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. Bacterial Genetics
Jill B Keeney, Juniata College, Huntingdon, Pennsylvania, USA
. Plasmids as Vectors for Amplifying Foreign DNA
ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net 1
Microorganisms: Applications in Molecular Biology
2 ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net
Microorganisms: Applications in Molecular Biology
are critical for survival in specific situations, such as genes 2. A selectable marker to select the bacteria which received
for antibiotic resistance, are exchanged between individu- the vector DNA during a bacterial transformation. The
als, including cross-species exchange. There are three main selectable marker is often an antibiotic resistance gene
mechanisms for exchanging DNA between individual bac- such as b lactamase, an enzyme that degrades
terial cells (see Bacterial Genetic Exchange for illustrations ampicillins.
of all 3 mechanisms): 3. An origin of replication so that the bacterial DNA
polymerase will replicate the plasmid, including the
. Transformation is the process by which bacteria pick up inserted foreign DNA. Origins of replication vary so
DNA from their immediate surroundings. In the natural that some vectors will be present in only a few copies,
environment, the source can be DNA fragments released while other vectors may have many copies within an
from dead bacterial cells. In the laboratory, this is the individual cell.
most common method used in molecular biology to
clone and replicate genes in bacteria (see later).
. Transduction is the exchange of genetic information me- Circular vector DNA can be cut, or linearized, at one
diated by bacteriophages (discussed later). Bacterio- or two of the restriction sites in the multiple cloning site.
phages are also commonly used tools for cloning and Foreign DNA is then combined with the linearized vector
manipulating DNA. and an enzyme (ligase or topoisomerase) joins the ends
. Conjugation is the process by which bacteria exchange together, forming a circular plasmid containing foreign
DNA through specialized protein structures called sex pili. DNA (Figure 2), and restriction enzymes.
A plasmid can be put into a bacterial cell by transfor-
mation, as outlined in Figure 2. In the laboratory this is
Each of these mechanisms of DNA transfer is employed
easily accomplished by placing freshly grown cells into
in a variety of molecular biology techniques to manipulate
calcium chloride solution, adding the plasmid DNA and
DNA. Some applications of transformation and phage in-
allowing the DNA to adsorb to the bacterial membrane.
fection are discussed later. See also: Bacterial Reproduc-
Following a brief heat shock, some of the bacterial cells will
tion and Growth
take up the DNA. The cells are then plated and allowed to
grow overnight to form bacterial colonies. Electroporation
Plasmids as Vectors for Amplifying is another common transformation process, in which a
quick electrical pulse is delivered to cells. During the tem-
Foreign DNA porary disruption of the cellular membrane, DNA enters
the cell. See also: Electroporation
In addition to the circular chromosome, bacteria may carry The transformation process is inefficient, so that only a
smaller extrachromosomal circular DNA, called plasmids, small fraction of the bacterial cells actually take up the
which are replicated in the cell but not necessarily carried plasmid DNA. The selectable marker on the plasmid al-
along in cell division. Often these plasmids carry nones- lows for selection of these cells by plating the cells on media
sential genes that allow bacteria to grow in special envi- containing antibiotic. For example, after transformation of
ronmental conditions, such as genes to degrade unique vector DNA carrying the b-lactamase gene, the bacterial
nutrients in the environment (e.g. oil) or genes to break cells are plated on to media containing ampicillin. Bacterial
down toxins in the environment (e.g. antibiotics). Over the cells that have obtained the plasmid begin producing the
past few decades, bacterial geneticists have isolated and enzyme that degrades ampicillin and are able to begin cell
altered these plasmids, and now hundreds of different spe- division, forming colonies after about 16 h. The ampicillin
cialized plasmids exist. Plasmids are used by molecular inhibits growth of all bacterial cells lacking the plasmid.
