Week 18 Introduction to Mini-project 2

Introduction to Mini-Project 2

Investigation of Lactose Intolerance Supplements

Practical Skills

• Accurate pipetting: Mastery of pipetting techniques to ensure precise measurements is crucial for experimental success.

• Planning and executing experimental work: Utilize a range of techniques, including:

  • Protein standard assay: Understanding protein concentration and quality.

  • Spectrophotometer: Measuring the absorbance of light by solutions to determine the concentration of substances.

  • Protein electrophoresis: Visualizing and separating proteins based on their size and charge.

  • Reaction kinetic assay: Analyzing the rates of enzyme reactions to inform about enzyme activity.

• Reporting experimental work: All findings must be compiled in a well-structured written report, adhering to scientific standards.

• Demonstrating computer skills: Data analysis and presentation skills using software like Excel, R, or GraphPad Prism are essential for summarizing findings.

• Critical thinking and problem-solving: Ability to interpret results, troubleshoot experiments, and devise solutions to unforeseen problems.

• Independent research skills: While collaboration with peers is encouraged, individual research on various lactase supplements is necessary to understand differing results and methodologies.

• Data Submission: Results must be submitted across three sessions for class aggregation, affecting final grades by 7.5%.

Project Outline: Investigation of Lactose Intolerance Supplements

• Aim: The mini-project aims to refine skills in:

  • Planning and executing investigations into lactase activity: Understanding the functionality of lactase supplements.

  • Data handling and presentation: Effectively summarizing and visualizing experimental results.

  • Formal report writing: Adhering to the scientific reporting structure.

Methodology:

• Recording Work: Maintain a meticulous lab book documenting all experimental procedures and observations.

• Data Analysis: Perform iterative data analysis and seek feedback from lab demonstrators to refine studies continuously.

Expected Outcomes:

• Independent Experiment Execution: Ability to conduct experiments autonomously.

• Enhanced Problem-solving Abilities: Improved capacity to address and resolve challenges encountered during research.

• Preparation of Formal Report: Culminating in a written report paralleling the comprehensive format of BIOS6000 final reports.

Understanding Lactose Intolerance

• Prevalence: Affects approximately 65% of the global population, varying significantly by ethnicity and geography.

• Pathophysiology: Results from insufficient breakdown of lactose due to low lactase enzyme activity, leading to gastrointestinal symptoms.

• Lactase Enzyme: A crucial enzyme for lactose digestion, characterized as a 160-kDa protein located in the brush border of intestinal epithelial cells.

Lactase Biosynthesis

• Genetic Encoding: Lactase is encoded by the LCT gene found on chromosome 2q21. The enzyme undergoes several processing stages:

  • Pre-pro-lactase: Initial inactive precursor.

  • Pro-LPH: The processed form post-endoplasmic reticulum cleavage, eventually maturing into functional lactase in intestinal cells.

Lactase Expression in Humans

• Developmental Regulation: High lactase levels present at birth decrease after weaning, resulting in varied levels of lactase in adults.

• Lactase Persistence vs Non-Persistence: Around one-third of adults retain lactase activity (lactase persistence), influenced by genetics and variations in milk consumption among populations.

Genetics of Lactase Persistence (LP)

• Inheritable Trait: Lactase persistence is an autosomal dominant trait influenced by genetic factors, particularly single nucleotide polymorphisms (SNPs) located near the LCT gene.

• Monitoring Lactase Activity: Can be measured through the increase of galactose in urine after lactose consumption.

Lactase Non-Persistence (LNP) and Lactose Intolerance

• Symptoms and Effects: Individuals lacking lactase mutations experience gastrointestinal discomfort, characterized by:

  • Diarrhea: Resulting from osmotic imbalances due to undigested lactose.

  • Fermentation: Leads to symptoms such as bloating, cramps, and flatulence due to microbial fermentation in the colon.

Clinical Management of Lactose Intolerance

• Nutritional Strategies: Historically included the avoidance of dairy; however, contemporary methods suggest moderated dairy intake is beneficial for nutrient retention (calcium and proteins).

• Exogenous Lactase: Supplementation with exogenous lactase (available in pills and capsules) can aid in the digestion of lactose during consumption.

Investigating Lactose Intolerance Supplements

• Project Aim: To evaluate the efficacy of commercially available lactase supplements using biochemical assays and to get comparative data from four selected products purchased online.

Weekly Investigation Plan

• Schedule:

  • Week 24: Extraction and assessment of lactase activity from supplements.

  • Week 26: Investigating the effects of substrate concentration on lactase activity.

  • Week 28: Observing the effects of pH on lactase activity.

  • Write-up Deadline: 21st March 2025 (Week 33). Preparatory exercises for practical sessions and documentation are mandatory.

Important Lectures

• Scheduled Topics: Key lectures are interspersed with practical exercises covering:

  • Techniques for optimizing lactase assays

  • Frameworks for running independent investigations

  • Strategies for effective report writing and data analysis.

Data Collection and Analysis

• Detailed Recording:

  • Concentration and molecular weight of protein extracts must be logged.

  • Use SDS-PAGE for visualization and to develop standard curve graphs.

  • Analyze enzyme kinetics to derive Vmax and Km values.

  • Weekly checklists need to be maintained for tracking progress towards the completion of the write-up.

Writing a Formal Report

• Data Organization: Maintain organized data throughout experimental phases.

• Report Structure: Final reports should include:

  • Abstract, Introduction, Methods, Results, Discussion, Presentation, References.

• Word Count: Maximum of 2500 words, excluding references.

• Weighted Contribution: Report is worth 55% of the module grade; significant time commitment estimated at 82.5 hours is necessary.

Report Breakdown

• Components:

  • Abstract (10%)

  • Introduction and Aims (15%)

  • Methods (10%)

  • Results (30%)

  • Discussion (20%)

  • Presentation and References (9%)

  • Data submission during labs (6%)

Preparation Before Practical Labs

• Essential Actions:

  • Watch all pre-practical training videos and carefully review the practical manual.

  • Conduct optional research on lactose intolerance and the specific assays to be used during the project.

  • Consider initiating the introduction for your report ahead of the practical sessions to save time later on.