UNIT 2.

PLASMIDS
WHAT IS A PLASMID?
WHAT IS A PLASMID?

• Plasmids are DNA molecules that can be found in essentially all types of bacteria

• Most of them are circular molecules of dsDNA but some of them are linear

• Plasmids encode proteins and RNA molecules but generally do not encode functions
essential to bacterial growth

• They replicate as the cell grows, and the copies are usually distributed into each
daughter cell when the cell divides

• They provide gene products that can benefit the bacterium under certain circumstances
(i.e. antimicrobial resistances, virulence factors, metabolism genes...) playing a
significant role in bacterial adaptation and evolution

• They also serve as important tools in studies of molecular biology

PLASMID REPLICATION

Rolling-circle (left) and Theta (right) plasmid replication (TEM)

PLASMID REPLICATION
• To exist free of chromosome, plasmids must have the ability to replicate
independently.

• Plasmids must have at least one origin of replication, named oriV (ori Vector),
where replication begins.

• Most plasmids encode only one of the elements needed for the initiation of
their replication at the oriV.

• All of the other proteins, including DNA polymerases, ligases, primases,

helicases, etc. are borrowed from the host.

• Each type of plasmid replicates by one of two general mechanisms:

Rolling-circle and Theta replication.
 ROLLING-CIRCLE REPLICATION
oriV
• In the first stage, the double-stranded circular plasmid DNA Rep
replicates to form another double-stranded circular DNA and a
single-stranded circular DNA:

-A plasmid-encoded Rep protein recognizes and binds to a

palindromic sequence at the oriV making a nick to initiate replication.

-After the nick, the Rep protein remains covalently attached to the
phosphate at the 5’ end through a Rep’s tyrosine.
DNA pol III
-Host DNA polymerase III uses the 3’ OH end as a primer to replicate
around the circle, displacing one of the strands.

-Once the circle is complete, the 5’ phosphate is transferred from the

tyrosine on the Rep protein to the 3’ OH end of the displaced strain,
producing a circular ssDNA.

-This stage is analogous to the replication of some ssDNA phages and

to DNA transfer during plasmid conjugation.

dsDNA ssDNA
ROLLING-CIRCLE REPLICATION

-In some phages, DNA replicates by a rolling-

circle making a longer molecule with
individual genomes in a structure called a
concatamer.

-However, during plasmid replication, Rep

makes a nick in the newly created strand concatamer
avoinding the concatamer formation. This
nick will be sealed by the DNA ligase.
dsDNA dsDNA
RNA pol
• In the second stage, the complementary strand DNA pol III & I
Ligase Hoste
is synthesized on the ssDNA to make another
dsDNA:

-The host RNA polymerase makes a primer and the DNA pol III synthesizes the new strand of DNA.
-The 5’ exonuclease activity of host DNA polymerase I removes the RNA primer, replacing it with DNA.
-Host DNA ligase joins the ends making the second double-stranded plasmid.
THETA REPLICATION
• Plasmids begin replication by opening the two strands of DNA at the oriV,
creating a structure that looks the Greek letter theta (Ɵ).

• To initiate theta replication it is necessary the opening of the strands through:

-A plasmid-encoded Rep protein and/or

-Host DnaA initiator proteins or
-A plasmid-encoded RNA

• The theta mechanism is the most common form of DNA replication.

• This mechanism is used not only by most plasmids, including ColE1, R1, F,
pSC101, RK2, and P1, but also by the chromosome.

• Replication can proceed in one (unidirectional replication: ColE1, R plasmids) or

both directions around the plasmid (bidirectional replication: most common).
PLASMID COPY NUMBER
The ori region and plasmid size determine the plasmid copy number (the average
number of a particular plasmid per cell):

-High copy number: >15 plasmids/cell

-Medium copy number: 4-15 plasmids/cell

-Low copy number: 1-3 plasmids/cell

Ori i tamany depenen de nº de copies

PLASMID COPY NUMBER
Regulation of replication

-Antisense RNA that inhibits the RNA involved in the initiation of replication
(ex. RNA I of ColE1-derived plasmids)
-Antisense RNA that inhibits the translation of the protein essential for replication
(ex. CopA of R1 plasmid)
-Proteins which are transcriptional repressors of the plasmid rep gene
(ex. CopB of R1 plasmid)
-Proteins which bind to repeated DNA sequences (iterons) in oriV
(ex. RepA of pSC101 plasmid)

Stability

-Toxin-antitoxin (ColE1, CcdAB of F plamid, ParB and ParD of R1 plasmid)

-Segregation (ParA of plasmid R1)

ColE1-Derived Plasmids
• The ColE1 plasmid was isolated from E. coli and carries a gene for the pore-
forming protein ColE1, destabilizing the outer membrane of target cells.