biologists in all fields of research as vectors for cloning and A single colony, or clone, containing the plasmid can then
amplifying DNA from many different organisms. The be transferred into a large volume of liquid media, and
structure and composition of DNA is universal among allowed to grow overnight. This culture will yield large
all living organisms. Thus, DNA from any other ‘foreign’ quantities of the purified plasmid for further study and
organism, if inserted or cloned into a bacterial vector, can analysis.
be amplified and then isolated from a bacterial cell. This Bacterial vectors can efficiently replicate inserts of DNA
process allows scientists to produce large quantities of a of about 10 000 bp in length. While this is adequate for
specific DNA sequence for experimental study. See also: some genes and sequences, often scientists need to work
Antibiotic Resistance Plasmids in Bacteria; Bacterial with longer fragments of DNA. Specialized vectors called
Plasmids; Bioremediation BACs, for bacterial artificial chromosomes, have been de-
Most standard bacterial vectors have three essential veloped which can allow cloning of inserts 100–300 000 bp
components: in length. The vectors are essential for cloning very large
genes and transforming these genes into other cell types,
1. A multiple cloning site containing several unique such as insect, plant or mammalian cells. BACs are
restriction enzyme sites, allowing foreign DNA to be essential to the sequencing of large genomes, as they
easily inserted into the plasmid. allow for sequencing of much larger continuous pieces of
ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net 3
Microorganisms: Applications in Molecular Biology
Origin Selectable
of replication marker Production
(ampicillin of enzyme
resistance) to degrade
ampicillin
(a)
Figure 2 Ligating, transforming and selecting plasmid DNA. (a) Linearized vector is ligated to a fragment of foreign DNA, forming a circular plasmid. When
this plasmid is transformed into bacteria, the ampicillin resistance gene is expressed, producing an enzyme that degrades ampicillin. (b) When the bacteria are
plated on to media containing ampicillin, cells containing the plasmid (and thus ampicillin-degrading enzyme) grow and form colonies (large, dark circles).
Many of the cells do not contain the plasmid and fail to divide (represented as small, faint circles, although they would not be at all visible).
DNA, significantly reducing the time-consuming task of then be transcribed and translated, producing large
piecing together many small segments of DNA. See also: quantities of the protein for purification. This process is
Artificial Chromosomes used to isolate large quantities of proteins for use in bio-
technology and medicine. See also: Protein Production for
Expressing genes in bacteria Biotechnology
4 ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net
Microorganisms: Applications in Molecular Biology
Yeast promoters
The promoters of yeast and other eukaryotes are more
complex than prokaryotic promoters. Eukaryotic promot-
Figure 3 Bacteriophage life cycle. The bacteriophage, a capsule of proteins ers often contain a ‘TATA box’ sequence near the site
(called a phage particle) surrounding the phage DNA, anchors on to the
surface of the bacterial cell (1) and injects its DNA into the cell (2). Once inside
where transcription initiates, but must also have other,
the bacterial cell, the bacteriophage DNA is transcribed into RNA (3), and also more distant, sequences to direct RNA polymerase to begin
replicated to produce many copies of the phage DNA (4). The RNA is transcription at the proper location and to control how
translated by the bacterial ribosomes (5), so that numerous phage particles, often transcription initiates. These are called upstream
containing phage DNA, can be assembled (6). The phages then signal lysis of
regulatory sequences and are targets of nuclear proteins
the bacterial cells, releasing a multitude of new phages (7), which can then
infect neighbouring bacteria. (transcriptional regulators) that regulate the activity of
RNA polymerase at the promoter. The function of these
sequences is often studied by cloning the promoter
Phagemids are common vectors that contain the re- (including the regulatory sequences) next to a reporter gene,
quired sequences both for replicating in bacteria and for an enzyme for which activity can be easily assayed. The
packaging DNA into phage particles. When transformed cloned promoter is mutated to determine what sequences
into bacterial cells, the bacterial replicating sequence are important for activation and suppression of gene
allows for selection of clones. When a clone is infected expression. Like bacteria, yeast regulate when each gene is
with an M13 helper phage, the M13 phage sequences direct expressed in the cell, so specific yeast promoters can be used
replication and packaging of the DNA into phage particles, to regulate the expression of other genes for genetic studies.
allowing isolation of single-stranded DNA. See also: For example, a natural gene promoter can be replaced with
Bacteriophages in Industry the promoter of a gene that is turned on in the presence of
copper; the presence or absence of copper in the media
allows the researcher to control expression of that gene.
