Download Lab_6_Part3

Lesson 4 Extension Activity: Calculate Transformation
Efficiency
Your next task in this investigation will be to learn how to detem~nethe extent to
which you genetically transformed E. coli cells. This quantitative measurement is referred
to as the transformation efficiency.
In many experiments, it is important to genetically transform as many cells as possible. For
example, m some types of gene therapy, cells are collected from the patient, transformed in the
laboratory, and then put back into the patient. The more cells that are transformed to produce
the needed protein, the more likely that the therapy will work. The transformation efficiency is
calculated to help scientists determine how well the transformation is working.
The Task
You are about to calculate the transformation efficiency, which gives you an indication
of how effective you were in getting DNA molecules into bacterial cells. Transformation
efficiency is a number. It represents the total number of bacterial cells that express the
green protein, divided by the amount of DNA used in the experiment. (It tells us the total
number of bacterial cells transformed by one microgram of DNA.) The transformation
efficiency is calculated 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 )
Therefole, before you can calculate the efficiency of your transfoimation, you will
need two pieces of information,
(I) The total number of green fluorescent colonies growing on your LBlamplara
plate.
(2) The total amount of pGLO plasmid DNA in the bacterial cells spread on the
LBlamplara plate.
1. Determining the Total Number of Green Fluorescent Cells
Place your LB/amp/ara plate near a W light. Each colony on the plate can be assumed to
be derived 6-om a single cell. As individual cells reproduce, more and more cells are
formed and develop into what is termed a colony. The most direct way to determine the
total number of green fluorescent cells is to count the colonies on the plate.
Enter that number here =,
L
Totzl number of cells =
2. Determining the Amount of pGLO DNA in the Bacterial Cells Spread on
the LBIamplara Plate
Wy need two pieces of information to find out the amount of pGLO DNA in the bacterial
ceUs spread on the LB/amp/ara plate in this experiment. (a) What was the total amount of
DNA we began the experiment with, and @) What fraction of the DNA (in the bacteria)
actually got spread onto the LB/amp/ara plates.
Once you calculate this data, you will need to multiply the a t a l amount of pGLO DNA
used in this experiment by the fraction of DNA you spread on the LB/amp/ara plate. The
answer to this multiplication will tell you the amount of pGLO DNA in the bacterial cells
that were spread on the LB/amp/ara plate.
a. Determining 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, or
(DNA in pg)
=
(concentration of DNA in ~ g / p l )x (volume of DNA in pl)
In this experiment you used 10 p1of pGLO at concentration of 0.08 pg/pl. This means that
each microliter of solution contained 0.08 pg of pGLO DNA. Calculate the total amount
of DNA used in this experiment.
Enter that number here 3
Total amount of pGLO DNA (pg)
used in this experiment =
How will you use this piece of information?
I
b. Determining the fraction of p a 0 plasmid DNA (in the bacteria) that actually got
spread onto the LBlamplara plate: Since not aU the DNA you added to the bacterial cells
willbe transferred to the agar plate, you need to find out what k t i o n of the DNA was actually
spread onto the LBIamplara plate. To do this, divide the volume ofDNA you s p a d on the
LBIarnplara plate by the total volume of hquid in the test tube containing the DNA. A formula
for this statanent is
Fraction of DNA used
=
v01UITle spread on LBIanlp plate (pl)
Total sample volume in test tube (pl)
You spread 100 p1 of cells contatning DNA from a test tube containing a total volime of
5 10 p1 of solution. Do you remember why there is 510 p1 total solution? Look in the laboratory
procedure and locate all 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 you spread
on the LB/amp/ara plate.
Enter that number here =.
How will you use this piece of information?
1
Eraction of DNA =
So, how many micrograms of pGLO DNA did you spread on the LBIamplara plates?
To answer this question, you will need to multiply the total amount of pGLO DNA
used in this experiment by the fraction of pGLO DNA you spread on the LB/amplara plate.
pGLO DNA spread in pg
=
Total amount of DNA used in pg x fiaction of DNA used
Enter that number here +
What will this number tell you?
r7J
pG-LO DNA spread (pg) =
Look at all your calculations above. Decide which of the numbers you calculated
belong in the table below. Fill in the following table.