• ColE1 toxin kills bacteria that compete for scarce resources that do not carry
this same plasmid.

• Many vectors have been derived from the closely related pMB1, including
pBR322, the pUC plasmids, and the pET series of plasmids.

• All these engineered plasmids, known as cloning vectors, have been modified
but all of them have the ori region of the original ColE1-like plasmid.

• ColE1-derived plasmids are able to replicate in E. coli and other enterobacteria

such as Salmonella and Klebsiella species.

• These plasmids have a high-copy number and a Theta mechanism of

replication.
ColE1 plasmid
Encodes a protein that protects Encodes a protein required for colicin transport
the cells carrying the plasmid
from the colicin ColE1 toxin
Promoter for the primer RNA II
Antitoxina

Encodes RNA I

Encodes the
colicin ColE1 toxin Origin of replication
ColE1
plasmid OriT
(6,646 bp)
Encodes a protein that
helps regulate
copy number

Encodes functions required for mobilization

 ColE1 plasmid replication

• The replication of this plasmid is not initiated by a

protein, the initiator is a molecule of RNA called RNA II

• The RNA II forms an RNA-DNA hybrid at the replication

origin

• RNA II is cleaved by the host endonuclease RNase H,

releasing a 3’ OH that serves as the primer for
replication first catalysed by host DNA polymerase I

• Replication is carried out entirely by host proteins

(RNase H and DNA polymerases)
ColE1 copy number control
• Replication is regulated mostly through the effects of a
small plasmid-encoded RNA called RNA I Rop

• RNA I and RNA II are complementary to each other and

form a double-stranded RNA helix interfering with the
replication

• The plasmid-encoded Rop protein maintains the copy

number since stabilizes dsRNA helix RNA I-RNA II

• More RNA I and Rop will be made when the

concentration of plasmid is high, the inhibition of
replication is complete when the plasmid reaches about
15-20 copies per cell

PArant la traducció

HOW TO INCREASE THE ColE1 COPY NUMBER??

ColE1 copy number control

Bacteriostatics inhibiting protein synthesis (such as chloramphenicol) can

increase the plasmid copy number:

-The cell will not be able to divide

-The translation of Rop protein will be inhibited

-The plasmid replication will not be inhibited, because it is RNA-dependent

 The R1 Plasmid
• The R1 Plasmid was first isolated from Salmonella enterica Paratyphi

• It is a low-copy number, theta replication (unidirectional) and broad host range

• Four properties can be distinguished in R1, all of which contribute to plasmid survival:

-Basic replicon

-Horizontal DNA transfer genes

-Antibiotic-resistance genes

-Stability (par systems)

• Low copy number could reduce the

chances of R1 being transmitted to
daughter cells. To counteract this, the
plasmid has evolved three stability
systems: parA, parB and parD R1-plasmid derivatives:
pEBs, pKNs, pSLs…
 The R1 Plasmid: Replication
• The R1 plasmid encodes a Rep protein called RepA, required for the initiation
of replication. The repA gene can be transcribed from 2 promoters:

-PcopB: transcribes copB and repA

-PrepA: transcribes only repA

• CopB protein represses PrepA so the RepA from PrepA is only produced
immediately after the plasmid enters the cell and before any CopB is made

Replication occurs
after plasmid enters
the cell because
there are not CopB
proteins repressing
PrepA, and RepA is
produced from the
two promoters
 The R1 Plasmid: Replication
• Once the plasmid reaches its copy number: ↑CopB → repression PrepA

• Now RepA is only produced from PcopB and regulated from CopA

• CopA is an RNA that hybridises the mRNA made from PcopB forming a dsRNA

• The host RNase III cleaves the dsRNA→ No translation of repA→ No replication

• ↑plasmid → ↑CopA → ↓RepA → Stabilization of the plasmid copy number

The R1 Plasmid: Stability (parA)

