Download Lecture 4 - E. coli Hosts and Plasmid Biology (AMG text pp. 31-42)

Bioc 471a/571a - Applied Molecular Genetics
Fall 2001 - Dr. Roger Miesfeld
Lecture 4 - E. coli Hosts and Plasmid Biology (AMG text pp. 31-42)
August 30, 2001
Biology of E. coli K-12
E. coli is a Gram-negative, rod-shaped bacterium about 2.5 micrometers long that
contains flagella and a genome of 4,639,221 base pairs encoding at least 4000 genes (see
chapter 4). The K12 strain was first isolated in 1921 from the stool of a malaria patient
and it has been maintained in laboratory stocks as a pure strain for the last 75 years. E.
coli K12 was the bacterial strain of choice in biochemistry labs because it was easy to
grow and amenable to metabolic studies. Like all Gram-negative bacteria, E. coli have
no nuclear membrane and the chromosome is a large circular duplex molecular with a
membrane attachment site and a single origin of replication. E. coli cells can support the
replication of DNA plasmids, many of which encode antibiotic-resistance genes.
Genetic variants of E. coli K-12 have led to an improved strain for recombinant DNA
methods:
1. Bacterial restriction modification systems have been removed.
2. DNA recombination systems are modified to prevent rearrangements.
3. Endonuclease activity has been mutated to increase plasmid yields.
Restriction modification systems have been removed from E. coli K-12 strains because
they will interfere with the replication of foreign DNA in bacterial cells - the bacteria
will degrade foreign DNA the same way bacteriophage DNA is degraded as a defense
against invasion!
The EcoK restriction system encodes proteins that degrade foreign DNA that is not
properly methylated a the sequence 5'-AAC-(N)5-GTGC-3' which would occur in
cloned DNA.
The second type of modification system is that of the mcrA/mcrB/mrr complex which
degrades foreign DNA that is not properly methlyated, such as methylated DNA
obtained from mouse and human cells which contains CpG methylated DNA.
Rearranged or deleted insert DNA is equally deleterious to successful gene cloning
experiments. Most E. coli K12 strains used for cloning contain mutations in the recA
gene which encodes a well-characterized recombination protein. recA mutations also
enhance the biosafety of E. coli K12 since recA- strains are sensitive to ultraviolet light.
Most E. coli K12 strains contain a mutation in the endA gene encoding DNA specific
endonuclease I. Loss of this endonuclease greatly increases plasmid DNA yields and
improves the quality of DNA that is isolated using standard biochemical preparations.
Taken together, the standard bacterial host for most recombinant DNA cloning
experiments is an E. coli K12 strain with mutations in 1) endogenous restriction
modification system (hsdR-), 2) homologous DNA recombination function (recA-), 3)
endonuclease I activity (endA-). An example of commercially available E. coli strains
are the XL-blue cells from Stratagene.
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Bioc 471a/571a - Applied Molecular Genetics
Fall 2001 - Dr. Roger Miesfeld
What is the main difference between the E. coli K-12 strain used in recombinant DNA methods,
and the E. coli strain 0157:H7 which is highly virulent and the cause of human disease?
Why was E. coli K-12 chosen as the model strain for the development of recombinant DNA
methods? Would other strains of bacteria have been just as good or even better?
Why would it be difficult to develop a single strain for all recombinant DNA methods
The Lac Operon of E. coli
The lac Operon of E. coli is a very useful system in applied molecular genetics for two
reasons:
1) Transcription of the lac operon is tightly regulated and can be exploited as a gene
expression system.
2) The enzyme beta-galactosidase provides a convenient enzyme marker in molecular
genetic studies.
The lac operon encodes three enzymes required for lactose metabolism in E. coli . The
first gene in the operon is the lacZ gene encoding beta-galactosidase, an enzyme that
cleaves lactose to produce galactose and glucose. The set of genes in the lac operon are
normally turned off by a repressor protein binding to the promoter when lactose levels
are low. However, in the presence of lactose, the repressor is inhibited and transcription
is induced. The beta-galactosidase enzyme is then able to metabolize lactose. Once
lactose levels fall, the repressor is able to bind the lac operon promoter again and turn
off the genes.
E. coli lac operon consists of a promoter, a transcriptional regulatory site called the
operator (o), a CAP binding site (c), and three structural genes (lacZ, lacY and lacA) that
are transcribed as a single polycistronic mRNA. Transcription of the lac operon is
regulated by the lac repressor protein (lacI) which is encoded on a gene physically
linked to the lac operon. lac operon inducers, such as IPTG, inactivate the lac repressor
protein resulting in transcriptional de-repression of the lac operon.
The lac repressor protein is a ~38 kd protein that binds to the lac operator site as a
tetramer (two dimers). lac repressor binding to the operator sequence is extremely tight
(Kd=10-13 M) and specific (the Kd for non-specific DNA binding is only 10-6 M),
causing transcriptional repression through a mechanism involving repressor
interference with RNA polymerase binding to the lac promoter.
