Transformation with pGREEN Plasmid

          

 

Background Information

 

Bacterial transformation refers to the rare event when a bacterial host cell takes up outside DNA, and then maintains that DNA and reproduces it when the host cell DNA replicates.  The foreign DNA can be expressed to produce new proteins which can alter the traits of the bacterial host cell.

 

In this transformation experiment, we are using a plasmid pGREEN as a vector to carry DNA into the bacterial host cell.  The bacterial host cell is E. coli MM294 which is normally sensitive to (growth inhibited by) ampicillin.  Hence, if you plate E. coli MM294 on an LB/amp agar plate (LB agar with ampicillin added to medium), the cells would not grow.  However, if the bacterial cells are transformed by the plasmid pGREEN, the bacteria can grow on LB/amp because the plasmid carries a gene for resistance to ampicillin.  Hence, the LB/amp plates “select for” transformed cells.  In addition, this plasmid pGREEN has been engineered to carry the GFP gene (green fluorescent protein).  GFP gene is derived from the glowing jellyfish Aequorea victoria.  Thus when bacterial cells are transformed by the pGREEN plasmid, they acquire two new traits:  they can grow on LB/amp agar and they produce large amounts of GFP, detectable by a green glow to the colonies, especially when exposed to black light.

 

In our experiment, we will show that MM294 bacterial cells without the plasmid cannot grow on LB/amp and are colored white.  After the transformation experiment, we will plate on LB/amp and if cells take up the pGREEN plasmid, they will be able to grow on LB/amp and they should have a green glow as well.

 

Transformation is not just a tool for genetic engineers; it occurs in nature.  For instance, it is known that bacteria that enter the human body may carry plasmids with antibiotic resistance genes, and these plasmids may transform other bacteria in the body, thus “spreading” antibiotic resistance.  The widespread use of antibiotics “selects for” antibiotic resistant bacteria which have become a challenge in today’s hospitals.

 

 

 

 

Reagents                                                        Equipment and Supplies

E. coli MM294                                                inoculating loops

50 mM cold sterile calcium chloride                  micropipettors

pGREEN plasmid, 0.005 mg/mL                       15 mL Falcon tubes, sterile

LB broth                                                          beaker of ice

2 LB plates                                                       95% ethanol and hockey stick spreader (or sterile glass beads)

2 LB/amp plates                                               42 degree C. water bath

 

Method:  The plasmid pGREEN and the method are derived from the Genomic Biology workshop of David Micklos, Dolan DNA Learning Center of Cold Spring Harbor Laboratory:  www.dnalc.org

 

Procedure:

 

Colony Lift Method:

  1. Mark one sterile 15 mL Falcon tube +GFP and the other –GFP.  Plasmid pGREEN DNA will be added to the + tube; none will be added to the – tube.
  2. Add 250 mL of cold sterile 50mM calcium chloride to each tube, using sterile tip and aseptic technique.
  3. Place both tubes on ice.
  4. Use a sterilized inoculating loop to transfer 3 to 4 large (3mm) colonies from the starter plate of E. coli MM294 to the + GFP tube by doing the following steps:
    1. Sterilize your loop in the Bunsen burner flame till it glows red hot.  Then flame the rest of the wire.
    2. Cool the loop – stab into the side of agar plate to cool.
    3. Scrape up as much cell mass as possible, but don’t transfer agar with it.
    4. Immerse loop in + tube and vigorously tap against wall of tube to dislodge cell mass.  Make sure the cell mass is not left on the loop or the side of the tube.  Tube contents should look cloudy because of added cells.
    5. Reflame loop before putting it down on the lab bench.
  5. Mix cells in the + tube well by pipetting in and out repeatedly, using a 100-1000 mL pipettor.  Do this promptly – cells don’t resuspend well if left untouched in the cold solution.  Hold the solution up to the light to make sure the solution is homogenous and no clumps remain.
  6. Return +GFP tube to ice.
  7. Repeat steps 4-6 with –GFP tube.
  8. Add 10 mL of 0.005 mg/ mL of pGREEN plasmid solution directly into the cell suspension in +GFP tube ONLY.  Tap with finger to mix.  Don’t splash or make bubbles.
  9. Return +GFP tube to ice.  Incubate both tubes on ice for 15 minutes if time is available.
  10. Label two LB plates and two LB/amp plates with your name and date.
    1. Label one LB/amp plate +.  This is experimental plate
    2. Label second LB/amp plate -.  This is negative control.
    3. Label one LB plate +.  This is positive control.
    4. Label one LB plate -.  This is positive control.
  11. Following 15 minute incubation, heat shock the cells in both tubes.  It is critical that cells receive a sharp and distinct shock by doing the following:
    1. Carry the ice beaker with tubes to the 42 degree C. water bath.  Remove tubes from ice and immediately immerse in 42 degree C. water bath for 90 seconds.
    2. Immediately return both tubes to ice for at least one minute, optimally for 5-30 minutes.  Gentle shaking aids recovery.
  12. Use micropipettor to transfer 250 mL of LB broth to each tube.  Gently tap tubes to mix.
  13. Spread cells on plate as follows:
    1. From +GFP tube, take 100 mL and dispense onto LB plate and spread as described in #14a or 14b.
    2. From + GFP tube, take 100 mL and dispense onto LB/amp plate and spread as described in #14.
    3. From –GFP tube, take 100 mL and dispense onto LB plate and spread as described in #14.
    4. From – GFP tube, take 100 mL and dispense onto LB/amp plate and spread as described in #14.