• ParA is a partition system that segregates

copies of R1 to daughter cells

• ParA is organized as a bicistronic operon

that encodes ParM and ParR

• ParR binds to the R1 plasmid DNA

sequence parC

• ParM is an ATPase that interacts with ParR

and polymerizes itself into filaments

• These filaments push plasmids to opposite

cell poles in order to ensure their
segregation to both daughter cells.
 Toxin-antitoxin (TA) systems
• The toxins (poison) of all known TA systems are proteins while the antitoxins (antidote) are either
proteins or non-coding RNAs

• TA genes were initially discovered in two conjugative plasmids of E. coli, F (ccdAB) and R1 (parB
and parD), as cassette of two genes involved in stable maintenance of these plasmids

• In F plasmid, CcdB is a toxin that targets

the E. coli DNA gyrase and CcdA is the
unstable antidote that interacts with CcdB
to neutralise its toxicity.

• Killing of plasmid free segregants was

termed post-segregational killing (PSK)
and shown to be due to the decay of the
more unstable antitoxin in plasmid-free
cells and to the subsequent activation of
the toxin in these cells
Toxin-antitoxin (TA) systems

CcdB
CcdA

DNAGyr

Antidote Poison
The R1 Plasmid: Stability (parB)
ParB is a TA system that generates the Hok (Host killing) toxin that kills cells that
lose the plasmid by damaging their membrane
Hok
toxin

NO HOK

snRNA
(unstable) NO HOK HOK

PMID: 17471262
The R1 Plasmid: Stability (parD)
ParD is a TA system that encodes the toxin Kid (Killing determinant) and the
antitoxin Kis (Killing suppressor).

Appropiate
R1 copy
number

(host protease)

Low R1 copy number

PMID: 17471262
 THE ITERON PLASMIDS
• Many plasmids use a protein (RepA) to regulate their replication and some of
them contain in their ori region several repeats (3 to 7) of a certain set of DNA
bases (approximately 20 bp long) called an iteron sequence

• pSC101: First cloning vector used in 1973, from Salmonella, about 5 copies/cell,
its plasmid-encoded RepA protein :

-Initiates the replication allowing the binding of host proteins: DnaA (binds to
dnaA boxes and opens helix), DnaC (loads DnaB helicase), and DnaG (primase)

-Represses its own synthesis by binding to its promoter (Inverted Repeats IR1-2)

-Regulates the copy number through three repeated iteron sequences (R1-3)

In pSC101 the host range is narrow

because the initiation of replication also
depends on the host DnaA protein Iterons
Regulation of replication in pSC101
• TRANSCRIPTIONAL AUTOREGULATION:

-The higher the concentration of plasmid, the more RepA protein will be
made and the more it will repress its own synthesis

• COUPLING OR HANDCUFF MODEL:

R1 R2 R3

-At low concentrations of plasmid, the

RepA protein only binds to the iterons of
one plasmid at a time, initiating replication.

-At high concentrations of plasmid, the RepA

protein binds to the iterons of two plasmids
simultaneously, hand-cuffing them and
inhibiting replication.
 Plasmid cloning vectors
• A cloning vector is an autonomously replicating DNA into which other DNAs can
be inserted, so that many copies of the original piece of DNA can be obtained

• Most naturally occurring plasmids are not convenient cloning vectors but they can
be engineered to make a cloning vector

• Desirable features of plasmid cloning vectors:

-Small
-High number copies
-Selectable
-Unique sites for restriction

• Plasmids with the same ori (same Inc group) are incompatible: they will compete
for the same machinery, creating an unstable and unpredictable environment
 Plasmid cloning vectors: pBR322

• The pBR322 plasmid is small (4,360 bp) and

has relatively high copy number (about 15-20
copies per cell), making it easy to isolate

• The vector was constructed by removing all

except the essential ori region from pMB1, a
ColE1-like plasmid, and adding two resistance
genes for the antibiotics tetracycline and
ampicillin, which were taken from pSC101
and transposon Tn3, respectively

• It also has several unique sites for restriction

endonucleases, including BamHI, EcoRI, and
PstI
Plasmid cloning vectors: pUC
Plasmids pUC (ColE-1 like) are small ( ̴3 kb), high copy number, confer ampicillin
resistance, and encode the α-subunit of LacZ (unfunctional)
Plasmid cloning vectors: pUC
 Plasmid cloning vectors:
Expression vectors (pET plasmids)
• A pET vector is a bacterial plasmid designed to enable the quick production of a
large quantity of any desired protein when activated

-lacI gene (repressor protein)

