Download Plasmid Sex Introduction .....In most bacteria there are several

Plasmid Sex
Introduction
.....In most bacteria there are several pieces of DNA. .. One is the
somatic genome - a huge circle of double-stranded DNA that actually
measures about 2 mm in length, and is all crammed into the little cell.
..This large piece of DNA is what defines the type of bacterium it is. ..
The cell cannot live without this circle of DNA. In addition, there are
various optional smaller circles of DNA, which are usually called
plasmids... To repeat: ..these are 'optional' and the cell can get along
without them unless the genes on those plasmids allow it to survive
under unusual conditions such as when a particular antibiotic is in the
neighborhood, and the plasmid contains a gene that protects the cell
from that antibiotic by any one of several different mechanisms.
.....A cell can duplicate a plasmid and then send one copy over to
another cell via a thin tube called a pilus. ..Seemingly, each type of
plasmid codes for its own special type of pilus.
.....A widely used plasmid of E. coli is one called "F" (for fertility). .. Cells
that possess "F" are called male (F-donors or "F+"), these cells usually
possess two F-pili for the transport of the F into cells lacking it. .. Those
without F are called females (potential recipients, F-). .. In this exercise
you will mix a very few F+ with a much larger number of F-, and show
that the F- cells are converted to F+ at a rather rapid pace. ..The rapidity
is because because it is a chain-reaction as the newly form F+ cells are
added to the pool of donors. ..(You can change this exercise into an
experiment if you can design an experiment from which you can
determine the time it takes to convert an F- into an F+.)
.....First, you must have a strain of donor that possesses a somatic
genome that is different from that of the recipient. ..That way you can
"select against" the original donor cells by using a mating medium in
which they cannot grow, but in which the recipients can. ..For this we
will use donors that are normal with respect to sensitivity to the antibiotic
streptomycin, while the recipients will be streptomycin resistant (strR).
..We will add streptomycin to the medium, and none of the original
donors will be able to grow up to form colonies. ..(Always keep in the
back of your mind a very important word: "controls"!)
.....Second, you must have a way to tell whether or not the recipients
have acquired the F. ..We will do a little trick here, and use an F that
has the lactose operon inserted into it. ..This is called "F-lac."
..(Whenever other genes are incorporated into F, the F is now called a
type of F-prime (F'). ..Thus F-lac is a type of F'. ..We will therefore start
with F- cells that do not have a functional lac-operon (lac-), and so when
they acquire the F-lac, they will be able to use lactose sugar to grow. ..
(This is not a genetically engineered product, but one that can be made
naturally, and therefore doesn't not need to be approved by an
institutional recombinant DNA committee. ..Indeed, no part of this
exercise is considered genetic engineering. ..It is all genetic
recombination using natural means. ..That is one of the important things
about this exercise - this happens naturally around you all the time in
the genetically mobile and fluid microbial world.)
.....Back in the first decade of the 1900's, Dr. MacConkey concocted a
medium bearing his name. ..On MacConkey agar (page 61), E. coli that
possess the ability to ferment lactose (lac+) grow up as red colonies,
while lac- mutants grow up as pale colonies. ..Thus what do we need?
..Yes! ..MacConkey agar into which we have added some streptomycin.
(Controls?)
THE EXERCISE
1. Make two separate overnight cultures of the donors and recipients
in 0.7% tryptone (a la page 2). .. (Please don't follow the
directions on the bottles! ..Use TAP-water as these little critters
need minerals to grow.) .. Do NOT add any sugars to the medium!
2. Into a new sterile bottle of 20 ml of tryptone add 20 drops of the
recipient F-, and 1 drop of the donor F-lac. ..(Here strict aseptic
technique is not required because the product of this reaction will
be plated on antibiotic plates.)
3. At timed intervals make plate counts (page 112) of the culture
using the MacConkey agar PLUS streptomycin.* ..(The next day,
your results should show many pale colonies on the plates, but as
time proceeds there will be more and more red ones, which are
clones of the recombinants.)
* Making antibiotic medium is a little tricky as most antibiotics are
destroyed in heat, and therefore cannot be autoclaved. ..Of
course you are wondering why worry about sterilizing an antibiotic
in the first place. .. Because they are not free of bacteria! ..They
are either made by a type of bacteria or fungi, which will then
contaminate the powder. ..Thus the antibiotic powder must be
dissolved in water at the desired concentration, and then
sterilized. ..The most common way to do so is by "ultra-filtration"
through sterile filters that have such small holes that bacteria
cannot get through while the antibiotic molecules easily fit. .. The
filtrate is collected in a cool pre-sterilized container. ..The most
common devices are little filters that fit onto the ends of large
hypodermic syringes. .. The syringe (without the filter) is filled with
the antibiotic solution, and then the sterile filter device is screwed
onto the bottom of the syringe... The liquid is forced through the
filter by pushing down on the syringe piston. ..The stream or drops
of sterilized antibiotic solution is then allowed to flow into a
sterilized bottle that can the be screw capped tight. .. A popular
brand of such filter assemblies are 0.2nm AcroDisks®, and are
sold through most laboratory supply houses. .. (Virus suspensions
and heat labile vitamins are also sterilized in this same way.)
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Mechanisms of Evolution
Evolutionary stages of resistance
There are several stages in the development of resistance; full antibiotic resistance is
not necessarily conferred by an immediate change in the bacterial genome. Tolerance is
the ability of bacteria to survive in the presence of antibiotics, but not to continue cell
division. Resistance, on the other hand, is when bacteria can both survive and duplicate
when antibiotics are present (Novak, 1999). For example, there is concern that
vancomycin tolerant strains are now appearing, with documented cases in Streptococcus
pneumoniae (Novak, 1999). Because tolerance is often a direct precursor to resistance,
this observation is of serious concern. Resistance to vancomycin might leave no
alternative for patients.
It is hypothesized that tolerance may favor the development of resistance in bacteria.
Because tolerant bacteria can survive in the presence of antibiotics, they have the
opportunity to develop resistance. Since a selective pressure exists for bacteria that can
survive antibiotic treatment, tolerant strains often become resistant. Thus, it is possible
that tolerance may help lead to resistance by encouraging survival of cells tolerant of
antibiotics.
Species evolution
Vertical genetic exchange is when genetic information is passed down through
generations as cells divide. Horizontal genetic exchange is the "movement of genetic
information between bacteria other than by descent" (Maiden, 1998). Horizontal genetic
exchange is the primary mechanism of the evolution of antibiotic resistance. It is a
sexual process, which can take place through conjugation, transduction, or
transformation.
Conjugation is the transfer of DNA from one cell to another by direct cell-cell
contact. Transduction is when DNA is transferred from one cell to another by a
bacteriophage. Transformation is a process in which "a DNA molecule is taken up from
the external environment and incorporated into the genome" (Hartl and Jones, 1998,
p308). The three processes yield DNA that can be replicated and passed on to progeny.
Therefore, these three mechanisms will be described in further detail, as applied to the
evolution of antibiotic resistance. Figure 2 diagrams an overview of the three processes.
Figure 2: Transfer of Genetic Material through transformation, translation, and
conjugation
figure taken from Levy, 1998
see reference in text
Conjugation
Conjugation plays a large role in the spread of antibiotic resistance through bacteria.
This process involves direct cell-to-cell contact of two bacterial cells, and the
