Week 27 BIOS5320 Pre-practical for Week 28

Mini-Project Overview

Title: Mini-Project 2 Pre-practical for Week 28

Week 26 Practical Summary

Key Focus Areas:

• Overview of Week 26 practical and comprehensive data analysis related to enzyme kinetics.

• Data analysis techniques:

  • Michaelis-Menten Plot: A graphical representation that helps determine kinetic parameters of enzymes.

  • Lineweaver-Burke Plot: A double-reciprocal graph used to derive the values of Km and Vmax, facilitating easier calculations of enzyme kinetics.

Preparation for Week 28 Practical

Importance of Record-Keeping:

• Maintaining up-to-date and accurate records in the lab book is crucial for reproducibility and tracking experimental progress.

• Demonstrators will conduct periodic checks of lab books during attendance to ensure compliance with record-keeping guidelines.

Record-Keeping Guidelines:

• Lab Book Usage:

  • It is mandatory to document every procedure and result in the lab book.

  • Students must use their lab books in every session to ensure completeness and accuracy of information.

Week 26 Experiments Completed

Summary of Experiments Conducted:

1. Determined the optimal volume of extract needed for supplements to achieve an initial reaction rate of 0.25 OD/min.

2. Varied substrate (o-nitrophenyl-β-D-galactopyranoside, ONPG) concentration in a lactase activity assay to investigate its effects on enzymatic activity.

Importance of Investigating Substrate Concentration

Purpose of Variation:

• Allows for the generation of kinetic graphs which facilitate the calculation of vital kinetic parameters:

  • Km (Michaelis Constant): Represents the substrate concentration at which the enzyme operates at half its maximum velocity, providing insights into the enzyme's binding affinity for the substrate. A low Km indicates high affinity, while a high Km indicates low affinity.

  • Vmax: The maximum rate of reaction achieved by the enzyme when the substrate is saturating, indicating the effectiveness of lactase supplements and their potential applicability in dietary contexts.

Experiments and Initial Rate Determination

Initial Rate Found:

• The initial reaction rate was established at 0.25 OD/min during the lactase activity assays, confirming enzyme activity under the tested conditions.

• Ongoing experiments involved adjusting the concentration of ONPG to further study its effect on enzyme kinetics.

Week 26 Data Analysis

During the laboratory session, collected data was uploaded pertaining to OD/min for all ONPG concentrations, which contributes to 2% of the overall mark.

• Students accessed and shared class data via Moodle under Section 4 – Mini Project 2.

At Home Tasks:

• Prepare an Excel-generated Lineweaver-Burke plot, ensuring the best-fit equation crosses the x-axis for accurate parameter estimation.

• Construct a Michaelis-Menten plot using the curve fitting program available on Moodle.

• Calculate Vmax and Km for the lactase supplements from both plots to compare efficiencies.

• Produce comparative graphs that illustrate class averages for Vmax and Km to assess overall class performance.

Checklist for Week 26 Data and Analysis

Items to Complete:

• Determine the concentration of lactase extracts for supplements A, B, C, and D, ensuring a thorough understanding of each supplement's performance.

• Identify molecular weights of lactase extract proteins for all supplements to evaluate their characteristics.

• Compile an annotated SDS-PAGE gel image for each supplement to visualize protein separation.

• Create a standard curve graph of molecular weights, including the equation and error bars for statistical accuracy, enabling further analysis of experimental results.

• Conduct Vmax and Km determinations for each supplement at varying pH levels to investigate optimal conditions for enzyme activity.

• Generate separate graphs that illustrate the effects of pH on lactase activity, highlighting how varying conditions impact enzyme efficiency.

Supplement Evaluation

Class Poll for Supplemental Choice:

• Students are invited to participate in a class poll via vevox.app with ID: 189-898-861 to discuss their preferred supplemental options.