Yeast: Eukaryotic Unicellular In yeast and other eukaryotes, cellular DNA is organized
by extensive wrapping of the DNA around proteins called
Organisms histones, which collectively form a structure called chro-
matin. The study of the yeast genome (see later) has re-
Yeast, like bacteria, is a unicellular organism; however, vealed an extensive network of enzymes that chemically
yeast is characterized as eukaryotic because cellular func- modify histones located near promoter sequences to reg-
tions are compartmentalized into distinct organelles, such ulate gene expression. See also: Transcription Activation in
as the nucleus, the endoplasmic reticulum and the mito- Eukaryotic Cells
chondria. As with bacteria, scientists study a number of
different yeast species, many of them pathogenic. Some Yeast genome
yeast species are easy to grow, replicate quickly and are
easy to manipulate genetically. Yet, because yeasts are In 1996, S. cerevisiae earned the distinction of being the first
eukaryotic, they can also be used as a tool to study complex eukaryote to have its genome entirely sequenced, a total of
cellular functions such as the cell cycle, chromosome seg- 12 million bases. Table 2 compares the genome properties
regation, transcription, intracellular signalling and protein for some common research organisms. The sequence data
modification. Many processes that occur in larger eukar- of the yeast genome has been analysed to determine puta-
yotic cells, such as human cells, can be more readily studied tive gene coding regions. It contains 6000 putative genes,
in yeast. The most commonly used species are the budding and a significant number of these probable gene sequences
yeast Saccharomyces cerevisiae (also known as brewer’s or are of unknown function. A major component of the yeast
baker’s yeast) and the fission yeast Schizosaccharomyces genome project is developing strategies to determine the
pombe. This discussion deals with S. cerevisiae, the most cellular function of each putative gene. The Saccharomyces
ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net 5
Microorganisms: Applications in Molecular Biology
Genome Database (www.yeastgenome.org) is a compre- haploids and diploids can be cultured for genetic analysis.
hensive, public database that lists each gene with extensive See also: Yeast Mating Type
annotations to sequence data, protein data, cellular local-
ization studies, known functions, published references, Yeast plasmids
gene expression data (i.e. from DNA microarrays) and
interactions with other genes. The Saccharomyces Genome Yeast plasmids, like bacterial plasmids, also have a mul-
Deletion Project (http://sequence-www.stanford.edu/ tiple cloning site, a selectable marker, and often, but not
group/yeast_deletion_project/deletions3.html) is an inno- always, an origin of replication. The selectable marker may
vative resource created by scientists in the yeast community be a drug effective against yeast cells, but is most commonly
working together to construct a collection of yeast strains, an auxotrophic marker. Yeast does not have any essential
each containing a different gene deletion. This collection is amino acids and can synthesize all the amino acids from
used by many scientists to study the phenotypes of strains carbon and nitrogen supplied in growth medium. Muta-
harbouring gene deletions and to decipher genetic interac- tions in enzymes needed to synthesize certain amino acids,
tions between genes. Essential genes, which are lethal if called auxotrophic markers, are important selection tools
deleted, can also be studied by deleting these genes in for yeast geneticists. For example, a yeast strain carrying a
diploids and combining them with other gene deletions to mutation in a gene for synthesizing histidine (his3) cannot
recover viability. grow on media lacking histidine. However, if a plasmid
The comparative ease of working with yeast has also containing the missing gene (HIS3) is transformed into the
allowed scientists to use high-throughput techniques to yeast cell, the individual cells that obtain the plasmid can
map the physical interactions between the yeast proteins. now grow and form a colony on media lacking histidine.