-T7 promoter (specific to phage T7 RNA polymerase)
-lac operator (LacI binds blocking transcription)
-polylinker (Multiple cloning site)
-Ampicillin resistance gene
-ColE1-like origin of replication

• The host cell for this expression system is an E. coli which has been genetically
engineered to incorporate the gene for the phage T7 RNA polymerase

• The system is repressed by LacI (protein overexpression can be toxic for the cell)
Plasmid cloning vectors:
TA vectors
5’ 3’
3’ 5’
• When DNA fragments are generated by Taq 3’ 5’
DNA polymerase*, extra adenine is added 5’ 3’
onto the 3’ ends of DNA

• Several commercially available kits take

adventage of this ability (ex. pGEM-T vector) Ligation

• These kits use a plasmid vector with

thymidine residues linked onto the 3’ ends of
linearized plasmid DNA

• As the insert and vector the vectors have Transformation

compatible ends, the ligase will be able to join
them together Screening of colonies, colony PCR,
plasmid extraction, restriction
and sequencing
*Taq DNA polymerase without activity exonuclease 3’→5’,
unlike high fidelity DNA polymerases (which generate PCR products with blunt ends)
 Plasmid cloning vectors:
Mobilizable vectors

• Many of the common E. coli cloning vectors such as pBR322, pUC, and pET
plasmids have been constructed with the pMB1 oriV (ColE1-like) and their host
range are E. coli and close relatives

• Other cloning vectors have been derived from the broad host-range plasmids
RSF1010 and RP4, which can replicate in most gram-negative bacteria

• These cloning vectors sometimes contain the mob site (which includes OriT),
allowing them to be transferred into other bacteria by conjugation

• Mobilizable plasmids are very useful, because other ways of introducing DNA may
be difficult for many types of bacteria or strains
 Plasmid cloning vectors:
Shuttle vectors
• Shuttle vectors contain more than one type of origin replication and so can be
replicated in unrelated microorganisms

• Also must contain selectable genes that can be expressed in both organisms

• In most cases, one of the organisms in which the shuttle vector can replicate is
E. coli: the genetic test can be performed in the other organism, but the plasmid
can be manipulated and purified by the methods already optimized for E. coli

• Some shuttle vectors can replicate in gram-positive bacteria and E. coli, whereas
others can be used in lower or even higher eukaryotes

• Shuttle vectors have been even also developed for mammalian cells, they contain
the ColE1 origin of replication and the replication origin of simian virus SV40
 Plasmid cloning vectors:
Suicide vectors
• Suicide plasmids cannot replicate when are introduced into non-permissive hosts
and/or under determinate conditions

• For example, plasmids containing a ColE-1 like oriV are able to replicate in E. coli
but are suicide for unrelated gram-negative hosts such as Pseudomonas species

• One of the most common strategies to generate knockout mutants in bacteria

are suicide vectors for allelic exchange

• A suicide plasmid may be also conditional for replication to allow selection for
integration into the chromosome (ex. a plasmid that is temperature sensitive for
replication)

• A plasmid lacking its Rep protein is suicide for all bacteria except for those that
has been modified to express it
Plasmid cloning vectors:
Suicide vectors

• Replication of the R6K plasmid and

derivatives harbouring its origin of
replication is dependent on the pir gene-
encoded π protein (a Rep protein)
(ApR)
• Originally encoded by R6K, this protein
is usually provided in trans in hosts
engineered to support replication of
plasmids harbouring the R6K oriV but
lacking the pir gene (GmR)

• In E. coli this is commonly achieved by

chromosomal integration of pir either
via lysogenization with a λ pir phage or
homologous recombination at a pre-
determined locus.
Plasmid cloning vectors:
Integrative vectors
• Integrative plasmids are in most cases
suicide vectors that are unable to
replicate in the destination host and
therefore must either integrate or
disappear

• They take advantage of the

bacteriophages to integrate into the The plasmid pMC1 is based on
Lactobacillus bacteriophage mv4
chromosome: present an integrase and and is able to integrate into the
an attP region associated chromosome of a wide range of
gram-positive hosts:
• These vectors can be incorporated into -Lactobacillus casei
a specific site of the attB host genome -Lactobacillus plantarum
-Lactococcus lactis
-Enterococcus faecalis
-Streptococcus pneumoniae
Genotypes of most used E. coli
host strains
STRAIN GENOTYPE (most relevant features)
DH5a F– endA1 thi-1 recA1 gyrA96 lacZΔM15 hsdR17(rK–mK+), λ–
BL21(DE3) F– ompT lon hsdSB (rB–, mB–), dcm, λ-DE3, Cmr