subsequent transfer of DNA. Conjugation can occur between species that are unrelated;
for this reason, a large gene pool is available from which bacteria can exchange and
acquire new genetic material. (Guiney, 1984). Sex pili make contact between the donor
and the recipient cell. Once the two cell walls are in contact, this allows a mating bridge
to form. The plasmid DNA in the donor, possibly containing antibiotic resistance genes, is
nicked in one strand; this strand proceeds into the recipient cell by undergoing rollingcircle replication (Hartl and Jones, 1998, p316). Figure 3 demonstrates the process of
rolling-circle replication. Complementary copies of the DNA are produced in both the
donor and the recipient cells. Finally, the linear plasmid in the recipient becomes circular
and is ligated, and then both of the cells have a copy of the plasmid.
Figure 3: Rolling Circle Replication
see reference in text
There are certain barriers to the process of conjugation, which often play a role in the
evolution of resistance. First, interactions at the cell surface are involved. Contact must
occur for mating to take place. Next, foreign DNA is susceptible to host restriction
modification systems which target and cleave foreign DNA. Third, the new plasmid be
unable to replicate in the new host. An appropriate origin of replication in the new host
may not be available, thus preventing replication.
For example, transferred DNA is more likely to be stable in a new host if it contains
fewer restriction enzyme sites. This makes it less likely that the DNA will be degraded
by restriction enzymes which attack foreign DNA. A selective advantage exists for
plasmids with fewer restriction sites. Selective advantages which aid in stability can
parallel the beginnings of the evolution of strains that are resistant to antibiotics.
Antibiotic resistance will spread more readily if the resistance genes can be transferred
into non-resistant cells. The ability to both invade many types of cells and to be
maintained in these cells are traits that are advantageous for survival and transfer of
plasmids.
Transduction
Transduction occurs when a bacteriophage carries DNA from one species to another.
When a bacteriophage destroys its current host and invades a new one, it may carry
pieces of chromosomal DNA or plasmids from the previous host. An occasional phage may
carry some bacterial DNA. Recombination can then occur between the phage (carrying
bacterial DNA) and the new host's bacterial DNA. Transfer of DNA thus occurs from
one bacterial cell to another, carried by a bacteriophage. ( Lacey, 1984 and Hartl and
Jones, 1998, p308). This provides another method for the spread of resistance among
bacteria.
Transformation
Transformation is another method of acquiring resistance. During transformation,
bacterial cells take up DNA from the surrounding environment. Certain requirements
exist in order for transformation to take place. First, exogenous DNA must be present in
the immediate environment. Bacteria must have mechanisms that allow the DNA to be
taken up through the bacterial cell walls. Also, the DNA must be incorporated into the
chromosome of the host, often by homologous recombination. During homologous
recombination, parts of the chromosome are replaced with related DNA (Maiden, 1998).
Restriction modification systems play a role in transformation as well as in conjugation.
However, it is thought that since these modification systems generate both DNA ends
and smaller fragments, restriction modification may actually increase the chance of
recombination with incorporated fragments. This could occur because recombination
occurs more frequently if the ends are homologous.
Possible origin of antibiotic resistance genes
So now we have some mechanisms by which transfer and uptake of resistance can
occur. But where did this resistance originate? The answer is complicated; some
resistance is actually the result of random mutations that provide a selective advantage.
However, there are other suspected sources of resistance.
Many resistance genes may have come from bacteria in soil and water. Soil and water
bacteria must live and survive along with fungi/actinomycetes, which produce antibiotics.
These bacteria must possess a certain level of resistance in order to survive in their
natural environment. Some organisms can exchange plasmids with those in soil and water,
as well as with one another. Therefore, antibiotic resistance genes may have originated
from bacteria in this environment (Guiney, 1984).
When antibiotics are isolated from sources such as fungi, some resistance genes may
be isolated as well. Therefore, ironically, antibiotic preparations may be contaminated
with DNA encoding resistance genes. This increases the chance that genetic exchange
can occur with the bacteria they are trying to kill - the close proximity increases the
chance of transformation of DNA (Davies, 1994).
There is also some clue into the origin of resistance to -lactam antibiotics. Resistance
genes were detected even before the use of antibiotics began. For example, a strain of
Staph aureus was found which produced -lactamase--before penicillin was even
discovered. It is believed that lyzozyme, which is present in nasal secretions, and is a
"natural bactericidal agent," played a role (Lacey, 1984). Lyzozyme may have imposed a
selective pressure, which helped strains to emerge with antibiotic resistance, even
before our discovery of antibiotics.
Genetic mechanisms
The basic mechanisms of transformation, transduction, and conjugation provide a
starting point for more complex mechanisms of the development and spread of antibiotic
resistance. Some common mechanisms are outlined below and illustrated in figure 4
Point mutations
Point mutations are also involved in the development of antibiotic resistance.
Spontaneous changes in single nucleotides have produced resistance to some antibiotics.
(Lacey, 1984). Point mutations are usually random, and thus occur before exposure to
antibiotics. For example, one base change in the -lactamase gene (which cleaves lactam antibiotics such as penicillin) can change this enzyme's substrate specificity
(Davies, 1994). Such point mutations may cause changes in the target molecule of the
antibiotic. Often however, several random point mutations must occur in order to confer
resistance; therefore, this type of resistance development is somewhat less common.
Intragenic recombination
When smaller fragments of DNA are incorporated through transformation, intragenic
recombination and mosaic genes result. Mosaic genes contain DNA of the original allele in
some locations, but from different genes or organisms in other locations. This is a result
of intragenic recombination. These genes may express proteins that have new phenotypes
(Maiden, 1998). Most mosaic genes are lost because they represent only a low frequency
of the genes. However, some mosaic genes may express a phenotype that helps the
organism to survive. An example of this phenomenon is a mosaic gene encoding an altered
penicillin binding protein with low affinity for -lactam antibiotics. This mosaic gene
allows survival in the presence the antibiotic, and may be favored by selection. Thus, the
selective pressure of antibiotics may be promoting the maintenance and spread of mosaic
genes (Maiden, 1998).
Transposons
Transposable elements are small regions of DNA that can move from one place to
another in the genome. Therefore, these play a role in evolution of antibiotic resistance
as well, by providing yet another method of genetic exchange. Transposable elements
that contain genes in the central region are called transposons. This central sequence
may contain resistance to one or more antibiotics for example (making multiple antibiotic
resistance probable). Genes in transposons can be transferred between bacterial hosts
by transposition into bacterial plasmids, which can then undergo conjugation (Hartl and
Jones, 1998, p347).
Transposons make multiple antibiotic resistance not only possible, but very likely; this
mechanism provides an easy and efficient way for the transfer of resistance to several
antibiotics to be spread at one time.
Figure 4: Mutations which may Confer Antibiotic Resistance
see reference in text
Several mechanisms exist for the transfer of resistance between bacteria. When the
selective pressure of antibiotics is imposed, bacteria have a large population of
resistance genes available to them (Davies, 1994). This provides an environment where
