Title: Lab: Purification of GFP
Course: BTEC-1000-301-302-303-F21 > Section: Protein Purification

Module 3 Purification of GFP

Lab

You’ll follow the protocol in the GFP Purification Student Manual to grow up some of the cells you transformed in Module 2. Recall that these E. coli have been transformed with the plasmid pGLO, allowing them to express GFP in the presence of the sugar arabinose. Starting on the page labeled 29 (or page 4 within this document), you will need to grow up two cultures of your E.coli transformed with pGLO, one from the LB/amp plate, and another from the LB/amp/ara plate.

Quick Check

Let’s test your knowledge: which plate should have functional GFP?

LB/amp

Go back and review the pGLO transformation lab.

LB/amp/ara

Yes! Arabinose is required to turn on expression of GFP

After you start your cultures, you will need to let them grow approximately 24 hours in the shaking 32 degree incubator. Write a note on the “Help, please!” board, and one of our staff will take out the culture and put it in the fridge for you.

On your second day in the lab, you will move on to page 33 in the GFP Purification Student Manual. Here, you will harvest the bacteria by centrifugation, resuspend them, and then add lysozyme to lyse (break open) the cells. (In the videos about industrial GFP production and purification, lysis was accomplished in a high pressure homogenizer.) On page 34 it says to freeze your tubes until the next class period, but if you’d like to continue on the same day, it’s also acceptable to freeze them for just 20 minutes, then proceed with the protocol. The rest of the protocol can be completed on the same day if you have time, or you can store your samples as directed until your next lab period.

As you complete this lab, keep in mind that you are doing a simplified version of the process that is used to purify human insulin and other drugs from E. coli!

Objectives

- Describe how protein purification is used in biotechnology.
- Purify a protein of interest.