Currently, a project is underway to construct a collection of Several different common auxotrophic markers exist and
strains containing each of the possible double gene deletion thus a given yeast strain can harbour several plasmids at
combinations. Using this information, yeast geneticists are once. Since the auxotrophic marker is not essential to cell
collaborating with computational biologists to work on growth in rich medium, these plasmids can also easily be
constructing a map of the network of protein interactions removed from the cell, in a process called plasmid shuffling.
in the cell, with the eventual goal of a comprehensive Using various auxotrophic markers, plasmids have been
understanding of cellular function. constructed as tools for studying the function of yeast genes
and cellular processes. As with E. coli, plasmids can be
easily transformed into yeast. Yeast plasmids can exist as
extrachromosomal DNA, replicating as an independent
unit, if they contain an origin of replication. Yeast molec-
Yeast Genetics ular biologists also use integrating plasmids or simply
transform a fragment of DNA containing a selectable
Yeasts generally exist as haploid organisms of two different marker. The DNA being transformed will insert into the
mating types, termed ‘a’ and ‘a’ in S. cerevisiae. Two strains chromosome that has DNA sequences matching those on
of opposite mating type can be cultured together to form a the transformed DNA, in a process called homologous
diploid cell. The diploid cell normally undergoes rapid recombination. This characteristic of yeast has proven to
meiosis and sporulation, producing four haploid spores. be a very powerful tool, as researchers can easily replace a
During this process, genetic information is randomly wild-type yeast gene with any mutation, or remove it com-
shuffled and sorted so that each spore produced contains pletely to create a gene deletion. Yeast plasmids also allow
genetic information from each parent. Yeast strains for selection of important mutations in studying gene
commonly used in research have been altered so that both function. Many mutations are introduced into a cloned
6 ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net
Microorganisms: Applications in Molecular Biology
gene, which is then transformed into yeast, and the resulting incubators and media that mimic these conditions are re-
colonies selected for a particular phenotype. The plasmid quired for growing mammalian cells. The media, in addi-
from the selected colonies can be isolated and transformed tion to carbon and nitrogen sources, must also contain
into E. coli for DNA sequencing to determine which other growth factors and serum components. These are
mutations affect gene function. The ability to introduce generally supplied by bovine (cow) serum that is added to
and select for specific mutations in yeast genes and to move the media. In our bodies, our skin and immune system keep
plasmids easily in and out of yeast for genetic studies has bacteria and yeast out of our blood and tissues. In tissue
resulted in significant advances in our understanding of culture, the media and cells are in direct contact with the
eukaryotic cellular processes. See also: Yeast as a Model environment of the laboratory. Work with these cultures
Genetic Organism must be done in sterile cabinets, called laminar flow hoods.
The persons culturing the cells must be sure their hands are
scrubbed of all microorganisms before working with the
Cloning large pieces of DNA cultures. Without these precautions, the faster replicating
Sometimes it is desired to have a very large piece of microorganisms will overrun the culture. See also: Safety
DNA cloned into a yeast cell. This can be accomplished by Considerations in the Tissue Culture Laboratory
constructing a yeast artificial chromosome, or YAC.
