Genetic Transformation

 ESSP 241 L Kibak
Expression of a foreign protein in E. coli  
 
   
 		  
In this three part laboratory procedure you will introduce a DNA plasmid into a bacterium, grow a culture of the transformed bacteria, and purify the protein encoded on that foreign DNA.
 
About fifteen years ago, a group of scientists were looking for a way to see which DNA sequences upstream from genes were important in modulating the expression of genes at different times and in different tissues during development.  They came up with the possiblity of using a fluorescing protein from a jelly fish (Aequorea victoria) as a "reporter gene" in the system they were looking at (a small translucent roundworm).  You can read the original research paper here. Since that time green fluorescent protein (GFP) has been used in hundreds of experiments and in Taiwan you can actually buy "glowing" aquarium fish that have been transformed with this gene from jellyfish. Today we will take a plasmid with sequence that codes for GFP, and cause bacteria in a culture to take up the DNA. We will then start plates of transformed bacteria (and some controls) growing for use in the expression phase of this procedure.
 
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.

 

 

*small ring of DNA containing an origin of replication, cloning sites, and a few special genes

 

  
It is important not to cross-contaminate the "+" and "-" tubes today... use good sterile technique.
 
  •  Label one closed eppendorf tube "+" and the other "-"
  • Using a sterile blue pipet tip, transfer 250 uL transformation solution (CaCl2) into each of the tubes and place the tubes on ice.
  • Use a sterile loop to pick up a single colony of bacteria from your starter plate and suspend it in the transformation solution in you "+" tube.  Make sure the colony is completely dispersed in the transformation solution by using your loop as an egg beater. Do the same for the "-" tube.
  • Using a sterile pipet tip transfer 5uL of liquid from the plasmid DNA stock tube ONLY INTO THE "+" tube.  DO NOT ADD plasmid suspension to the "-" tube!!!  Why?
  • The tubes need to incubate in the ice for ten minutes. 
  • While your tubes are incubating, label your four agar plates on the bottom (no the lid):
    1. +pGLO/LB/amp
    2. +pGLO/LB/amp/ara
    3. -pGLO/LB/amp
    4. -pGLO/LB

*****The next step is fairly exacting, so do not proceed until you understand what you are doing.*****

  • You will now "heat shock" your bacterial cell suspension for exactly 50 seconds.  Take your ice bucket over to the waterbath and using the rack, set both your tubes into the 42° water bath for exactly 50 seconds and then right back into the ice.  This ice-warmwater-ice step must be done rapidly. Note the time you put them back in the ice because they are only staying there briefly.
  • Bring the bucket with the tubes back to your bench and after two minutes are up, take the tubes out of the ice.
  • Add 250 uL of LB nutrient broth to the tube, mix, and reclose it. Repeat with a new sterile tip for the other tube.
  • After incubating for ten minutes on the bench top at room temperature, mix them again and pipet 100 uL of the "+" tube to the two +pGLO plates and 100 uL of the "-" tube to the two -pGLO plates.  Use a different sterile pipet tip for each of the tubes.
  • Using a turntable and a sterile spreader bar for each of the plates spread the 100 uL suspension evenly across the surface of the agar. Don't leave them open too long...
  • Parafilm the edges of the plates, stack them up and tape them with your group name.  Place them upside down in the incubator.

The colonies were incubated overnight (~16 hours) at 37° C and placed in the refrigerator.