Download OVERVIEW OF THE BIO208 GFP LABORATORY PROJECT

BIO208: GENETICS
Bacterial Transformation with pGLO plasmid: Cloning of GFP gene
Objectives:
Complete the pre-lab assignment due before the laboratory
Record title purpose, steps, observations, and data in the laboratory notebook.
Answer questions as indicated
I.
General Laboratory Skills
Sterile Technique
With any type of microbiology technique it is important not to introduce contaminating bacteria into the
experiment. Bacteria are ubiquitous and are found on fingertips, bench tops, etc., it is important to avoid
contaminating surfaces. Using sterile technique is an issue of human cleanliness and safety.
Working with E. coli
The host organism in this kit, an E. coli K-12 strain, the vector containing the recombinant
GFP protein and the subsequent transformants created by their combination are not pathogenic.
However, handling E. coli K-12 requires the use of Standard Microbiological Practices. These practices
include but are not limited to the following. Work surfaces are decontaminated once a day and after any
spill of viable material. All contaminated liquid or solid wastes are decontaminated before disposal.
wash hands: (i) after handling materials involving organisms containing recombinant DNA molecules,
and (ii) before exiting the laboratory. All procedures are performed carefully to minimize the creation of
aerosols. Mechanical pipetting devices are used; mouth pipetting is prohibited. Eating, drinking,
smoking, and applying cosmetics are not permitted in the work area. Wear gloves.
Decontamination and Disposal
Contaminated disposables should be placed in the biohazard container. Wash lab benches with
disinfectant and hands with soap and water.
Ultraviolet (UV) Lamps
Ultraviolet radiation can cause damage to eyes and skin. Short-wave UV is more damaging
than long-wave UV light. The Bio-Rad UV lamp recommended for this module is long-wave. If
possible, use UV rated safety glasses or goggles.
II.
Introduction
A. pGLO plasmid DNA
bla
ori
GFP
araC
Encodes resistance to ampicillin
Origin of replication allows plasmid to be copied by bacterial DNA replication enzymes
Gene encoding green fluorescent protein
Encodes repressor to prevent transcription (and expression) of the GFP gene. Arabinose sugar
binds the repressor to allow GFP expression. GFP expression can be turned on by addition or
removal of arabinose from the bacterial growth medium.
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B. Transformation
With pGLO transformation, E. coli are transformed 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 DNAs called plasmids. Plasmids may
confer beneficial traits to a bacterium. 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
results in green glowing colonies. Those bacteria that do not take up the plasmid DNA cannot produce
GFP.
III.
Procedure
A. Equipment and Reagents
microcentrifuge tube racks and sterile microcentrifuge tubes
p1000 pipettemen/tips and 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
B. Transformation
*Wear gloves
Record the title, purpose, procedure including units, microliters, micrograms, times, temperatures
etc, observations, and answers to questions in the laboratory notebook
1. Complete the BioRad Transformation Kit quick guide
2. Answer the following in the laboratory notebook in your own words.
a. Why doesn’t the pGLO plasmid itself glow?
b. Why is pGLO referred to as a plasmid “vector”?
c. What is the role of the heat shock in transformation?
d. What are competent cells?
e. What is the role of ampicillin in the transformation process?
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f. 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?
g. What is the role of arabinose in the expression of GFP in the pGLO transformation system?
h. Predictions: For each of the following, indicate if colonies will grow and if yes, will they
glow?
LB plate with non-transformed bacteria
LB plate with transformed bacteria
LB/amp with non-transformed bacteria
LB/amp with transformed bacteria
LB/amp/ara with non-transformed bacteria
LB/amp/ara with transformed bacteria
C. Analysis of Transformation Results
Record the following observations and analyses in the laboratory notebook.
Analysis of plates
1. How much growth do you see on each plate? If there are too many colonies to count use "TNTC"
(too numerous to count). If not, count the colonies. Record for each plate in the laboratory notebook.
2. On which plate(s) did you expect to see NO bacterial growth and why? Do your observations
support your hypothesis? Use full sentences.
3. Examine the bacterial plates in the dark under long wave UV light. Are there glowing bacteria? On
which plate(s) are there glowing bacteria and how many glowing colonies do you observe?
Calculation of Transformation Efficiency
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 the LB/amp/ara plate under UV light. Determine the number of individual glowing colonies
and record in the laboratory notebook.
2. Calculate the amount ug pGLO by determining the ug plasmid used in the experiment – take into
account that you only plated a fraction of the transformation on the agar plate. See BioRad quick guide
steps 2, 9, and 11 to determine the total ug of pGLO used on the agar plate.
3. Determine the transformation efficiency. Use scientific notation to express the results (example
10,000 = 1X 105). Record the transformation efficiency in the laboratory notebook in colonies/ug
plasmid.
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?
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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. 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 explanations.
a. E. coli cells are dead
b. Arabinose is degraded
c. Ampicillin is degraded
d. Plasmid is degraded
e. E. coli were contaminated with another, ampicillin-resistant bacteria
If you obtained unexpected results, provide an explanation.
*Plates are disposed of in the biohazard trash
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 are killed by ampicillin.
Ampicillin
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. 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.
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 phospholipids of the cell membrane allowing DNA to
pass through the bacterial cell wall and enter the cell. Ca+2 may also neutralize the
repulsive negative charges of the phosphate backbone of the plasmid DNA facilitating
cellular uptake
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E. coli
The host organism in this experiment is E. coli strain K12 and is not pathogenic.
However, Standard Microbiological Practices should be used. E. coli 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.
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 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
Recovery
The 10 minute incubation following the addition of LB broth allows the cells to grow and
express the ampicillin resistance protein, beta-lactamase.
Transformation
Occurs when a bacterium takes up and expresses a new piece of genetic material (DNA).
Genes are often on a vector
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)