Genetic Transformation

 ESSP 241 L Kibak
Expression of a foreign protein in E. coli  
 
   
 		  
 
In this three part laboratory procedure you will first introduce a DNA plasmid into a bacterium, then grow a culture of the transformed bacteria and prepare the purification columns, and finally purify the protein encoded by that foreign DNA.
 
 
 
Today we will be working with human enteric bacteria. Wear gloves, and... ABSOLUTELY NO FOOD OR DRINK in the Lab
 
Overview:  The plasmid we are using is a ring of DNA with an origin of replication, an antibiotic resistence gene, a set of regualtory genes that allow us to turn on or off the production of GFP based on whether or not there is a sugar called arabinose in the cell's environment, and of course the gene for the GFP itself. We are going to prepare some E coli cells to take up DNA by immersing them in CaCl2 on ice, then add the plasmid and let them sit some more on ice.  We will then heat shock the cells briefly and put them back on ice for a short while. Finally we will feed the starving cells and let them start growing a bit.  The ones that took up the plasmid will begin to express the antibiotic resistance enzyme (beta-lactamase).  When we put them on the plates with ampicillin, only the plasmid-containing cells should continue to grow.  Furthermore, only the plasmid containing cells on the plate with arabinose should glow or grow.
 

Today you will first examine your four plates and record your observations. 

You will then pick one white colony from your LB/amp plate and one green colony from your LB/amp/ara plates and grow cultures of each at Room Temperature for two days before storing at 4° C until next week.

You will also prepare the columns you will use next week during the purification step.

 

Examine the plates you prepared last week.

  1. Remove the transformation plates from the refrigerator and untape them.  Examine the plates.  How many colonies do you see on the plates?
    • +pGLO/LB/amp
    • +pGLO/LB/amp/ara
    • -pGLO/LB/amp
    • -pGLO/LB
  2. Take the time to take notes and provide potential explanations for your observations.
  3. Complete the Lab 02 Worksheet.
  4. Identify several green colonies that are isolated (not touching) from other colonies on the LB/amp/ara plate. Also identify several white colonies on the LB/amp plate.


    5. Start the Protein Expression Cultures
  5. Label one tube containing LB/amp/ara growth media "G" and one "W". 
  6. Using a sterile loop, touch a green colony and immerse it in the "G" tube. 
  7. Using a new sterile loop, repeat for a white colony and immerse it in the "W" tube (be sure to pick only one isolated colony).  Both times spin the loop a bit to disperse the bacteria from the colony in the medium.
  8. Cap the tubes and let them grow as described. We will use these cultures next week. 


Set Up the Protein Purification Columns for Next Week
  1. Remove the cap and snap off the bottom of the pre-filled Hydrophobic Intereaction Column. Allow all of the liquid buffer to drain from the column (3-5 minutes) with a minimum of disturbance.
  2. Prepare the column by adding 2 x 1 mL aliquots of Equilibration Buffer to the top of the column carefully.  Drain the buffer to the 1 mL mark on the colum. 
  3. Cap the top and bottom and store the column in the refrigerator until next week.