A YAC contains an auxotrophic marker and a centromeric Primary lines and transformed lines
and a telomeric sequence found on a yeast chromosome;
the rest of the DNA can be from another organism. Cells that are normally part of a multicellular organism are
Whereas plasmids are generally not much bigger than programmed to stop dividing at a certain cell density and to
10 000 bp, YACs can be millions of base pairs, allowing die at a specified time through the cellular process of
very large pieces of DNA to be cloned into yeast. Since apoptosis. Thus, when cells are moved from an organism
many yeast and human genes are functionally related, into tissue culture, they normally will grow to a certain cell
YACs can be used to study large human genes in yeast. density and then die. In order to maintain cells in culture
See also: Yeast Artificial Chromosomes over long periods of time, cells must be immortalized or
transformed. Tumour or cancer cells have already acquired
this characteristic. If placed into culture and given a fresh
Expressing genes in yeast supply of media every few days they can be grown indefi-
Yeast plasmids are also used to express large quantities of a nitely. Scientists often use chemicals or viruses to immor-
specific protein for research and the biotechnology indus- talize certain cell types in the laboratory so that they can be
try. For example, the hepatitis B virus vaccine is commer- studied over long periods of time. As with microorganisms,
cially produced in yeast. A foreign gene to be expressed can cultured cells can be transformed with cloned DNA to
be cloned next to an inducible yeast promoter, like the study the effects of gene expression on cellular processes.
process used for expressing proteins in bacteria. Some See also: Primary Cell Cultures and Immortal Cell Lines
advantages of using yeast cells for protein production are Tissue culture is a routine procedure, and a central part
that the expressed protein will contain carbohydrate mod- of biomedical research, biotechnology and pharmaceutical
ifications specific to eukaryotic cells, is more likely to be production. Many drugs, vaccines, monoclonal antibodies
folded correctly, and will be free of potentially toxic bac- and other substances are produced from cell cultures.
terial cell components. See also: Saccharomyces cerevisiae: See also: Monoclonal Antibodies
Applications
ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net 7
Microorganisms: Applications in Molecular Biology
Scientists working with a given organism must make them- Lodish H, Berk A, Zipursky S et al. (2000) Chapter 6: Manipu-
selves familiar with the appropriate selections and shuttle lating cells and viruses in culture and Chapter 7: Recombinant
vectors available for that organism. DNA and genomics. In: Molecular Cell Biology. New York:
W. H. Freeman & Co. [available on-line at http://www.
ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books]
National Center for Biotechnology Information. [http://
Summary www.ncbi.nlm.nih.gov/]
Saccharomyces Genome Database. [http://www.yeastgenome.org/]
Researchers studying a particular aspect of molecular Sherman F (1998) An Introduction to the Genetics and Molecular
biology in any organism use the bacterium E. coli for clon- Biology of the Yeast Saccharomyces cerevisiae. [http://
ing, replicating and manipulating DNA. Yeasts, namely dbb.urmc.rochester.edu/labs/sherman_f/yeast/index.html]
S. cerevisiae and S. pombe, are also commonly used for
selecting specific genetic mutants useful in studying
eukaryotic cellular process. The knowledge gained from Web Links
studying cloned genes in yeast leads to a greater under-
standing of cellular processes in more complex eukaryotic http://www.ncbi.nlm.nih.gov/ NCBI (National Center for Bio-
organisms. Specialized yeast and bacterial expression vec- technology Information) provides public assess to vast amounts
of scientific information and tools, including scientific refer-
tors are used in the biotechnology industry to produce large
ences, genomic and gene sequences, human genetic diseases,
quantities of purified proteins. Shuttle vectors allow cloned
and nucleic acid and protein structures.
DNA to be easily replicated in bacteria and then trans-
www.yeastgenome.org The Saccharomyces Genome Database is a
formed into another host cell. comprehensive, public database that lists each yeast gene with
extensive annotations to sequence data, protein data, cellular
localization studies, known functions, published references,
Further Reading gene expression data (i.e. from DNA microarrays), and inter-
actions with other genes.
Alberts B, Johnson A, Lewis J et al. (2002) Manipulating proteins, http://sequence-www.stanford.edu/group/yeast_deletion_project/
DNA, and RNA. In: Molecular Biology of the Cell, chap. 8. deletions3.html The Saccharomyces Genome Deletion Project is
New York and London: Garland Science. [available on-line at a collaborative effort among many researchers studying yeast to
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books] create a library of yeast strains, each with a different gene deleted.
8 ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net