AP Biology
Bacterial Transformation
pGLO
Background: In this lab, you will be performing a procedure known
as bacterial transformation. A gene is nothing more than a piece of
DNA that codes for a protein. That protein gives the organism
some sort of trait. Genetic transformation literally involves
insertion of a gene into a new organism in order to change the
organism’s traits. In agriculture, genes coding for traits such as
frost resistance, pest resistance, or slower spoilage have been
genetically added to food crops. In bioremediation, bacteria can
be genetically transformed with genes enabling them to digest oil
spills. In medicine, diseases caused by defective genes are
beginning to be treated by gene therapy, where the sick person’s
cells are transformed with healthy copies of the defective gene.
Your task in this lab is to transform bacteria (E. coli) by adding a gene that codes for the Green
Fluorescent Protein (GFP). The source of the GFP is the bioluminescent jellyfish Aequorea
victoria. Following transformation, E. coli will “express” or make the GFP protein and glow a
brilliant green color when placed under ultraviolet light.
In addition to one large circular chromosome, bacteria naturally contain one or more circular
pieces of DNA called plasmids. Plasmid DNA usually contains genes for one of more traits that
may be beneficial to bacterial survival. As an example, some plasmids contain genes that confer
resistance to antibiotics. Under the right conditions, plasmids are freely exchanged between
bacterial cells.
In this procedure, Carolina Biological supply has engineered a plasmid that
contains both the gene for GFP and the gene for resistance to the antibiotic
Ampicillin. Carolina has also engineered a “regulation” system, which allows
expression of the new GFP gene only in the presence of the sugar arabinose—
which must be added to the cell’s nutrient medium. Transformed cells will
therefore appear white on agar plates without arabinose (because they can’t
express the GFP gene), but will appear green under UV light on agar plates
which have had arabinose added.
Pre-Lab Questions (to be completed before coming to class Tuesday):
1.
2.
Why do scientists use bacteria in transformation instead of other organisms?
What is a “control” plate? What purpose does it serve? Which plate is the control
plate?
Hypothesis: Based on the information that you have read and what we have discussed, Create
your own hypothesis that describe what is going to on each plate and why (make sure to write
about all four plates, and explain the why as well).
Data Table (to be copied into lab notebook before lab)
Make sure it is not too small!!!!
Drawing of Plate
Written Observations
+pGLO
LB/amp
+pGLO
LB/amp/ara
-pGLO
LB/amp
-pGLO
LB
The Act of Transformation:
The process of transformation involves three main steps. The steps are intended to
introduce the plasmid DNA into the E. coli cell and provide an environment for the cells to
express their newly acquired genes. The process consists of:
1.
Moving the pGLO plasmid DNA through the cell membrane by:
a.
Using a transformation solution of CaCl2
b. Heat shocking the cell
2.
To grow the transformed cells in the presence of Ampicillin, you must
a.
Provide them with nutrients and enough time (incubation period) that they can
express their newly acquired genes.
Procedure:
1.
Follow the procedure on the “Quick Guide” (will be provided)
Data Collection:
(Day two)Observe the plates you streaked during the transformation lab
under normal room lighting. Then turn the lights out and observe them under ultraviolet
illumination. Carefully observe and draw what you see (in color) on each of the four plates. Place
your drawing and observations in the table below. Include observations on how much bacterial
growth you see on the plates, the color of the bacteria (under normal and UV light) and how many
bacterial colonies (spots) you can see (count) on each plate.
Analysis
1.
2.
3.
Which of the traits that you originally observed in E. coli did not seem to be altered by
the transformation?
Which of the traits of E. coli did appear to be significantly different after performing
the transformation?
If the genetically transformed cells have acquired the ability to live in the presence of
Ampicillin, what other trait have they acquired? How do you know this?
Conclusion
Complete this section as usual (rejecting or not your hypothesis- this need to be at least a
paragraph, points of error, design improvements, apply to real life situation, connect to
Big Idea) Then you will complete the following in your lab notebook.
