yeast, focusing on the use of different promoters to regulate expression levels.

• Functional principles of the yeast two-hybrid and split-ubiquitin systems.
• Molecular cloning of bait and prey constructs using various promoters (TEF1, SSA1, SSB1).
• Analysis of protein interaction via yeast growth assays on selective media.
• Troubleshooting and optimization strategies for split-ubiquitin based protein interaction studies.

Excerpt from the Thesis

Summary of Chapters

1. Introduction: Provides an overview of autophagy, the Rab GTPase family, and the theoretical background of the yeast two-hybrid and split-ubiquitin systems.

2. Material and Methods: Details the experimental procedures including yeast transformation, polymerase chain reaction (PCR), cloning strategies, and DNA preparation techniques.

3. Results: Documents the successful construction of vectors with different promoters and the subsequent yeast two-hybrid assays conducted to test protein interactions.

4. Discussion: Evaluates the experimental outcomes, addresses potential reasons for the lack of observed interaction growth, and suggests further optimizations for the system.

Keywords

Split-Ubiquitin System, Yeast Two-Hybrid, Protein-Protein Interaction, GLO-1, C35B1.2a, Rab GTPase, TEF1, SSA1, SSB1, Molecular Cloning, Yeast Transformation, Autophagy, HIS3, Reporter Gene, N-end rule

Frequently Asked Questions

What is the core subject of this bachelor thesis?

The work focuses on developing and optimizing a reverse split-ubiquitin system to identify and characterize protein-protein interactions, specifically between the Rab GTPase GLO-1 and its effector C35B1.2a.

What are the primary research themes?

The main themes include protein interaction screening methods, molecular cloning techniques, the influence of different promoters on protein expression, and the troubleshooting of yeast-based interaction assays.

What is the central research question?

The study aims to confirm the interaction between GLO-1 and C35B1.2a and to determine whether replacing a strong promoter (TEF1) with weaker chaperonin promoters (SSA1, SSB1) can optimize the specificity of the split-ubiquitin system.

Which scientific methods were applied?

The research employed molecular cloning (PCR, ligation, bacterial transformation), yeast transformation, and yeast two-hybrid assays on selective media to detect protein interactions.

What is covered in the main section?

The main section describes the theoretical framework of the split-ubiquitin system, the materials and methods used for vector construction, the results of the test digestions and yeast growth assays, and a critical discussion of the results.

Which keywords characterize this thesis?

Key terms include Split-Ubiquitin System, Yeast Two-Hybrid, GLO-1, Rab GTPase, Protein-Protein Interaction, and promoter optimization.

Why was the TEF1 promoter replaced?

The TEF1 promoter was replaced because it proved to be too strong, leading to overexpression of the C-terminal ubiquitin fusion and preventing the proper degradation of the reporter gene, which caused false-positive growth signals.

What were the results regarding the interaction testing?

The interaction between GLO-1 and C35B1.2a could not be demonstrated in this study, as no growth was observed on selective plates containing 3-AT, suggesting potential issues with the system's sensitivity or the experimental setup.