Genetic Transformation

READ CAREFULLY...

Last week you picked a white colony from an LB/amp/plate and a green colony from an LB/amp/ara/plate and started two liquid cultures. After growing overnight at 37 C they were placed in the refrigerator until now.

The E. coli you grew have settled at the bottom of the culture tubes so, vortex the tubes vigorously to resuspend the bacteria.  Use the UV light to examine the cultures in relative darkness. Record any observations... What color does the "green" tube fluoresce? What color does the "white" tube fluoresce?

Label two microfuge tubes with your group intitials.

Using a P1000, transfer 1mL of your liquid culture into each microfuge tube. Remember, this blue tip is highly contaminated! Where should you dispose of it?

Centrifuge the microfuge tubes for 5 minutes at the maximum rpm (probably 13K). 

Remove the supernatant (biohazard) and observe the pellet under UV light. 

Using the P200, add 125 uL TE buffer to each tube.  Resuspend the pellets thoroughly by chopping it with the yellow pipet tip and pipetting the mixture up and down a few dozen times until there are no chunks of bacterial pellet left. Combine the two suspensions in one tube and vortex vigorously for 30 seconds. Save the remaining TE buffer for use later as the Elution Buffer.

Using a P200, add 60 uL of lysozyme (50 mg/mL) to the tube with the pellet suspension to initiate enzymatic digestion of the bacterial cell wall.  Mix the tube well initially and again every five minutes during the 15 minute incubation at 30° C.

After incubating 15 minutes at Room Temperature, pipet the mixture up and down carefully several times using a P1000 set to 0.4 mL.

Now place the tube in the -20°C freezer for 15 minutes.  The freezing hopefully causes the bacteria with their weakened cell walls to rupture completely.

Remove the microtube from the freezer, thaw it using hand warmth, and then vortex vigorously for 30 seconds.

Next centrifuge the tube for 10 minutes at maximum rpm to pellet the insoluble bacterial debris.

While the broken E. coli are spinning, prepare a fresh microfuge tube with 250 uL of Binding Buffer [4M (NH4)2SO4 ] and label it BB.

Also, use this centrifugation time to set up the column you prepared last week. You will not need a rack this time since the column is designed to fit into a culture tube which can be supported in an ordinary test tube rack.

Remove caps from top and bottom of the column and place the column in a culture tube labeled "waste" and let the buffer drip out. Label three more culture tubes #1-3. When the last of the buffer has reached the surface of the column bed, gently move the column over to tube #1.

After the 10 minute spin is finished, remove your tube from the centrifuge and examine it with the UV light. Again, record observations.  Hopefully the bacterial debris is a tight pellet at the bottom of the tube leaving the soluble proteins in the supernatant.

Using a p1000, transfer 250 uL of the ruptured E. coli supernatant into the microfuge tube labelled "BB" and mix well with the ammonium sulfate by pipetting up and down several times. Discard the tip in biohazard as some of the bacteria may not have ruptured.

Allow the solution to equilibrate 5 minutes.

Using a new pipette tip, carefully load 500 uL of the supernatant that has equilibrated with Binding Buffer into the top of the column by resting the tip of the pipette against the side of the column wall... and dialing the supernatant slowly out of the pipette so that it can drip down the side of the column minimally disturbing the column matrix.  Examine the column with UV and record observations.  Let the entire volume of supernatant flow into collection tube #1.

When it has finished dripping, transfer the column to tube #2.  Again, carefully add 250 uL of Wash Buffer [ 1.3M (NH4)2SO4 ] and let the entire amount flow into the column.  What do you predict will happen?  Record your observations.

When the Wash Buffer has finished dripping, transfer the column to tube #3.  This time, carefully add 750 uL of Elution Buffer (TE) (10 mM Tris/EDTA ) and let the entire amount flow into the column.  What do you predict will happen?  Record your observations. It can be fun at this stage to shine the UV light on each drop as it exits the column!

Save 250 uL samples of your four fractions (W), (1), (2), & (3) in labelled microfuge tubes in the freezer.

See the "Lab Write-ups for Bio 241L" handout.

Your Title could look something like this: "A prokaryotic system for transformation, expression, and purification of eukaryotic proteins."

Your Introduction should describe how transformation is accomplished, the arabinose operon and control of foreign protein expression from a plasmid, and the principle of Hydrophobic Interaction Chromatography.

Your materials and methods should be as the handout describes.

In this case your results will be a list of your observations at each step of the way.  This will be short and easy if you actually recorded your observations each time the handouts said, "Record your observations." :-) One extra step would be to describe how to calculate transformation efficiency.

The only discussion required will be your explanation of why the white non-fluorescing colonies on the plates could ultimately give rise to green fluorescence when grown in a tube at the next stage of the proceedure.