Analyzing Raw Data

Conversion Process:

• Convert OD/min data into micromoles/min or nanomoles/min (reaction rate) utilizing appropriate equations based on the Beer-Lambert Law.

  • Beer-Lambert Law: Convert OD values to concentration using the formula:( c = \frac{OD \ component}{\epsilon l} )

  • Calculate the number of moles/min as follows:( \text{Number of moles/min} = c \times \text{total assay volume in liters} )

• Utilize ONPG as the artificial substrate, with a molar extinction coefficient for o-nitrophenate of 3055 M-1cm-1, and a standard pathway length (l) of 1 cm.

Velocity vs Substrate Plots

Michaelis-Menten Considerations:

• It is advisable to select substrate concentration values near the level that achieves ½ Vmax for optimal accuracy in kinetic measurements.

Curve Fitting for Michaelis-Menten

Use of Excel for Graph Creation:

• Conduct necessary calculations with caution, ensuring correct units are consistently used throughout all calculations.

Lineweaver-Burke Plot Creation

Graphing Steps:

1. Convert substrate concentrations and velocity values to their reciprocals (i.e., 1/[S] and 1/[V]).

2. Plot the graph; apply the line of best fit ensuring the regression line crosses the x-axis at the appropriate point.

3. Determine Km and Vmax from the equation of the regression line for accurate interpretations of enzyme kinetics.

Investigating pH Effects - Week 28

Objective:

• The primary goal is to determine the optimal pH for lactase enzyme activity, setting the foundation for further experimental recommendations.

Research Requirement:

• Conduct thorough investigations into existing literature focusing on the effects of pH on lactase activity to inform experimental designs.

Experiment Planning:

• Define buffer pH conditions, number of experimental repeats, and necessary controls for the upcoming investigations to ensure robust and reliable results.

Experiment Resemblance to Week 26

Methodology:

• Employ similar techniques used in Week 26, which involve examining substrate concentration using ONPG and measuring reaction rates via absorbance of o-nitrophenol.

• Convert OD/min values to moles/min for comprehensive analysis of enzyme performance.

Optimal pH Considerations

Experimental Design:

• Plan to alter the pH of a 50 mM sodium phosphate buffer (starting at pH 7.0) and select four additional pH levels ranging from 4 to 10 to compare lactase activity across a broad spectrum of conditions.

Chemicals for pH Change:

• Required reagents include 1M NaOH and 1M HCl for basicity and acidity adjustments, as well as 5M NaOH and 5M HCl, which must be handled in a fume hood for safety considerations.

• Ensuring consistency in conditions is vital by maintaining uniform ONPG concentrations across all trials to accurately assess the impacts of pH modifications.

Key Experimental Reminders

• Ensure all enzyme tests maintain a constant assay volume of 1 ml for reliable comparisons.

• Store enzyme solutions on ice prior to use to preserve enzymatic activity and prevent denaturation.

• Establish appropriate controls, including zero substrate and enzyme controls, to monitor background effects on measured values.

• Conduct all assays in triplicate and promptly verify results for accuracy and reliability.

Data Analysis for pH Effects

Graphs to Produce:

• Generate a series of graphs depicting pH vs. rate of reaction for each individual supplement as well as aggregated class averages to visualize overall trends in enzyme activity.

Handling Data Variability

• It is essential to comprehend class data variability during analysis, contributing to the development of robust data handling skills specific to the biosciences field.

Practical Report Preparation

Upcoming Week 29 Discussion:

• Key focus will be on in-depth data analysis derived from Week 28 findings, with practical write-up guidelines available on Moodle. The deadline for submission is Week 33 (21st March 2025).

• Note that drop-in sessions are scheduled for Weeks 30 and 31 for additional support and guidance as needed.

Week 20 Summary

Recap of Key Points:

• Overview of Week 26 practical and its data analysis highlights.

• Discussion surrounding the objectives for Week 28 practical, specifically focusing on pH investigations and comprehensive experimental planning.