the development and spread of antibiotic resistance is likely to continue indefinitely,
both due to the selective pressure of antibiotics and to the large existence of resistance
in the population currently. In other words, discontinuing the use of antibiotics would
probably not stop the spread of resistance entirely. The selective pressure would be
lower, but the resistance could still be spread throughout the population in ways such as
those described above. In addition, continued use of antibiotics will only worsen the
problem. For this reason, close attention to the mechanisms of antibiotic resistance is
necessary in order to gain an understanding of how to develop more effective antibiotics.
Plasmid
From Wikipedia, the free encyclopedia
(Redirected from Plasmids)
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Figure 1: Schematic drawing of a bacterium with plasmids enclosed. (1)Chromosomal
DNA. (2) Plasmids
Plasmids are (typically) circular double-stranded DNA molecules that are separate from
the chromosomal DNA (Fig. 1). They usually occur in bacteria, sometimes in eukaryotic
organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae). Their size varies
from 1 to over 400 kilobase pairs (kbp). There are anywhere from one copy, for large
plasmids, to hundreds of copies of the same plasmid present in a single cell.
Contents
[hide]
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1 Antibiotic resistance
2 Episomes
3 Vectors
4 Types of plasmid
5 Applications of plasmids
6 Plasmid DNA extraction
7 Conformations
8 See also
[edit]
Antibiotic resistance
Figure 2: Schematic drawing of a plasmid with antibiotic resistances
Plasmids often contain genes or gene-cassettes that confer a selective advantage to the
bacterium harboring them, e.g., the ability to make the bacterium antibiotic resistant.
Every plasmid contains at least one DNA sequence that serves as an origin of replication
or ori (a starting point for DNA replication), which enables the plasmid DNA to be
duplicated independently from the chromosomal DNA (Fig. 2)
[edit]
Episomes
Episomes are plasmids that can integrate themselves into the chromosomal DNA of the
host organism (Fig. 3). For this reason, they can stay intact for a long time, be duplicated
with every cell division of the host, and become a basic part of its genetic makeup. This
term is no longer commonly used for plasmids, since it is now clear that a region of
homology with the chromosome such as a transposon makes a plasmid into an episome.
[edit]
Vectors
Figure 3: Comparison of non-integrating plasmids (top) and episomes (bottom). 1
Chromosomal DNA. 2 Plasmids. 3 Cell division. 4 Chromosomal DNA with integrated
plasmids
Plasmids used in genetic engineering are called vectors. They are used to transfer genes
from one organism to another and typically contain a genetic marker conferring a
phenotype that can be selected for or against. Most also contain a polylinker or multiple
cloning site (MCS), which is a short region containing several commonly used restriction
sites allowing the easy insertion of DNA fragments at this location. See also
'Applications of plasmids', below.
[edit]
Types of plasmid
One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative
plasmids contain so-called tra-genes, which perform the complex process of conjugation,
the sexual transfer of plasmids to another bacterium (Fig. 4). Non-conjugative plasmids
are incapable of initiating conjugation, hence they can only be transferred with the
assistance of conjugative plasmids, by 'accident'. An intermediate class of plasmids are
mobilisable, and carry only a subset of the genes required for transfer. These plasmids
can 'parasitise' another plasmid, transferring at high frequency in the presence of a
conjugative plasmid.
It is possible for several different types of plasmids to coexist in a single cell, e.g., seven
different plasmids have been found in E. coli. On the other hand, related plasmids are
often 'incompatible', resulting in the loss of one of them from the cell line. Therefore,
plasmids can
Figure 4 : Schematic drawing of bacterial conjugation. 1 Chromosomal DNA. 2
Plasmids. 3 Pilus.
be assigned into incompatibility groups, depending on their ability to coexist in a single
cell. These incompatibility groupings are due to the regulation of vital plasmid functions.
An obvious way of classifying plasmids is by function. There are five main classes:
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Fertility-(F)plasmids, which contain tra-genes.
They are capable of conjugation.
Resistance-(R)plasmids, which contain genes that
can build a resistance against antibiotics or poisons.
Historically known as R-factors, before the nature
of plasmids was understood.
Col-plasmids, which contain genes that code for
(determine the production of) colicines, proteins
that can kill other bacteria.
Degrative plasmids, which enable the digestion of
unusual substances, e.g., toluene or salicylic acid.
Virulence plasmids, which turn the bacterium into a
pathogen.
Plasmids can belong to more than one of these functional groups.
Plasmids that exist only as one or a few copies in each bacterium are, upon cell division,
in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have
systems which attempt to actively distribute a copy to both daughter cells.
Some plasmids include an addiction system. These plasmids produce both a long-lived
poison and a short-lived antidote. Daughter cells that retain a copy of the plasmid survive,
while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate
because of the lingering poison from the parent cell. This is an example of plasmids as
selfish DNA.
[edit]
Applications of plasmids
Plasmids serve as important tools in genetics and biochemistry labs, where they are
commonly used to multiply (make many copies of) or express particular genes. There are
many plasmids that are commercially available for such uses. Initially, the gene to be
replicated is inserted in a plasmid. These plasmids contain, in addition to the inserted
gene, one or more genes capable of providing antibiotic resistance to the bacterium that
harbors them. The plasmids are next inserted into bacteria by a process called
transformation, which are then grown on specific antibiotic(s). Bacteria which took up
one or more copies of the plasmid then express (make protein from) the gene that confers
antibiotic resistance. This is typically a protein which can break down any antibiotics that
would otherwise kill the cell. As a result, only the bacteria with antibiotic resistance can
survive, the very same bacteria containing the genes to be replicated. The antibiotic(s)
will, however, kill those bacteria that did not receive a plasmid, because they have no
antibiotic resistance genes. In this way the antibiotic(s) acts as a filter selecting out only
the modified bacteria. Now these bacteria can be grown in large amounts, harvested and
lysed to isolate the plasmid of interest.
Another major use of plasmids is to make large amounts of proteins. In this case you
grow the bacteria containing a plasmid harboring the gene of interest. Just as the bacteria
produces proteins to confer its antibiotic resistance, it can also be induced to produce
large amounts of proteins from the inserted gene. This is a cheap and easy way of massproducing a gene or the protein it then codes for--for example, insulin or even antibiotics.
[edit]
Plasmid DNA extraction
As alluded to above, plasmids are often used to purify a specific sequence, since they can
easily be purified away from the rest of the genome. For their use as vectors, and for
molecular cloning, plasmids often need to be isolated.
There are several methods to isolate plasmid DNA from bacteria, the archaetypes of
which are the miniprep and the maxiprep. The former can be used to quickly find out
whether the plasmid is correct in any of several bacterial clones. The yield is a small
amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and
for some cloning techniques. In the latter, much larger volumes of bacterial suspension
are grown from which a maxi-prep can be performed. Essentially this is a scaled-up
miniprep followed by additional purification. This results in relatively large amounts
(several ug) of very pure plasmid DNA.
In recent times many commercial kits have been created to perform plasmid extraction at
various scales, purity and levels of automation.
[edit]
Conformations
When performing DNA_electrophoresis, plasmid DNA may appear in the following five
conformations:

"Supercoiled" (or "Covalently Closed-Circular")
DNA is fully intact with both strands uncut.
 "Relaxed Circular" DNA is fully intact with both
strands uncut, but has been enzymatically "relaxed"
(supercoils removed).
 "Supercoiled Denatured" DNA, is not a "natural"
form present in vivo. It is a contaminent often
produced in small quantities following excessive
alkaline lysis; both strands are uncut but are not
correctly paired, resulting in a compacted plasmid
form.
 "Nicked Open-Circular" DNA has one strand cut.

"Linearized" DNA has both strands cut site at only
one site.
The relative electrophoretic mobility (speed) of these DNA conformations in a gel are as
follows:
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Nicked Open Circular (slowest)
Linear
Relaxed Circular
Supercoiled Denatured
Supercoiled (fastest)
The rate of migration for small linear fragments is directly proportional to the voltage
applied at low voltages. At higher voltages, larger fragments migrate at continually
increasing yet different rates. Therefore the resolution of a gel decreases with increased
voltage.
At a specified, low voltage, the migration rate of small linear DNA fragments is a
function of their length. Large linear fragments (over 20kb or so) migrate at a certain
fixed rate regardless of length. This is because the molecules 'reptate', with the bulk of the
molecule following the leading end through the gel matrix. Restriction digests are
frequently used to analyse purified plasmids. Enzymes specifically break the DNA at
certain short sequences. The resulting linear fragments form 'bands' after gel
electrophoresis.
[edit]
See also

Bacterial artificial chromosome
Retrieved from "http://en.wikipedia.org/wiki/Plasmid"
Category: Molecular biology
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Bacterial artificial chromosome
From Wikipedia, the free encyclopedia
Jump to: navigation, search
A bacterial artificial chromosome (BAC) is a DNA construct, based on a fertility
plasmid, used for transforming and cloning in bacteria, usually E. coli. Its usual insert
size is 150 kbp, with a range from 100 to 300 kbp.
BACs are often used to sequence the genetic code of organisms in genome projects, for
example the Human Genome Project. A short piece of the organism's DNA is amplified
as an insert in BACs, and then sequenced. Finally, the sequenced parts are rearranged in
silico, resulting in the genomic sequence of the organism.
Retrieved from "http://en.wikipedia.org/wiki/Bacterial_artificial_chromosome"
Category: Molecular biology
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Category:Molecular biology
From Wikipedia, the free encyclopedia
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Molecular biology is the study of biology at a molecular level. The field overlaps with
other areas of biology, particularly genetics and biochemistry. Molecular biology chiefly
concerns itself with understanding the interactions between the various systems of a cell,
including the interrelationship of DNA, RNA and protein synthesis and learning how
these interactions are regulated.
The main article for this category is Molecular
biology.
Subcategories
There are 6 subcategories to this category.
E
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Electrophoresis
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Gene expression
G
M
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Molecular biologists
Molecular genetics
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Peptides
Proteins
P
Articles in category "Molecular biology"
There are 153 articles in this section of this category.
*
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G cont.
P cont.
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Molecular biology
List of molecular
biology topics
A
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Affymetrix
H
Agarose gel
electrophoresis
Amplified fragment
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length polymorphism
Antisense
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Bacterial artificial
I
chromosome
Bacterial conjugation
Bonnie Bassler
Biochip
Biopolymer
Blot (biology)
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C-myc
CDNA library
Central dogma of
molecular biology
Chromosome
Immunoelectrophoresis
Immunomagnetic
separation
Insertion sequence
International Nucleotide
Sequence Database
Collaboration
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Protein-fragment
Complementation
Assay
Proteinoid
Pyrenoid
Pyrosequencing
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C
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Helicase-dependent
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amplification
Henderson limit
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Hybridisation (molecular
biology)

Hybridization probe


B
Gene copy number
Gene gun
Gene therapy
Genetic fingerprinting
Genetically modified
organism
Knockout mouse





Q-PCR
RNase H
RT-PCR
Real-time PCR
Replica plating
Reporter gene
Restriction digest
Restriction
enzyme
Restriction
fragment length
polymorphism
Restriction sites
Retrotransposon
Retrovirus
Reverse
transcriptase






walking
Cloning
Cloning vector
Competent cell
Complementarity
(molecular biology)
Cosmid
Cycling probe
technology
D

Krüppel associated box
S





L




Library (biology)
Lipofection
List of proteins
List of publications in
biology


M












DNA bank

DNA computing

DNA electrophoresis

DNA extraction

DNA ligase
DNA microarray
N
DNA sequencing
DNA topology

DNA-DNA

hybridisation

DNase footprinting

assay

Dideoxynucleotides

Downstream

(molecular biology)
N-formylmethionine
Neuropeptide
Nick translation
Nonribosomal peptide
Northern blot
Nucleic acid
Nucleosome
O
E









EMBO Journal
EcoRI
P
Edman degradation
Electrophoresis

Electrophoretic

mobility shift assay

Electroporation

Endogenous

retrovirus

Expression profiling
F


F1 generation
Fluorescence
resonance energy












Minipreparation
Molecular genetics
Molecular lesion
Molecular modelling
Optical tweezers
T








