Creating a Cell Culture of Escherichia coli

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Objective

Students will be able to use the technique of cell culturing to produce a greater number of bacterial cells which carry the gene for a mutant florescent protein.

Big Idea

In cell culturing genes don't "lie" they "multiply"... and create PROTEIN!

Introduction

20 minutes

All of the lab investigations in this unit are based on nothing less than authentic Nobel Prize–winning science. Kary Mullis received the Nobel Prize for his discovery of the Polymerase Chain Reaction or PCR.  Werner Arbor, Daniel Nathans and Hamilton Smith received the Nobel Prize for their work with restriction enzymes. Stanley Cohen, Paul Berg and Herb Boyer received the highly esteemed prize for making the first recombinant DNA molecule. Actually, the recombinant DNA molecule that students create during this lab series extends beyond the scope of these honored Nobel Laureates original work as it incorporates a gene from a eukaryotic rather than prokaryotic organism into a plasmid.  It would be impossible to explain the effect these advancements and breakthroughs have had on the development of biotechnology, medicine, forensic science, and research in general however perhaps by experiencing the same triumphs as these giants the significance will become apparent. 

In this laboratory investigation, we will select one of the red colonies transformed with the p-ARA-R plasmid from the LB/amp/ara plates from Lab 5 Transforming E.coli with a Recombinant Plasmid and use the sample to inoculate an overnight culture.

The purpose of this lab is to start a bacterial culture that will produce a sufficient quantity of mutant fluorescent protein to enable your lab group to isolate and purify the protein.

I begin this lab by setting the context of the work we will be doing and providing background information as illustrated on SLIDES 12 thru 17 of the AMGEN Recombinant DNA Lab Series PowerPoint Presentation. At the end of this segment of our prelab discussion I check for understanding using a Pre Lab Quiz as well as visit the community lab area in our laboratory classroom in order to walk students through staging their workspaces or "bench" as shown in this photo. As we move about the community lab area and either stage or review staging that has already been completed, I explain the significance of each piece of equipment and how it will be used in our work. Students record notes from our discussion in their laboratory notebook as oftentimes the information provided appears on our Post Lab Quiz and Conclusion Questions.

Materials

10 minutes

REAGENT

LB/amp/ara plate with transformed colonies (from Lab 5 Transformation)

Sterile flask with LB/amp/ara broth

EQUIPMENT & SUPPLIES

Shaker

Clean toothpick or inoculating loop

Methods

45 minutes

For the Student:

Because we will be working with bacteria aseptic technique is critical. It was important that you and your labmates work quickly to avoid contamination.

1. Use a clean toothpick to transfer a few cells from a mFP-expressing colony (red) into a flask containing LB/amp/ara broth.

2. The flask will be placed on a shaker and shaken overnight to encourage both cell division and rfp expression.

Results

30 minutes

The purpose of this overnight cell culture is to clone the bacteria expressing the rfp gene and to have the organisms produce sufficient mFP to purify the protein from the other proteins in the cell. The result is a flask that appear red due to the mutant fluorescent protein located within structures in the cytoplasm of the bacterium. 

Conclusion

30 minutes

To end this lab investigation students individually answer the Lab 6 Conclusion Questions in preparation for a whole group discussion on the topics presented in this experiment.