Number of colonies on
LBIamplara plate =
Micrograms of pGLO DNA
spread on the plates
I
Now use the data in the table to calculate the efficiency of the pGLO transformation
Transformation efficiency = Total number of cells growing on the agar plate
Amount of DNA spread on the agar plate
Enter that number here
Transformation efficiency =
-transformants/pg
Analysis
Transformation efficiency calculations result in very large numbers. Scientists often use
a mathematical shorthand referred to as scientific notation. For example, if the calculated
transformation efficiency is 1,000 bacterialpg of DNA, they often report this number as:
lo3 transformantslpg
(1O3 is another way of saying 10 x 10 x 10 or 1,000)
How would scientists report 10,000 transformantslpg in scientific notation?
Carrying this idea a little farther, suppose scientists calculated an efficiency of 5,000
bacterialpg of DNA. This would be reported as:
5 x lo3 transformantslpg
(5 times 1,000)
How would scientists report 40,000 transformantslpg in scientific notation?
One final example: If 2,600 transformantslpgwere calculated, then the scientific notation
for this number would be:
2.6 x lo3 transformants/pg
(2.6 times 1,000)
Similarly:
5,600 = 5.6 x lo3
271,000 = 2.71 x lo5
2,420,000 = 2.42 x lo6
How would scientists report 960,000 transfomants/pg in scientific notation?
Report your calculated transformation efficiency in scientific notation.
Use a sentence or two to explain what your calculation of transformation efficiency
means.
Biotechnologists are in general agreement that the transformation protocol that you
have just completed generally has atransfonnation efficiency ofbetween 8.0 x lo2 and 7.0
x lo3 transformants per microgram of DNA.
How does your transforn~ationefficiency compare with the above?
In the table be!ow, report the transformation efficiency of several of the teams in the
class.
1
Team
-
Efficiency
How ddcs your transformation efficiency compare with theirs?
Calculate the transformation efficiency of the following experiment using the information and the r e s u l ~listed below.
DNA plasmid concentration: 0.08 pglpl
250 pl CaCL, transformation solution
10 pl pGLO plasmid solution
250 p1 LB broth
100 pl cells spread on agar
227 coloniesof transformants
Fill in the following chart and show your calculations to your teacher:
Number of colonies on LBIamplara plate =
Micrograms cf DNA spread on the plates =
.I-
Transformation efficiency =
Extra Credit Clallenge:
If a particular experiment were known to have a transformation efficiency of 3 x lo3
bacterialpg of DNA, how many transformant colonies would be expected to grow on the
LBlamplara plate? You can assume that the concentration of DNA and fraction of cells
spread on the LB agas are the same as that of the pGLO laboratory.
4. For which fragment sizes was your graph most accurate? For which fragment sizes was it least
accurate? What does this tell you about the resolving ability of agarose-gel electrophoresis?
Analysis
1. Discuss how each of the following factors would affect the results of electrophoresis:
a. Voltage used
b. Running time
c. Amount of DNA used
d. Reversal of polarity
2. Two small restriction fragments of nearly the same base pair size appear as a single band,
even when the sample is run to the very end of the gel. What could be done to resolve the
fragments? Why would it work?
Questlons
1. What is a plasmid? How are plasmids used in genetic engineering?
2. What are restriction enzymes? How do they work? What are recognition sites?
3. What is the source of restriction enzymes? What is their function in nature?
4. Describe the function of electricity and the agarose gel in electrophoresis.
5. A certain restriction enzyme digest results in DNA fragments of the following sizes:
4,000 base pairs, 2,500 base pairs, 2,000 base pairs, 400 base pairs. Sketch the resulting
separation by electrophoresis. Show starting point, positive and negative electrodes,
and the resulting bands.
6. What are the functions of the loading dye in electrophoresis? How can DNA be prepared
for visualization?
7. Use the graph you prepared from your lab data to predict how far (in cm) a fragment of
8,000 bp would migrate.