P element
Percentage solution
Phage display
Phagemid
Phosphodiester bonds
Phosphodiesterase
Pilus
Plant breeding
Plant virus
Plasmid
Polyhistidine-tag
Polyketide
SDS-PAGE
STING
Salvage synthesis
Sequencing
Serial Analysis of
Gene Expression
Shotgun
sequencing
Single nucleotide
polymorphism
Southern blot
Southwestern blot
Standard curve
Structural biology
Subcloning
Superhelix






Tachykinin
peptides
Taffazin
Temperature
gradient gel
electrophoresis
Thermal cycler
Tilling
Transduction
(genetics)
Transfection
Transformation
(genetics)
Transgenic plants
Transposase
Transposon
Triparental
mating
Two-dimensional
gel
electrophoresis
Two-hybrid
screening



transfer
Fluorescent in situ
hybridization
Fluorescent tag
Functional genomics





G




Gel electrophoresis
Gel extraction
Gel filtration
chromatography
Gene




Polymerase chain
U
reaction
Post transcriptional gene

silencing

Primer (molecular
biology)
Protein
Protein Information
W
Resource
Protein Misfolding

Cyclic Amplification
Protein electrophoresis Y
Protein microarray
Protein tag
UniProt
Upstream
(molecular
biology)
Western blot

Yeast artificial
chromosome

Zinc finger
protein
Zymography
Z

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Category:Electrophoresis
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Electrophoresis is a method of moving charged particles through a medium by using an
electric field induced by electrodes. It is also used to separate molecules with different
physical characteristics using electrical charges.
The main article for this category is
Electrophoresis.
Articles in category "Electrophoresis"
There are 13 articles in this section of this category.
A
E

P


Agarose gel
electrophoresis
Electrophoresis
Electrophoresis
(disambiguation)

Protein electrophoresis

Serum protein
electrophoresis

Temperature gradient
gel electrophoresis
Two-dimensional gel
electrophoresis
S
C
G

Capillary
electrophoresis

Gel electrophoresis
T
D
I


DNA
electrophoresis
Difference gel
electrophoresis


Iontocaine
Iontophoresis

Retrieved from "http://en.wikipedia.org/wiki/Category:Electrophoresis"
Categories: Molecular biology | Analytical chemistry
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Category:Gene expression
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Gene expression is the multi-step process by which a gene's information is converted
into the structures and functions of a cell, following the central dogma of molecular
biology.
The main article for this category is Gene
expression.
Subcategories
There is 1 subcategory to this category.
T

Transcription factors
Articles in category "Gene expression"
There are 67 articles in this section of this category.
A



I cont.
R cont.




Regulatory sequence
Repressor
SECIS element
STAT protein
Shine-Dalgarno
sequence
Signal peptide
Silencer (DNA)
Sox2
Spliceosome
Splicing (genetics)
Activator (genetics)
Alternative splicing
Anisomycin
Inducible gene
Intron
K
S
B

Basic-helix-loophelix

CCAAT box
Cis-acting element
Coactivator
(genetics)
Constitutive gene
Krüppel associated box





Lac operon
Lac repressor




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
MSin3 interaction
domain
Messenger RNA
Monocistronic
L
C

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M


D


DNA microarray
Dicistronic
N

E





Enhancer
Exon trapping
O
Expressed sequence
tag

Expression vector

G
P




Gene expression
Gene regulatory
network
Gene silencing
Genetic code


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
H

Nanog (transcription
factor)
Noncoding DNA
Oct-4
Operon
T




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




Paramutation
U
Polyadenylation
Polycistronic

Posttranslational
modification
Preliminary messenger
Z
RNA
Pribnow box
TATA box
Three prime
untranslated region
Trans-acting factor
Transcription
(genetics)
Transcription Factor
II D
Transcription factor
Transfer RNA
Translation
(genetics)
Translational errors
Transvection
(genetics)
Upstream
transcription factor


Housekeeping gene
Hypoxia inducible
factors
I



Promoter
Protein biosynthesis
Protein synthesis


RNA interference
Regulation of gene
expression

Zif268
R


Icsbp
Imprinting
(genetics)
Retrieved from "http://en.wikipedia.org/wiki/Category:Gene_expression"
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Category:Molecular genetics
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Molecular genetics is the field of biology which studies the structure and function of
genes at a molecular level. Molecular genetics employs the methods of genetics and
molecular biology.
The main article for this category is Molecular
genetics.
Subcategories
There are 4 subcategories to this category.
D


DNA repair
DNA replication

Gene expression

Repetitive DNA sequences
G
R
Articles in category "Molecular genetics"
There are 55 articles in this section of this category.
*

C cont.
M cont.



Mitotic crossover
Molecular clock



Non-coding RNA
Nuclear localization
signal
Nucleotide diversity

Open reading frame

PMal-C2


RNA interference
RNA polymerase
Molecular genetics
A
CpG site
D
N



AP site
Array comparative
genomic
hybridization
Auxotrophy



E

B


Biopolymer
C
DNA barcoding
DNA repair
Dyad symmetry
Electrophoretic
mobility shift assay
Euchromatin
G





C7 protein
C7.GAT protein
CAAT box
CCAAT box
Central dogma of
molecular biology
O
P






Gene duplication
Gene family
Gene gun
Genetic code
Genetic engineering
Genetic screen
R








Chromatid
Chromatin
Chromosomal
crossover
Coactivator
(genetics)
Comparative
genomic
hybridization
Conservation
(genetics)
Consortium for the
Barcode of Life
CpG island

