Title: Graded Lab: BglA Expression, Purification, and Kinetics
Course: BTEC-2020-301-302-303-F21 > Section: Final Project and Exam

Module5Graded Lab: BglA Expression, Purification, and Kinetics

Instructions

As a final project for this course, you will express, purify, and determine kinetic parameters for the enzyme BglA. (You cloned the BglA gene in BTEC 1100 in preparation for this process.) BglA is from the soil bacterium Bacillus halodurans. It encodes a β-glucosidase, also known as cellobiase, which is involved in the breakdown of cellulose into glucose. Cellulose is found in plant materials, including inedible grasses, stems, etc., and glucose from cellulose can be fermented to make biofuels. For biofuel production, optimization of the breakdown of cellulose to glucose is desirable. In your experiments, you will test various conditions to see which are best for BglA’s efficiency.

There are four major steps in the final project.

1. Expression of BglA - inducing E. coli to make lots of BglA
2. Verification of BglA expression - by SDS-PAGE and western blot
3. Purification of BglA - involves cell lysis,  affinity chromatography (BglA’s his tag binds to a nickel column, performed on the FPLC), and dialysis
4. Kinetics of BglA - you will determine the turnover rate of BglA under a set of standard conditions, and some other condition of your own choosing.

Protocols for the major steps are below.

Submit a document that includes the following items. All gels, graphs, etc. must be well-labeled (including sizes of ladder bands) and clear.

1. SDS-PAGE that shows expression of BglA in uninduced cells, and 1, 2, and 3 hours after induction
2. Western blot version of the gel above
3. FPLC profile with BglA-containing peak identified
4. SDS-PAGE of FPLC fractions
5. Bradford assay standard curve and the concentration of your BglA
6. Kinetics data
  1. Day 1 Abs vs. time, all 6 cuvettes on one graph, with linear curve fits and R^2 values
  2. Day 1 V vs. [E], with linear curve fit and R^2 value
  3. Day 2 Abs vs. time for standard conditions, all 6 cuvettes on one graph, with linear curve fits and R^2 values
  4. Day 2 Abs vs. time altered condition, all 6 cuvettes on one graph, with linear curve fits and R^2 values
  5. Day 2 V vs. [S], both conditions on one graph so they can be compared (do not fit a curve, as they should be hyperbolic and Excel can’t fit that)
  6. Day 2 1/V vs. 1/[S], both conditions on one graph so they can be compared, with linear curve fits and R^2 values
  7. Calculations for V_max and k_cat for both conditions, with appropriate units
  8. A written statement of how the condition affected k_cat, and what that implies for using BglA in biofuel production

You must achieve a 80% or better on the final exam and 80% for each criteria in the grading rubric on the final lab to continue in the next biotechnology course.

Objectives

- Express protein of interest.
- Determine the turnover rate of an enzyme in particular conditions.