8. How can a mutation that alters a recognition site be detected by gel electrophoresis?
GFP Purification-Quick Guide
Lesson 2 Inoculation
Growing Cell Cultures
1. Remove the transformation plates from
the incubator and examine using the UV
light. Identify several green colonies that
are not touching other colonies on the
LB/amp/ara plate. Identify several white
colonies on the LB/amp plate.
2. Obtain two culture tubes containing the
growth media LB/amp/ara.Label one "+"
and one "-". Using a sterile loop, lightly
touch the loop to a green colony and
immerse it in the "+" tube. Using a new
sterile loop, repeat for a white colony and
immerse it in the "-" tube (it is very important to pick only a single colony). Spin the
loop between your index finger and
thumb to disperse the entire colony.
3. Cap the tubes and place them in the shaking incubator or on the shaking platform
and culture overnight at 32 OC or 2 days
at room temperature.
or
Cap the tubes and shake vigorously by
hand. Place in the incubator horizontally
at 32 OC for 24-48 hours. Remove and
shake by hand periodically when possi-
ble.
+
Incubate at 32 "C overnight
or
2 days at room
temperature
Lesson 3 Purification Phase 1
Bacterial Concentration
1. Label one microtube "+" with your name
and class period. Remove your liquid cultures from the shaker and observe with the
UV light. Note any color differences
between !he two cultures. Using a new
pipette, transfer 2 ml of "+" liquid culture
into the "+" rnicrotube. Spin the rnicrotube
for 5 minutes in the cenhifuge at maximum
speed. The pipette used in this step can be
repeatedly rinsed in a beaker of water and
used for all f~llowingsteps of this laboratory period.
+
+
I
2. Pour out the supernatant and observe the pellet under W light.
3. Using a rinsed pipette, add 250 yl of TE
solution to the tube. Resuspend the pellet
thoroughly by rapidly pipetting up and down
several times..
4. Using a rinsed pipette, add 1 drop of
lysozyme to the resuspended bacterial pellet to initiate enzymatic digestion of the bacterial cell wall. Mix the contents gently by
flicking the tube. Observe the tube under the
W light.
5. Place the microtube in the freezer until the
next laboratory period. The freezing causes
the bacteria to rupture completely.
1 drop lysozyme
Lesson 4 Purification Phase 2
Bacterial Lysis
1. Remove the microtube from the freezer
and thaw using hand warmth. Place the
tube in the centrifuge and pellet the insoluble bacterial debris by spinning for 10
minutes at maximum speed.
2. While your tube is spinning, prepare the
chromatography column. Remove the cap
and snap off the bottom from the prefilled
HIC column. Allow all of the liquid
buffer to drain from the column (-3-5
minutes).
3. Prepare the column by adding 2 ml of
Equilibration Buffer to the top of the column. This is done by adding two 1 ml
aliquots with a rinsed pipette. Drain the
buffer to the 1 ml mark on Lhe column.
Cap the top and bottom and store the column at room temperature until the next
laboratory period.
4. After the 10 minute spin, immediately
remove your tube from the centrifuge.
Examine the tube with the UV light.
Using a new pipette, transfer 250 ~1of the
"+" supernatant into a new microtube
labeled "+". Again, rinse the pipette well
for the rest of the steps of this lab period.
5. Using a well rinsed pipette, transfer 250 pl
of binding buffer to the "+" supernatant.
Place the tube in the refngerator until the
next laboratory period.
Thaw
I
I
Centrifuge
0
Equilibration buffer (2 ml)
I
Lesson 5 Purification Phase 3
Protein Chromatography
1. Label 3 collection tubes 1-3 and place the
tubes in the foam rack or in a rack supplied
in your laboratory. Remove the caps from
the top and bottom of the column and place
the column in collection tube 1. When the
last of the buffer has reached the surface of
the HIC matrix proceed to the next step
below.
250 pl) + supernatant
2. Using a new pipette, carefully and gently
load 250 ~1 of the "+" supernatant onto the
top of the column. Hold the pipette tip
against the side of the column wall, just
above the upper surface of the matrix and let
the supernatant dnp down the side of the column wall. Examine the column using a TJV
light. Note your observations. After it stops
dripping transfer the column to collection
tube 2.