Germinal choice
technology



Recombinant DNA
Retrotransposon
Reverse genetics


Sigma factor
Site-directed
mutagenesis
Sp1 (biology)
Sp1C
Synthetic biology
H
S

Heterochromatin
I





Imprinting (genetics)
Intein
K
T

Krüppel associated
box

Transcription Factor
II D
M


MSin3 interaction
domain
Mitochondrial genome
Retrieved from "http://en.wikipedia.org/wiki/Category:Molecular_genetics"
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Bugs Fighting Back
Basics of Bacterial Resistance
How Bacteria Become Resistant
Once it was thought that antibiotics would help us wipe out
forever the diseases caused by bacteria. But the bacteria have
fought back by developing resistance to many antibiotics.
Bacterial resistance to antibiotics can be acquired in four ways:
1. Through a spontaneous change in the bacterium's DNA:
Changes like this are called mutations. Mutations happen at
random in all living things, and they can result in all
kinds of changes in the bacterium. Antibiotic resistance is
just one of many changes that can result from a random
mutation.
2. Transformation: This happens when one bacterium takes up
some DNA from the chromosomes of another bacterium.
3. Plasmid exchange: Antibiotic resistance can be coded for in
the DNA found in a small circle known as a plasmid in a
bacterium. The plasmids can randomly pass between bacteria
(usually touching).
4. Sharing of mutations, some of which control resistance to
antibiotics. Two examples are:
 Plasmid transfer between different kinds of bacteria.
This can happen between similar bacteria and between
very dissimilar bacteria.
 Gene cassettes are a small group of genes that can be
added to a bacterium's chromosomes. The bacteria can
then accept a variety of gene cassettes that give the
bacterium resistance to a variety of antibiotics. The
cassettes also can confirm resistance against
disinfectants and pollutants.
The acquired genetically based resistance is permanent and
inheritable through the reproductive process of bacteria, called
fission.
Some bacteria produce their own antibiotics to protect themselves
against other microorganisms. Of course, a bacterium will be
resistant to its own antibiotic! But sometimes the DNA that gives
that bacterium resistance to its own antibiotic can be
transferred to a bacterium of another species. Then that other
bacterium could be resistant to the first bacterium's antibiotic!
Scientists think, but haven't proved, that the genes for
resistance in this case have been transferred between bacteria of
different species through plasmid or cassette transfer.
Laboratory analysis of commercial antibiotic preparations has
shown that they contain DNA from antibiotic-producing organisms.
The DNA includes the antibiotic-resistance gene sequence.
Genetic transfer may be induced by the bacteria involved, that is
the source and the destination bacteria. One model suggests that
when a DNA resistance plasmid released by one bacterium is
accepted by a different species of bacterium, the recipient may
be stimulated to release its own plasmid. The process is known as
retrotransfer.
Resistant genes occur not only in bacteria that carry disease,
but also in commensal bacteria (those living within the same
environment—soil, water, digestive tract—benefiting from each
other). Eating meat or milk from animals that have been exposed
to antibiotics, or plants that have been exposed to pesticides,
brings the antibiotic and/or resistant bacteria in contact with
bacteria in your digestive tract. The interaction between
bacteria can then allow for transfer of genes for antibiotic
resistance to the bacteria in your intestines.
How Resistance Works
Some mechanisms for resistance include:
o
o
o
o
o
Changing the target molecule: For example, if the
antibiotic attacks a certain enzyme in a bacterium, the
bacterium can adapt by using a different enzyme to
accomplish the same function.
Enzymatically inactivating or decomposing the antibiotic.
Sequestering (storing) the drug by creating alternative
pathways within the bacterium.
Preventing the drug from entering the bacterium.
Pumping out the antibiotic as quickly as it enters the
bacterium.
Recommended Actions for Consumers and Physicians to Limit Resistance
(from Levy, S.B. Scientific American, March 1998; p 53.)
Consumers:
o
o
o
o
o
Do not demand antibiotics.
Never use antibiotics unless they are prescribed by your
doctor.
When given antibiotics, take them exactly as prescribed and
complete the full course of treatment: continue taking the
antibiotic even after you start to feel well, and do not
hoard pills for later use.
Wash fruits and vegetables thoroughly; avoid raw eggs and
undercooked meat, especially in ground form.
Avoid antibacterial soaps and other products unless you are
caring for a sick person whose defenses are weakened.
Physicians (Who knows? You might be a doctor someday!):
o
o
Wash hands thoroughly between patient visits.
Do not give in to patients' demands for unneeded
antibiotics.
o
o
o
When possible, prescribe antibiotics that target only a
narrow range of bacteria.
Isolate hospital patients with multidrug-resistant
infections.
Familiarize yourself with local data on antibiotic
resistance.
For Further Reading
20.
Nordenberg, Tamar. “Miracle Drugs vs. Superbugs,” FDA
Consumer, November/December 1998; pp 22-25.
21.
Radetsky, Peter “Last Days of the Wonder Drugs,”
Discover, November 1998; pp 76-85.
22.
Levy, S.B. “The Challenge of Antibiotic Resistance,”
Scientific American, March 1998; pp. 46-53.
23.
Miller, R.V. “Bacterial Gene Swapping in Nature,”
Scientific American, January 1998.
For more information, at other Web sites...
Agricultural Antibiotic Use Could Contribute to Drug
Resistance — from Scientific American, 23 April 2002.
The Alliance for the Prudent Use of Antibiotics (APUA) — official site
with information for patients and health care professionals.
Ribosome Research Encompasses Early Life on Earth and Antibiotic
Resistance — news article from Argonne National Laboratory, 26
October 2001.
The Rise of Antibiotic-Resistant Infections — from FDA Consumer,
September 1995, published by the U.S. Food and Drug
Administration.
Back to:
Antibiotics in Action Directory | Site Map | Pharmaceutical Achievers Home
References
24.
Devitt, Terry, ed. Microbes: What Doesn't Kill them
Makes them Stronger, The Why Files, University of
Wisconsin, May 1997.
25.
Hawkey, Peter M. “The Origins and Molecular Basis of
Antibiotic Resistance,” British Medical Journal, September
5, 1998, pp. 657-660.
26.
Levy, S.B. "The Challenge of Antibiotic Resistance,"
Scientific American, March 1998; pp. 46-53.
Copyright ©2002 The Chemical Heritage Foundation
Recombinant
DNA and
Gene Cloning
Recombinant DNA is DNA
that has been created
artificially. DNA from two or
more sources is incorporated
into a single recombinant
molecule.






Index to this page
An Overview
Plasmids
An Example
o pAMP
o pKAN
o Ligation Possibilities
Transforming E. coli
Cloning other Genes
Recombinant DNA products for human therapy
Making Recombinant DNA (rDNA): An Overview

Treat DNA from both sources with the same restriction endonuclease (BamHI in
this case).

BamHI cuts the same site on both molecules
5' GGATCC 3'
3' CCTAGG 5'




