Download Cloning A population of cells produced from a single cell contains

BIO208: GENETICS
Bacterial Transformation with pGLO plasmid
Cloning of GFP gene
ASSIGNMENTS
Read the terms and concepts portion of this laboratory
Complete the pre-lab assignment due before the laboratory
Record purpose, all steps, observations, and data in the laboratory notebook.
Answer questions as indicated
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Transformation of E. coli with pGLO Plasmid DNA
With pGLO transformation, students transform bacteria with a gene that codes for a green fluorescent
protein (GFP). The natural source for the GFP gene is the bioluminescent jellyfish, Aequorea victoria.
The gene encodes for the GFP which allows the jellyfish to glow in the dark. The protein absorbs energy
when exposed to ultraviolet light and gives off some of this energy in the form of visible green light.
Genes can be moved into bacteria by the use of small circular pieces of DNA called plasmids. Plasmids
may confer beneficial traits to a bacterium, such as resistance to ampicillin or other antibiotics. The
pGLO plasmid encodes for resistance to ampicillin as well as a gene regulation system to control the
expression of GFP. Expression of GFP by transformed bacteria will result in green glowing colonies.
Those bacteria that do not take up the plasmid DNA will not produce GFP.
pGLO plasmid vector
bla
orf
GFP
araC
Encodes resistance to ampicillin for antibiotic selection
Open reading frame allows plasmid to be copied by bacterial DNA replication enzymes
Gene encoding green fluorescent protein
Encodes a repressor to prevent transcription (and expression) of the GFP gene. The arabinose
sugar binds the repressor to allow GFP expression. This allows the research to turn on and off
GFP expression by addition or removal of arabinose from the bacterial growth medium.
Transformation
E. coli bacterium
plasmids
E. coli
DNA
Bacterial cell wall and cell
membrane
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Terms and Concepts
Antibiotic
selection
The pGLO plasmid also contains the gene for ampicillin
resistance. This gene encodes for beta-lactamase (bla) which is secreted by the bacteria
harboring the plasmid. The beta-lactamase
inactivates the ampicillin contained on the LB agar plates thereby
allowing the bacteria to grow. Non-transformed cells (do not
the plasmid) are killed by ampicillin.
Ampicillin
Provided as a 10 mg/ml stock in sterile water Ampicillin is a derivative of penicillin.
This antibiotic kills bacteria by blocking the synthesis of a key cell membrane
component. Ampicillin-sensitive cells are killed as they divide. Bacteria often carry small
circular pieces of DNA called plasmids. The plasmid used in this lab contains an
ampicillin resistance gene that encodes for beta-lactamase, a protein which inactivates
ampicillin. Bacteria that carry the plasmid can be selected for by growing bacteria in the
presence of ampicillin.
Arabinose
Arabinose is a sugar that bacteria can use as a source of carbon for cellular respiration.
The enzymes needed to metabolize arabinose are only made in the presence of arabinose.
The genes for the enzymes are part of the arabinose operon, a series of genes whose
expression is coordinated and whose transcription is turned on by the presence of
arabinose. When the arabinose is used up, the genes are no longer expressed. A gene of
interest can be spliced onto the arabinose operon so that the expression of the gene is
turned on in the presence of arabinose. Provided as a 200mg/ml stock in sterile water
Beta-lactamase
see antibiotic selection
Cloning
A population of cells produced from a single cell contains
identical clones. The process of producing a clonal population is
called “cloning”. One can produce large quantities of a specific
DNA sequence or plasmid by cloning.
Colony
A group of identical bacterial cells (clones) growing on an agar
plate. The colony contains millions of individual cells.
Competent cells
E. coli that have been treated with calcium chloride more readily
accept plasmid DNA. Ca+2 ions are thought to neutralize the
phospholipids of the cell membrane allowing the DNA to pass
through the bacterial cell wall and enter the cell.
E. coli
The host organism in this experiment is E. coli strain K12. It is not pathogenic. However,
Standard Microbiological Practices should be used. These bacteria grow best with
aeration, provided by constant shaking. After 30 minutes of lag phase, E. coli enter a log
phase in which doubling occurs every 20 minutes. When nutrients are depleted (cells at a
concentration of 109/ml) cells enter a stationary phase. An overnight, stationary, culture
of cells is used to prepare a mid log phase culture. LB at room temperature is inoculated
with the overnight culture at a 1:100 dilution and allowed to incubate (with shaking at
37oC) for 1.75 hours. At this phase they exhibit an optical density of 0.4 at OD 550. Cells
are then stored on ice for up to 24 hours to arrest in mid log phase. 100 ml midlog cells
yield 10 ml competent cells. 200 ul cells are used for each transformation.
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Gene Regulation
Genes involved in the transport and breakdown of arabinose are
highly regulated. The genes encoding the bacterial enzymes
needed are only transcribed in the presence of arabinose. When
the arabinose runs out, the genes are turned off.
Green Fluorescent
Protein
GFP was originally isolated from the bioluminescent jellyfish,
Aequorea victoria. The gene for GFP has been cloned and plasmid
vectors containing the gene are in wide use. The 3D structure of
GFP causes it to give off energy when exposed to UV light in the
form of visible green light
Heat Shock
The heat shock increases the permeability of the bacterial cell
membrane to exogenous DNA. The time of the heat shock is
critical and is optimized for the tube type volume
LB media
Liquid and solid media are made from an extract of yeast and an enzymatic digest of
meat byproducts which provides a mixture of carbohydrates, amino acids, nucleotides,
salts, and vitamins – nutrients for bacterial growth. LB agar (made from seaweed)
provides a support on which bacterial colonies can grow
Plasmid
A small circular DNA molecule capable of autonomous replication
Many plasmids also contain antibiotic resistance genes and must
contain an origin of replication.
pGLO
pGLO is a plasmid which contains the GFP gene and the
ampicillin resistance gene which encodes for beta-lactamase
Recovery
The 10 minute incubation following the addition of LB broth
allows the cells to grow and express the ampicillin resistance
protein, beta-lactamase.