14a. Sterilize cell spreader (by dipping into alcohol and running through flame, letting alcohol burn off – take care not to let alcohol drip onto bench top or your arm).  Lift lid of plate, touch cooled spreader to cell suspension on plate, and drag spreader around plate surface to evenly distribute the cell suspension over entire surface of plate.

  1. Reflame spreader after last use.

14b.  Alternative method – take the 15 mL tube containing sterile glass beads.  On each plate from #13, remove plate cover, pour ¼ of beads onto agar surface, cover plate, and with plate on level surface, swirl the beads around the surface to spread the liquid.  Repeat for all four plates.  When spreading is done on all the plates, return the beads to the test tube container – do not handle beads, they are now contaminated with bacteria.

  1. Let plates sit for several minutes to let cell suspension soak into agar plate.  Tape set of four plates together.
  2. Incubate upside down in 37 degree C. incubator for 12-24 hours.
  3. Store in refrigerator after initial incubation.
  4. Disinfect your lab bench, properly dispose of contaminated materials, and wash your hands before leaving the lab.

 

 

 

 

 

 

 

 

 

Results and Discussion*

 

  1. Record your observations:  count the number of colonies and the appearance of colonies.  The colonies should look greenish, and this is more obvious if viewed under black light.  Sometimes tiny white “satellite” colonies may be observed on edges of large green colonies.  These satellite colonies represent non-transformed cells that grow in the shadow of transformed colonies where antibiotic in the medium has been broken down by transformed cells.  A “lawn” should be observed on positive controls where bacterial growth covers the surface and individual colonies can’t be counted.

 

+GFP

-GFP

LB/amp

 

 

LB

 

 

 

 

  1. Carefully think through and answer completely the questions below:
    1. Compare and contrast the growth on +LB and –LB plates – what does this pair tell you about the experiment?

 

 

 

 

    1. Compare and contrast the growth on –LB and –LB/amp plates:  what does this pair tell you about the experiment?

 

 

 

 

    1. Compare and contrast the growth on +LB/amp and –LB/amp plates: what does this pair tell you about the experiment?

 

 

 

 

 

    1. Compare and contrast the growth on +LB/amp and + LB plates:  what does this pair tell you about the experiment?

 

 

 

 

  1. Transformation efficiency is expressed as the number of antibiotic resistant colonies per microgram of plasmid DNA.  To calculate,
    1. Determine the total mass (in micrograms) of pGREEN used in step 8:

Concentration x volume = mass, 0.005 mg/ mL x 10 mL = 0.05 micrograms (mg).

    1. Determine the fraction of the cell suspension spread onto the LB/amp plate:

Volume of suspension spread/total volume of suspension: 100/500 mL = 1/5.

    1. Determine the mass of pGREEN actually spread onto the +LB/amp plate:

Total mass pGREEN (a) X  fraction spread (b) = mass pGREEN spread:

0.05 micrograms X 1/5 (or 1/10) = 0.01 micrograms (or .005).

d.  Finally, determine the number of colonies per microgram of pGREEN:

# colonies/ micrograms of plasmid = transformation efficiency.

Example: if you counted 31 colonies:  31/.01 = 3100 colonies/mg plasmid = 3.1 x 103 colonies/ mg plasmid.

 

Calculate the transformation efficiency for your experiment:

 

  1. What factors might influence transformation efficiency?

 

 

 

  1. Why are the satellite colonies a different color than the large transformed colonies?  Why are they smaller?

 

 

 

  1. Transformation efficiency problem:  You used 10 mL of plasmid DNA at a concentration of 0.0001 mg/ mL and obtained 32 colonies when you plated 100 mL of cells (out of total of 500 mL of cells) – what was the transformation efficiency of this experiment?

 

 

 

 

*Answer sheet available upon teacher request:  cmulvihill@occc.edu