The ends of the cut have an overhanging piece of single-stranded DNA.
These are called "sticky ends" because they are able to base pair with any DNA
molecule containing the complementary sticky end.
In this case, both DNA preparations have complementary sticky ends and thus can
pair with each other when mixed.
DNA ligase covalently links the two into a molecule of recombinant DNA.
To be useful, the recombinant molecule must be replicated many times to provide
material for analysis, sequencing, etc. Producing many identical copies of the same
recombinant molecule is called cloning. Cloning can be done in vitro, by a process called
the polymerase chain reaction (PCR). Here, however, we shall examine how cloning is
done in vivo.
Cloning in vivo can be done in



unicellular prokaryotes like E. coli
unicellular eukaryotes like yeast and
in mammalian cells grown in tissue culture.
In every case, the recombinant DNA must be taken up by the cell in a form in which it
can be replicated and expressed. This is achieved by incorporating the DNA in a vector.
A number of viruses (both bacterial and of mammalian cells) can serve as vectors. But
here let us examine an example of cloning using E. coli as the host and a plasmid as the
vector.
Plasmids
Plasmids are molecules of DNA that are
found in bacteria separate from the
bacterial chromosome.
They:




are small (a few thousand base
pairs)
usually carry only one or a few
genes
are circular
have a single origin of replication
Plasmids are replicated by the same machinery that replicates the bacterial chromosome.
Some plasmids are copied at about the same rate as the chromosome, so a single cell is
apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate
and a single cell may have 50 or more of them.
Genes on plasmids with high numbers of copies are usually expressed at high levels. In
nature, these genes often encode proteins (e.g., enzymes) that protect the bacterium from
one or more antibiotics.
Plasmids enter the bacterial cell with relative ease. This occurs in nature and may account
for the rapid spread of antibiotic resistance in hospitals and elsewhere. Plasmids can be
deliberately introduced into bacteria in the laboratory transforming the cell with the
incoming genes.
An Example
(courtesy of David Miklos and Greg Freyer of the Cold Spring Harbor Laboratory, who
used these plasmids as the basis of a laboratory introduction to recombinant DNA
technology that every serious biology student — high school or college — should
experience!)
pAMP




4539 base pairs
a single replication origin
a gene (ampr)conferring resistance to the antibiotic ampicillin (a relative of
penicillin)
a single occurrence of the sequence
5' GGATCC 3'
3' CCTAGG 5'
that, as we saw above, is cut by the restriction enzyme BamHI

a single occurrence of the sequence
5' AAGCTT 3'
3' TTCGAA 5'
that is cut by the restriction enzyme HindIII
Treatment of pAMP with a mixture of BamHI and HindIII produces:


a fragment of 3755 base pairs carrying both the ampr gene and the replication
origin
a fragment of 784 base pairs

both fragments have sticky ends
pKAN





4207 base pairs
a single replication origin
a gene (kanr) conferring resistance to the antibiotic kanamycin.
a single site cut by BamHI
a single site cut by HindIII
Treatment of pKAN with a mixture of BamHI and HindIII produces:



a fragment of 2332 base pairs
a fragment of 1875 base pairs with the kanr gene (but no origin of replication)
both fragments have sticky ends
These fragments can be visualized by subjecting the digestion mixtures to electrophoresis
in an agarose gel. Because of its negatively-charged phosphate groups, DNA migrates
toward the positive electrode (anode) when a direct current is applied. The smaller the
fragment, the farther it migrates in the gel.
Ligation Possibilities
If you remove the two restriction enzymes and provide the conditions for DNA ligase to
do its work, the pieces of these plasmids can rejoin (thanks to the complementarity of
their sticky ends).
Mixing the pKAN and pAMP fragments provides several (at least 10) possibilities of
rejoined molecules. Some of these will not produce functional plasmids (molecules with
two or with no replication origin cannot function).
One interesting possibility is the joining of