Although lactose is the normal substrate of the enzyme beta-galactosidase, the inducer
molecular that binds to the lac repressor and inactivates its DNA binding protein is
actually allolactose, a lactose related compound that is produced by basal levels of betagalactosidas. Allactose levels increase when lactose levels increase, leading to repressor
inactivation and transcription of the lactose operon. It is possible to artificially induce
the lac operon using a nonmetabolizable allolactose analogue, isopropylthiogalactoside
(IPTG), which binds to the lac repressor protein.
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Bioc 471a/571a - Applied Molecular Genetics
Fall 2001 - Dr. Roger Miesfeld
An IPTG-inducible expression system has been developed using components of the lac
operon. Expression of cloned genes can be regulated using plasmids containing the
lacUV5 promoter (a variant promoter that has a low basal activity) that are grown in E.
coli strains containing the lacIq receptor overexpression mutation.
Activity of the lac operon enzyme beta-galactosidase can be measured in live cells using
the chromogenic substrate 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-gal). An
example is identification of cell-specific expression using lacZ reporter genes in mouse
embryos as seen here for transcription of the nodal gene promoter during heart
development.
One of the many uses of the beta-galactosidase (lacZ) gene is to take advantage of the
observation that the enzymatic function can be reconstituted from two polypeptide
fragments. These protein subfragments are non-functional by themselves and consist of
the N-terminal (alpha fragment) and a C-terminal (omega fragment); reconstitution is
called alpha complementation. This complementation system has been used to monitor
insertional cloning into plasmids containing restriction sites within the ~150 amino acid
coding sequence of the a fragment as shown below. In the tetrameric form, it can be
seen the two alpha regions need to interact to form the functional complex. This
arrangment explains how a polypeptide fragment (alpha region) can be held in place to
permit enzymatic reconstitution.
Explain why IPTG is a more potent inducer of the lac operon than is lactose?
What does the molecular structure of the lac repressor-DNA complex tell you about the
arrangement of sequences in the lac repressor DNA binding site?
What would happen if the number of nucleotides in the spacer region (sequence between a pair of
binding sites) were increased or decreased?
What is X-gal and how is it used to identify cells that are expressing functional betagalactosidase?
Why does insertion of a DNA fragment into the lacZ coding sequence of a plasmid give white
bacterial colonies on an agar plate containing X-gal?
Why might a very small insertion, such as a short PCR product or double strand oligonucleotide,
into the plasmid lacZ gene still produce blue colonies?
Plasmid Biology
Three types of naturally occurring bacterial plasmids have been exploited for molecular
genetic applications; virulence plasmids encoding bacterial toxins such as colicins
(ColE1 plasmid ori is in cloning vectors), conjugation plasmids (F plasmid is used to
carry lacI gene and binding protein for M13 phage), and drug resistance plasmids (R
plasmids have been a source of antibiotic resistance genes).
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Bioc 471a/571a - Applied Molecular Genetics
Fall 2001 - Dr. Roger Miesfeld
How are antibiotic resistance genes used in applied molecular genetics?
Can you think of any reason why an ampicillin-resistant E. coli K-12 colony might not contain a
copy of the recombinant plasmid you think it should (you isolated DNA from amp-resistant cells
and found no plasmid)?
What accounts for the spread of antibiotic resistant bacteria in hospitals?
What strategies do you think pharmaceutical companies might be taking to try and stay ahead of
this problem?
Cloning vectors are DNA molecules that are used to "transport" cloned sequences
between biological hosts and the test tube (e.g., from bacterial cells to test tubes to plant
cells). Cloning vectors share four common properties:
1. Ability to promote autonomous replication.
2. Contain a genetic marker (usually dominant) for selection.
3. Unique restriction sites to facilitate cloning of insert DNA.
4. Minimum amount of nonessential DNA to optimize cloning.
A typical plasmid cloning strategy involves five steps:
1) Enzyme restriction digest of DNA sample.
2) Enzyme restriction digest of DNA plasmid vector.
3) Ligation of DNA sample products and plasmid vector.
4) Transformation of E. coli K-12 with the ligation products.
5) Growth on agar plates with selection for antibiotic resistance.
The process of transferring exogenous DNA into cells is call “transformation” and it
refers to any application in molecular genetics in which purified DNA is actively or
passively imported into host cells using non-viral methods. There are basically two
general methods for transforming bacteria. The first is a chemical method utilizing
CaCl2 and heat shock to promote DNA entry into cells. A second method is called
electroporation based on a short pulse of electric charge to facilitate DNA uptake. The
major difference between these two techniques is the efficiency with which they work.
Why is it difficult to come up with a single plasmid cloning vector for all recombinant DNA
methods?
What might be an explanation for the variety of commercially available cloning vectors?
How is the ligation reaction set-up to maximize the number of vector molecules with only one
insert, isn't it possible to get multiple inserts into a single vector molecule?
What explains the observation that single bacterial colonies contain genetically indentical
recombinant plasmids, what happens if more than one ligation product enters the same bacterial
cell?
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