Download pGLO MUTAGENESIS To be able to study any trait in any organism

Molecular Biology of Life Laboratory
BIOL 123
pGLO MUTAGENESIS
To be able to study any trait in any organism, one needs to compare the wild type (the
most common type) to the altered types (mutants). Mutations occur naturally in every gene but
at a very low rate and over a long period of time. If the mutation is advantageous to the
organism, it will get incorporated into the population. This is thought to be the basis of evolution.
However to be able to increase the rate of mutation in one or more genes, we need to have
other methods of inducing mutations over a short period of time.
Scientists are able to increase the rate of mutation by thousand to a million times over
the spontaneous rate by using artificial methods. There are three basic methods that one can
use to increase the rate of spontaneous mutation:
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The first method is by chemical means; i.e., treating subjects with chemicals that cause
mutations. Chemical mutagenesis methods are quite common and the chemicals used fall
into three main categories: base modifiers, base analogs and intercalating agents.
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Base modifiers: alter the structure of nucleotides or modify their side chains, e.g.,
alkylating agents (add CH2-CH3) or nitrous acid (changes NH2 to C=O).
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Base analogs: resemble bases and are therefore wrongly incorporated into the newly
synthesized DNA strand, e.g., azidothymidine (AZT).
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Intercalating agents: insert themselves between adjacent bases and cause a frame
shift mutation, e.g., ethidium bromide.
The second method is by using radiation. Some radiations have very short wavelength rays
(e.g., UV and X ray radiations) and are highly mutagenizing.
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UV rays: bond two adjacent thymine dimmers together such that the newly
synthesized complementary DNA strand would have a single base for every thymine
dimmer.
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X-rays: break DNA molecules, usually causing large deletions.
The third method is the use of transposons. A transposon is a short piece of DNA that can
move from one place on the genome to another. It can insert itself into the middle of a gene
and render that gene or its product non-functional, thereby producing a deficiency in some
enzyme or protein that may have an effect on the trait we are studying.
Out of the three methods mentioned above, the first two are harmful not only for the subjects
but also to the human experimenter. However, most transposons are usually quite speciesspecific and, if not of animal or human origin, are safe to work with.
We will be using the EZ-Tn5 <KAN-2> Insertion Kit from Epicentre (www.epicentre.com;
Cat# EZ1982K) that causes the formation of a stable complex (transposome) between the EZTn5 transposase and EZ-Tn5 transposon. Transposase is an enzyme that is responsible for the
movement (insertion as well as excision) of the transposon. Tn5 has a 19-bp Mosaic End (ME)
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Molecular Biology of Life Laboratory
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at each side, flanking a gene that confers resistance to kanamycin (KanR). The MEs are
inverted repeats.
Once the transposome is activated (i.e., the transposase is attached to the ME sites),
the EZ-Tn5 can insert itself randomly into the host’s genome or any other available piece of
DNA such as a plasmid, and will confer kanamycin resistance, if that piece of DNA is
transformed into kanamycin sensitive cells. The insertion of transposon into DNA is through a
cut and paste process accomplished by transposase without the need for any of the host’s
enzymes.
Tn5
Below are more specific data on Tn5 that will be useful later on.
-Size of EZ-Tn5 <KAN-2> transposon: 1221 bp. The exact sequence can be found at
Epicentre’s Website (http://www.epibio.com/technical-resources/dna-sequences/kan2).
-KAN-2 FP-1 Forward Primer: 5'-ACCTACAACAAAGCTCTCATCAACC-3'.
-KAN-2 RP-1 Reverse Primer: 5'-GCAATGTAACATCAGAGATTTTGAG-3'.
-Mosaic Ends: 5'-AGATGTGTATAAGAGACAG-3'.
In the experiment today, we are going to mutagenize a special plasmid called pGLO
(more formally named pBAD-GFPuv) with our transposon. We obtain our pGLO DNA
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commercially from Bio-Rad. You can obtain the complete nucleotide sequence of pGLO from
Bio-Rad's Website or search Enterez (http://www.ncbi.nlm.nih.gov/entrez/)
for pBAD-GFPuv.
R
This plasmid (shown below) contains the Amp gene (or bla for beta-lactamase gene).
Therefore, selection for cells that have been transformed with pGLO DNA can be accomplished
by growth on plates containing the antibiotic ampicillin. In addition, pGLO incorporates a special
gene regulation system that can be used to control expression of Green Fluorescent Protein
(GFP) in transformed cells. The GFP gene, originally found in the bioluminescent jellyfish
Aequorea victoria, causes the jellyfish to fluoresce and glow in the dark. This glowing property is
now used in research as a marker or tag to follow the movement of proteins within a cell in real
time.
The synthesis of GFP can be switched on in transformed cells by adding arabinose (a
simple sugar) to the nutrient medium (See Appendix: The ara Operon). Under UV light
transformed cells will appear white (wild type phenotype) on ampicillin plates not
containing arabinose, but fluoresce green when arabinose is included in the medium
along with ampicillin.
Since the GFP gene is only expressed under specific conditions, its expression is said to
be regulated. Gene expression in all organisms is carefully controlled to allow adaptation to
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different conditions and to prevent wasteful overproduction of unneeded proteins. The genes
involved in the transport and metabolism of food are good examples of highly regulated genes.
For example, the simple sugar arabinose is a source of both energy and carbon. The bacterial
genes needed to break down (digest) arabinose for food are not expressed in the absence of
arabinose, but are expressed in its presence. In other words, when arabinose is in the
environment, these genes are turned on. In the genetically engineered pGLO plasmid system,
the genes involved in the breakdown of arabinose have been replaced with the jellyfish gene for
GFP. Thais is the reason that when bacteria carrying pGLO are grown in the presence of
arabinose, the pGLO’s GFP gene is turned on, and the bacteria fluoresce a brilliant green color
under UV light.
As to the genotypes, we will designate cells transformed with pGLO as AR KS G+. A
stands for ampicillin, K for kanamycin, G for GFP, R for resistant, S for sensitive and +/- for
expressions of GFP. Therefore, if a mutagenized cell can grow on an L+Kan+Ara plate and is
fluorescent under long wave UV but cannot grow on an L+Amp+Ara, its genotype would be AS
K R G +.
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For the next session, read the material and do the calculations necessary for the Transposon Prep before
coming to the lab. For these calculations, note that the weight measurements in descending order are: g,
mg, µg, ng, and pg and for molarity, they are M, mM, µM, nM, and pM (pmol). Show your calculations to
your TA before starting the experiment.
Use of any section of this Lab Manual without the written consent of Dr. Eby Bassiri, Dept. of
Biology, University of Pennsylvania is strictly prohibited.
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