Calculating Transformation Efficiency
Your next task it to determine quantitatively how efficient the transformation process
was—in other words, to what extent you transformed E. coli cells. In transformation, the
more cells that are transformed during a procedure, the better. Higher transformation
efficiency ensures more product and better outcomes from therapies. ** If your group did
not get any colonies to transform, then you may use the plates of another group to complete
this portion. You will just need to indicate who’s data you are using**
The task:
To calculate the transformation efficiency, you need to do some simple mathematical
calculations. Efficiency is determined by using the following formula:
Transformation efficiency = Total number of cells growing on the agar plate
Amount of DNA spread on the agar plate (in g)
Before you begin your calculations, you need two pieces of information:
1.
The total number of GFP colonies growing on your LB/amp/ara plate.
2.
The total amount of pGLO plasmid DNA in the bacterial cells you spread on the
LB/amp/ara plate.
1.
To determine #1, the total number of GFP colonies, you need to understand that each
colony started as an individual cell. Simply place your LB/amp/ara plate under the UV
light and count the number of colonies that you see.
#Colonies =
2.
= # Cells
To determine the amount of pGLO in the bacterial cells you spread on the LB/amp/ara
plate, you need to know a.) the total amount of DNA that we started the experiment
with and b.) the fraction of that DNA that actually got spread onto the LB/amp/ara
plates.
a.) To determine the total amount of pGLO plasmid DNA: The total amount of DNA
we began with is equal to the product of the concentration and the total volume
used:
DNA (in g) = (Concentration of DNA in g/l) x (volume of DNA in l)
In this experiment, you used 10 l of pGLO at a concentration of 0.08 g/l. This
means that each microliter of solution contained 0.08 g of DNA. Record that
number below:
Total amount of pGLO DNA g used in the experiment =
b. To determine the fraction of pGLO plasmid DNA that actually got spread on the
LB/amp/ara plate you need to realize that not all the DNA that you added to the
bacterial cells will be transferred to the agar plate. You need to determine what
fraction was actually transferred. To do this, divide the volume of DNA you spread on
the LB/amp/ara plate by the total volume of liquid in the test tube containing the DNA.
The formula is below:
Fraction of DNA used = Volume spread on LB/amp/ara plate (l)
Total sample volume in test tube (l)
You spread 100 l of cells containing DNA from a test tube a total volume of 510 l of
solution. Do you remember why there is 510 l total solution? (Look in the laboratory
procedure and locate the steps where you added liquid to the reaction tube. Add the
volumes!)
Use the above formula to calculate the fraction of pGLO plasmid DNA that you
spread on the LB/amp/ara plate, and enter the number below:
Fraction of DNA =
3.
So, how do you determine how many micrograms of pGLO DNA you spread on the
LB/amp/ara plate? To answer this question, you will need to multiply the total amount of
pGLO DNA used in the experiment by the fraction of pGLO DNA you spread on the
LB/amp/ara plate. (You have already determined both of these quantities.)
pGLO DNA spread in g = Total amount of DNA used in g x fraction of DNA used
Enter the number below:
pGLO DNA spread (g) =
Now, fill in the following table:
Number of colonies on LB/amp/ara plate =
Micrograms of pGLO DNA spread on the plate
Use the data in the table to calculate the efficiency of the pGLO transformation using the
following formula:
Transformation efficiency = Total number of cells growing on the agar plate
Amount of DNA spread on the agar plate
Enter that number here (note: use scientific notation to express the efficiency):
Transformation efficiency =
transformants/g
Analysis: Biotechnologists are in general agreement that the transformation protocol that you
used has a transformation efficiency of between 8.0 x 102 and 7.0 x 103 transformants/g of DNA.
1.
How does your transformation efficiency compare?
2.
How does your transformation efficiency compare to other groups in the class? In the table
below, record several groups who did better, and several who did worse.
Transformation efficiency was better than ours
Transformation efficiency was worse than ours
3.
Now, practice your skill at calculating transformation efficiency by calculating the
transformation efficiency of the following experiment:
DNA plasmid concentration: 0.08 g/l
250 l CaCl2 transformation solution
10 l pGLO plasmid solution
250 l LB broth
100 l cells spread on agar
227 colonies of transformants
Fill in the following chart and hand the lab in:
Number of colonies on LB/amp/ara plate =
Micrograms of DNA spread on plate =
Transformation efficiency =