the 3755-bp pAMP fragment (with ampr and a replication origin) with the
1875-bp pKAN fragment (with kanr)
Sealed with DNA ligase, these molecules are functioning plasmids that are capable of
conferring resistance to both ampicillin and kanamycin. They are molecules of
recombinant DNA.
Because the replication origin, which enables the molecule to function as a plasmid, was
contributed by pAMP, pAMP is called the vector.
Transforming E. coli
Treatment of E. coli with the mixture of religated molecules will produce some colonies
that are able to grow in the presence of both ampicillin and kanamycin.
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A suspension of E. coli is treated with the mixture of religated DNA molecules.
The suspension is spread on the surface of agar containing both ampicillin and
kanamycin.
The next day, a few cells — resistant to both antibiotics — will have grown into
visible colonies containing billions of transformed cells.
Each colony represents a clone of transformed cells.
However, E. coli can be simultaneously transformed
by more than one plasmid, so we must demonstrate
that the transformed cells have acquired the
recombinant plasmid.
Electrophoresis of the DNA from doubly-resistant
colonies (clones) tells the story.
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Plasmid DNA from cells that acquired their resistance from a recombinant
plasmid only show only the 3755-bp and 1875-bp bands (Clone 1, lane 3).
Clone 2 (Lane 4) was simultaneous transformed by religated pAMP and pKAN.
(We cannot tell if it took up the recombinant molecule as well.)
Clone 3 (Lane 5) was transformed by the recombinant molecule as well as by an
intact pKAN.
Cloning other Genes
The recombinant vector described above could itself be a useful tool for cloning other
genes. Let us assume that within its kanamycin resistance gene (kanr) there is a single
occurrence of the sequence
5' GAATTC 3'
3' CTTAAG 5'
This is cut by the restriction enzyme EcoRI, producing sticky ends.
If we treat any other sample of DNA, e.g., from human cells, with EcoRI, fragments with
the same sticky ends will be formed. Mixed with EcoRI-treated plasmid and DNA ligase,
a small number of the human
molecules will become incorporated
into the plasmid which can then be
used to transform E. coli.
But how to detect those clones of E.
coli that have been transformed by a
plasmid carrying a piece of human
DNA?
The key is that the EcoRI site is within
the kanr gene, so when a piece of
human DNA is inserted there, the
gene's function is destroyed.
All E. coli cells transformed by the
vector, whether it carries human DNA
or not, can grow in the presence of
ampicillin. But E. coli cells
transformed by a plasmid carrying
human DNA will be unable to grow in the presence of kanamycin.
So,
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Spread a suspension of treated E. coli on agar containing ampicillin only
grow overnight
with a sterile toothpick transfer a small amount of each colony to an identified
spot on agar containing kanamycin
(do the same with another ampicillin plate)
Incubate overnight
All those clones that continue to grow on ampicillin but fail to grow on kanamycin (here,
clones 2, 5, and 8) have been transformed with a piece of human DNA.
Some recombinant DNA products being used in human
therapy
Using procedures like this, many human genes have been cloned in E. coli or in yeast.
This has made it possible — for the first time — to produce unlimited amounts of human
proteins in vitro. Cultured cells (E. coli, yeast, mammalian cells) transformed with the
human gene are being used to manufacture:
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insulin for diabetics
factor VIII for males suffering from hemophilia A
factor IX for hemophilia B
human growth hormone (GH)
erythropoietin (EPO) for treating anemia
three types of interferons
several interleukins
granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating
the bone marrow after a bone marrow transplant
granulocyte colony-stimulating factor (G-CSF) for stimulating neutrophil
production, e.g., after chemotherapy and for mobilizing hematopoietic stem cells
from the bone marrow into the blood.
tissue plasminogen activator (TPA) for dissolving blood clots
adenosine deaminase (ADA) for treating some forms of severe combined
immunodeficiency (SCID)
angiostatin and endostatin for trials as anti-cancer drugs
parathyroid hormone
leptin
hepatitis B surface antigen (HBsAg) to vaccinate against the hepatitis B virus
Many more examples are in the pipeline.
Welcome&Next Search
12 February 2006
Bacterial Transformation
Introduction:
This is a very basic technique that is used on a daily basis in a molecular biological
laboratory. The purpose of this technique is to introduce a foreign plasmid into a bacteria
and to use that bacteria to amplify the plasmid in order to make large quantities of it. This
is based on the natural function of a plasmid: to transfer genetic information vital to the
survival of the bacteria.
The plasmid:
A plasmid is a small circular piece of DNA (about 2,000 to 10,000 base pairs) that
contains important genetic information for the growth of bacteria. In nature, this
information is often a gene that encodes a protein that will make the bacteria resistant to
an antibiotic. Plasmids probably came about as a result of bacteria evolving in close
proximity to other heterotrophs. Bacteria often grow in the same environment as molds
and fungi and compete with them for food (complex organic material). As a result, molds
and fungi have evolved to make toxins that kill bacteria (which we now use as antibiotics
in medicine) in order to win in the competition for food. Bacteria, in turn, evolved to
make proteins that inactivate the toxins. The bacteria share this vital information by
passing it among themselves in the form of genes in plasmids.
Plasmids were discovered in the late sixties, and it was quickly realized that they could
be used to amplify a gene of interest. A plasmid containing resistance to an antibiotic
(usually ampicillin) is used as a vector. The gene of interest is inserted into the vector
plasmid and this newly constructed plasmid is then put into E. coli that are sensitive to
ampicillin. The bacteria are then spread over a plate that contains ampicillin. The
ampicillin provides a selective pressure because only bacteria that have acquired the
plasmid can grow on the plate. Therefore, as long as you grow the bacteria in ampicillin,
it will need the plasmid to survive and it will continually replicate it, along with your
gene of interest that has been inserted to the plasmid.
There are many different kinds of plasmids commercially available. All of them contain
1) a selectable marker (i.e., a gene that encodes for antibiotic resistance), 2) an origin of
replication (which is used by the DNA making machinery in the bacteria as the starting
point to make a copy of the plasmid) and 3) a multiple cloning site. The multiple cloning
site has many restriction enzyme sites (to be discussed in a later lab) and is used to insert
the DNA of interest. The multiple cloning site is usually in the middle of a reporter gene
like Lac Z. A commonly used plasmid is pBluescript:
Figure 1
The main differences among commercially available plasmids are the number of
restriction enzyme sites, their order in the multiple cloning site, the type of antibiotic
resistance that the plasmid confers, and some other genetic information that makes the
plasmid useful for a specific purpose. Bacteria transformed with pBluescript will survive
in ampicillin containing media and will replicate the plasmid, including any gene that is
placed in the multiple cloning site.
Competent Cells:
Since DNA is a very hydrophilic molecule, it won't normally pass through a bacterial
cell's membrane. In order to make bacteria take in the plasmid, they must first be made
"competent" to take up DNA. This is done by creating small holes in the bacterial cells
by suspending them in a solution with a high concentration of calcium. DNA can then be
forced into the cells by incubating the cells and the DNA together on ice, placing them
briefly at 42oC (heat shock), and then putting them back on ice. This causes the bacteria
to take in the DNA. The cells are then plated out on antibiotic containing media.
For a short animation on E. coli transformation click here.
Competency
The procedure to prepare competent cells can sometimes be tricky. Bacteria aren't very
stable when they have holes put in them, and they die easily. A poorly performed
procedure can result in cells that aren't very competent to take up DNA. A wellperformed procedure will result in very competent cells. The competency of a stock of
competent cells is determined by calculating how many E. coli colonies are produced per
microgram (10 -6 grams) of DNA added. An excellent preparation of competent cells will
give ~108 colonies per ug. A poor preparation will be about 10 4 / ug or less. Our preps
should be in the range of 10 5 to 10 6.
In this experiment you will be making competent cells, transforming them with a plasmid
and calculating their competency. There will be a lab report due for this lab.
Procedure:
Important
This procedure must be performed under sterile conditions. Use only autoclaved plasticware and always work with a flame in front of you. Also, bacteria are very labile in high
calcium, so keep the bacteria on ice and away from the flame at all times to keep them
viable.
Competent Cells:
1. (This step will be performed for you before you come into lab). Pick a single
colony from a freshly grown plate of E. coli and disperse it in 100 ml of LB media in a 1
L flask. Incubate the culture at 37oC with vigorous shaking for approximately 3 hours.
Cell density is monitored by determining OD600 and should be less than 10 8 cells / ml
(log phase of growth - the most healthy bacteria).
2. Transfer 50 ml of this culture to a 50 ml conical tube and centrifuge at 2,000 rpm for
10 min. (Sorvall HS-4 rotor) in room 312.
3. Decant the supernatant into waste beaker (this must be sterilized before being dumped
down the drain). Resuspend the pellet in 10 ml of ice cold 0.1 M CaCl2. This is most
easily done by resuspending in 1 ml, using the P1000 pipette and then adding 9.0 ml. Cut
about 0.5 cm from the end of the blue tip before pulling E. coli through it, since the cells
are fragile in this high calcium solution and may lyse if sheared. After they have been
resuspended, centrifuge at 2,000 rpm for 10 min. (Sorvall HS-4 rotor).
4. Decant the supernatant into waste beaker (to be autoclaved later). Resuspend the pellet
in 1.0 ml of ice cold 0.1 M CaCl2
Bacterial Transformation:
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1. Pipette 200ul competent cells into each of 3 ice cold Eppendorf tubes. Label the tubes Control, 1 ng, and 10 ng (1 ng is 10 ug, or
-9
10 milligrams). The unknown plasmid is at a concentration of 1 ng/ul. Add 1 ng of your unknown plasmid to one tube and 10 ng to
the other. Place the tubes on ice for 30 min.
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2. Put the tubes at 42 C for exactly 90 seconds. Return the cells to ice for 1-2 minutes.
3. Pipette the transformation mixtures onto labeled plates containing ampicillin and spread them around using a sterilized, bent glass
rod spreader.
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4. Place upside down in the 37 C incubator in room 305 overnight.
5. 16 - 20 hours later, count the number of colonies on the plate with well-isolated colonies. Put parafilm around the edge of a plate
and put it in a refrigerator for later use. Check the control plate to see that no colonies grew on it. Dispose of the plate and the
control plate in the biohazard bag.
Calculations:
To calculate the competency of your cells, divide the number of colonies on your plate by the amount of DNA (in ug) you added to the
transformation.
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