'n
Wash buffer (250 pl)
3. Using the rinsed pipette, add 250 pl of wash
buffer and let the entire volume flow into the
column. Examine the column using the UV
light. Note you- observations. After the column stops dripping, transfer it to tube 3.
,TE buffer (750 pl)
4. Using the rinsed pipette, add 750 @ of TE
Buffer and let the entire volume flow into
the column. Examine the column using the
UV light. Note your observations.
5. Examine all three collection tubes and note
any differences in color between the tubes.
Parafilm or Saran Wrap the tubes and place
in the refrigerator until the next laboratory
period.
Lesson 1 Finding the Green Fluorescent Molecule
Genetic Transformation Review
In Bio-Rad Kit 1, you performed a genetic transformation of E. coli bacterial cells. The
results of this procedure were colonies of cells that fluoresced when exposed to ultraviolet
light. This is not a normal phenotype (characteristic) for E.coli. You were then asked to figure out a way to determine which molecule was becoming fluorescent under UV light. After
determining that the pGLO plasmid DNA was not responsible for the fluorescence under the
W light, you concluded that it was not the plasmid DNA that was fluorescing in response to
the ultraviolet light within the cells. This then led to the next hypothesis that if it is not the DNA
fluorescing when exposed to the UV light, then it must be a protein that the new DNA produces w i t h the cells.
1. Proteins.
a. What is a protein?
b. List three examples of proteins found in your body.
c. Explain the relationshp between genes and proteins.
2. Using your own words, describe cloning.
3. Describe how the bacterial cloned cells on your LB/amp plate differ from the cells on
your LB/amp/ara plate. Can you design an experiment to show that both plates of cloned
cells behave similarly and do contain the same DNA?
4. Describe how you might recover the cancer-curing protein from the bacterial cells.
Lesson 2
Name
Review Questions
1. What is a bacterial colony?
2. Why did you pick one green colony and one white colony from your agar plate@)?Why
do you think you picked one of each color? What could this prove?
3. How are these items helpful in this cloning experiment?
a.
ultraviolet (UV) light -
b.
incubator -
c.
shaking incubator -
4. Explain how placing cloned cells in nutrient broth to multiply rslates to your overall goal
of purifying the fluorescent protein.
Lesson 3
Name
Review Questions
1. You have used a bacterium to propagate a gene that produces a green fluorescent protein. Identify the function of these items you need in Lesson 3.
a.
Centrifuge -
b.
Lysozyme -
c.
Freezer -
2. Can you explain why both liquid cultures fluoresce green?
3. Why did you discard the supernatant in this part of the protein purification procedure?
4. Can you explain why the bacterial cells' outer membrane ruptures when the cells are
frozen. What happens to an unopened soft drink when it freezes?
5. What was the purpose of rupturing or lysing the bacteria?
Lesson 4
Name
Review Questions
1. What color was the pellet in this step of the experiment? What color was the supernatant?
What does ;this tell you?
2. Why did you discard the pellet in this part of the protein purification procedure?
3. Briefly describe hydrophobic interaction chromatography and identify its purpose in this
lab.
Lesson 5
Name
Review Questions
1. List your predictions and observations for the sample and what happens to the sample
when the following buffers are added to rhe HIC column.
Observations
Under UV Light
Collection Tube Number
Tube 1
Sample in Binding Buffer
Prediction
(column and collection tube)
Tube 2
Sample with Wash Buffer
Tube 3
Sample with Elution Buffer
2. Using the data table above, compare how your predictions matched up with your obser-
vations for each buffer.
a.
Binding Buffer-
b.
Wash Buffer-
c.
Elution Buffer-
3. Based on your results, explain the roles or functions of these buffers. Hint: how does the
name of the buffer relate to its function.
a.
Equilibration Buffer-
b.
Binding Buffer-
c.
Wash Buffer-
d.
TE (Elution) Buffer-
4. Whlch buffers have the highest salt content and which have the least? How can you tell?
5. Were you successful in isolating and purifying GFP from the cloned bacterial cells?
Identify the evidence you have to support your answer.
(ii)
FEMALE
MALE