2 Overview of Laboratory Responsibilities Tentative Schedule

Week Overview of Laboratory Responsibilities Tentative Schedule
Refer to separate schedule for specific assignment due dates
1 Perform a literature search on enzyme, its genetic sequence, and potential ncAAs and sites of incorporation.

·         Students’ laptops will be needed to perform a literature search on their enzyme, its genetic sequence, and potential ncAAs and sites of incorporation

·         Download and study crystal structure of enzyme

·         Investigate the ncAAs and sites that will be studied throughout the term

 

To prevent bottlenecks using equipment for kinetic assays, three groups in each section have been assigned carbonic anhydrase and three groups catalase.

2 Express wild-type protein and continue to develop hypothesis and plan experiments.

·         Mix autoinduction media and obtain E. coli cells with appropriate expression plasmids to start overnight protein expression of wild-type protein.

·         Develop experimental hypothesis based on the ncAAs and sites that your team is selecting to study.

·         Tell professor by Thursday, Week 2 at 5 pm which protein sites and ncAAs will be expressed so starter cultures can be prepared for class week 3.

·         Begin a literature search for assay procedures.

·         Harvest cells 48 hours after induction and store at -80 °C.

·         Prepare SDS-PAGE solutions.

·         Analyze wild-type protein production by crude SDS-PAGE.

3 Express ncAA-mutant and wild-type proteins, purify wild-type protein, and continue to develop assay(s).

·         Mix autoinduction media and obtain E. coli cells with appropriate expression plasmids to start overnight protein expression of ncAA-mutant proteins and wild-type protein.

·         Prepare protein purification buffers.

·         Understand assay procedure.

·         Purify wild-type protein from E. coli cells.

·         Make assay/storage buffer(s).

·         Desalt wild-type protein sample into assay/storage buffer.

·         Harvest cells 48 hours after induction and store at -80 °C.

·         Analyze ncAA-mutant protein production by crude SDS-PAGE.

4 Purify ncAA-mutant proteins and preliminary assessment of purified wild-type protein.

·         Analyze purified wild-type protein by SDS-PAGE.

·         Determine protein concentration.

·         Determine if pure protein is active.

·         Purify ncAA-mutant proteins from cells.

5 Preliminary kinetics assay of wild-type protein and preliminary assessment of purified ncAA-mutant proteins.

·         Begin preliminary kinetic assay trials with wild-type protein.

·         Analyze purified ncAA-mutant proteins by SDS-PAGE.

·         Determine protein concentrations.

·         Determine if pure protein is active.

6-9 Continue to design and execute studies on pure protein including testing of wild-type and ncAA-mutant proteins with kinetic assays.
10 Lab clean-up and Presentations in lab sections.

·         Final paper and notebooks due Monday of Finals Week at 4 pm.

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Chemical Biology & Biochemistry Laboratory Using Genetic Code Expansion Manual Copyright © 2019 by Ryan Mehl, Kari van Zee & Kelsey Keen is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, except where otherwise noted.