2 Overview of Laboratory Responsibilities Tentative Schedule

·         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.

·         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.

·         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.

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

·         Determine protein concentration.

·         Determine if pure protein is active.

·         Purify ncAA-mutant proteins from cells.

·         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.

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