GC

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.