Sterile technique
It is important not to introduce contaminating bacteria into an experiment. Because
contaminating bacteria are found everywhere including fingertips and bench tops, it is
important to avoid these surfaces. Wear gloves. Do not touch the portion of sterile
toothpicks, inoculating loops, the pipette tip, and agar plates that will come into contact
with your bacteria.
Transformation
Occurs when a bacterium takes up and expresses a new piece of genetic material (DNA).
Genes are often on a vector
UV lamps
Ultraviolet radiation can cause damage to eyes and skin. Short wave UV is more
damaging than long wave (used in this experiment). Wear protective goggles.
Vector
An autonomously replicating DNA molecule into which
foreign DNA fragments are inserted and then propagated
in a host cell (a plasmid is a type of vector)
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Equipment and Reagents
*Record the procedure, observations, and answers to questions in the laboratory notebook
*Wear gloves
Reagents and equipment
microcentrifuge tube racks and sterile microcentrifuge tubes
p1000 pipettemen/tips
p20 pipettemen/tips
gloves
small ice bucket/ice
sterile toothpicks (autoclaved)
long wave UV lamps
42 degree heat block
37 degree incubator
LB broth (Luria broth) at room temperature
CaCl2 (transformation) solution on ice, 50 mM
1 LB plate
2 LB/amp plates (0.1 mg/ml ampicillin)
1 LB amp/ara plate (0.1 mg/ml ampicillin + 6 mg/ml arabinose)
Streaked plate of E. coli strain K12/HB101
pGLO plasmid DNA on ice 0.08 ug/ul stock solution
Procedure
Follow the Transformation Kit quick guide pg 14 - 15
Answer the following in the laboratory notebook
1. Why doesn’t the pGLO plasmid itself glow?
2. Why is pGLO referred to as a plasmid “vector”?
3. What is the role of the heat shock in transformation?
4. What are competent cells?
5. What is the role of ampicillin in the transformation process?
6. Why is the recovery period important? What would happen to cells if the recovery period was
omitted and they were spread immediately onto LB amp plates?
7. Predictions: For each of the following, a – f, indicate if colonies will grow and if yes, will they
glow?
a. LB plate with non-transformed bacteria
b. LB plate with transformed bacteria
c. LB/amp with non-transformed bacteria
d. LB/amp with transformed bacteria
e. LB/amp/ara with non-transformed bacteria
f. LB/amp/ara with transformed bacteria
8. What is the role of arabinose in the expression of GFP in the pGLO transformation system?
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BIO208
Analysis of Transformation Results
Record the following observations and analyses in the laboratory notebook.
I. Analysis of plates
1. How much growth do you see on each plate, relatively speaking? If there are too many colonies
to count use "TNTC" (too numerous to count). List all plates in the lab notebook and the growth
or lack of growth observed on each. Write neatly, you can express the data in table form.
2. On which plate(s) did you expect to see NO bacterial growth and why? Do your observations
support your hypothesis? Use full sentences and provide detail.
3. On which do you expect to see glowing bacteria and why?
4. Obtain the long wave UV light and examine the bacterial plates in the dark. Are there glowing
bacteria? How many glowing colonies do you observe?
5. For each of the following scenarios consider the LB/amp/ara plate only
*Discuss the expected outcome (what would be observed)
*Assume that all other conditions are normal.
* Provide clear and detailed explanations.
a.
b.
c.
d.
e.
E. coli cells are dead
Arabinose is degraded
Ampicillin is degraded
Plasmid is degraded
E. coli were contaminated with another, ampicillin-resistant bacteria
II. Calculation of Transformation Efficiency
Record the following observations and analyses in the laboratory notebook.
The transformation efficiency (TE) is the extent to which the bacterial cells were genetically
transformed. The TE is a # that represents the total # of bacterial cells that express the GFP divided by
the amount (in ug) of plasmid DNA used in the experiment.
Transformation efficiency =
Total number of transformed cells on plate
Amount of plasmid DNA spread on plate
1. Examine one LB/amp/ara plate under UV light. Each colony on the plate is composed of millions of
cells (clones). Determine the number of individual colonies.
Number of glowing colonies =
2. The concentration of plasmid DNA was ______ ug/ul (check with instructor). Look in your notes to
find out the volume of pGLO used and then:
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Calculate the total ug pGLO used in the transformation reaction = _______
ug
3. Determine the transformation efficiency. Use scientific notation to express the results (example
10,000 = 1X 105). Note the units = colonies/ug plasmid DNA
Transformation Efficiency = _______________
colonies/microgram plasmid DNA (colonies/ug)
4. When making a bacterial library, scientists aim for transformation efficiencies over 2.5 X 10 6
colonies per microgram. To accomplish this, highly competent bacteria requiring extensive preparation are
used. How does the transformation efficiency observed in this experiment compare?
5. Calculate. If an experiment had a transformation efficiency of 2 X 10 3 bacteria/ug DNA, how many
transformant colonies would be expected to grow on the LB/amp/ara plate? (Assume 5 ul of pGLO at a
stock concentration of 0.001ug/ul was used).
6. Based on your results in this experiment, what are your suggestions for future experimentation?
Drawings or flow charts can be included in the experimental design description. Based on your
observations, analyses, and conclusions what component or components should be changed or tested?
If you obtained unexpected results, provide an explanation.
*Plates are disposed of in the biohazard trash
III. Pick colonies for GFP expression