DH5α is widely used for DNA cloning

BL21(DE3) is used for protein expression from gene with T7 Promoter (pET plasmids)

F– Does not carry the F plasmid

endA1 Elimination of non-specific DNA digestion by Endonuclease I
thi-1 Auxotrophy (prevent survival of released bacteria outside the lab)
recA1 For reduced occurrence of unwanted recombination in cloned DNA
gyrA96 Nalidixic acid resistance
lacZΔM15 LacZα deletion required for blue/white screening on X-Gal plates
hsdR17/dcm Elimination of restriction-modification systems to avoid DNA degradation
ompT/lon Deficient in proteases to avoid protein degradation
λ-DE3 Lysogenic for λ phage encoding T7 RNA polymerase
Cmr Chloramphenicol resistance
DNA CLONING (overview)
1. Vector preparation
1. Vector preparation

5’ Nicks will be repaired

5’
by the cell after
transformation
5’
3’ 5’
3’
nick

nick

AP prevents vector self-ligation. Alkaline phosphatase (AP) is usually used with

digested vectors to eliminate phosphates at 5’ avoiding recircularization by DNA
ligase but then the insert has to be phosphorylated.
2. Insert preparation
DNA insert with 5’-P

• DNA is phosphorylated at 5’ when:

-It has been digested by restriction enzymes or S1 nuclease

-It has been treated with a kinase

-It has been amplified by PCR with primers phosphorylated at 5’

3. Ligation
4. Transformation
4. Transformation
5. Screening
5. Screening
5. Screening

False positive?
CONSTRUCTION OF
DNA LIBRARIES
What is a DNA library?
• A DNA library is a collection of DNA clones that includes all (or at least all) the DNA
sequences of an organism.

• First, the DNA is cut with a restriction endonuclease, and the pieces are ligated into a
cloning vector cut with a compatible enzyme.

• The mixture is then transformed into cells and the transformants are pooled.

• If the collection is large enough, every DNA sequence will be represented somewhere in
the pooled clones, and the library is complete.

• There are basically two kinds of libraries: genomic DNA and cDNA libraries.

A genomic library is a collection of overlapping segments of genomic DNA

Different types of libraries
Genomic DNA libraries:

• Contain large fragments of DNA

Genomic
• The genome is cut with restriction enzymes library

• Cloned in plasmids, bacteriophages or

bacterial artificial chromosomes

cDNA libraries:
Eukaryotic
• Are made with cloned, reverse-transcribed organism
mRNA (transcriptome)

• Lack DNA sequences corresponding to

genomic regions that are not expressed, such
as introns (in eukaryotes) and 5′ and 3′
noncoding regions.

• Smaller fragments than genomic DNA libraries cDNA

library
• Usually cloned into plasmid vectors
How many clones are necessary?
• The number of clones required to make a complete DNA library of an organism
depends on the complexity of the DNA of that organism.

• The minimum clones number required to make a library of E. coli cut with EcoRI is
4.5x106 / 4x103 ≈ 1,100 different clones, since E. coli DNA contains approximately
4.5x106 bp and EcoRI cuts the DNA about 1 every 46 bp.

• In contrast, a library of λ DNA should require a minimum of 5x104 / 4x103 ≈ 13 clones,

since λ DNA has only 50,000 bp.

• An important point is that these are minimum estimates of clones required to make a
library of DNA of the organism; not all clones will be equally represented in the library
because of random statistical fluctuation.

• Also, cloning efficiency has to be taken into account: some pieces may be easier to
clone, for example, because they are smaller or contain no genes whose products are
toxic to the cell.
How many clones are necessary?
• Some sequences will be included in the library more than once due to overlaps and
some sequences will be missed if only the minimum number of clones is used. To
circumvent these limitations, the number of clones must be greater.

• Clarke and Carbon (1976) devised a formula which calculates the probability (P) of any
DNA sequence being included in a library of random fragments:

ln(1-P) N= number of clones needed

N= P= probability to find a sequence
ln(1-1/n) n= genome size (bp) /average insert size (bp)
(minimum estimates of clones required)

Ex. How many clones are needed to make a library from a 4Mb genome to be 99%
sure to find a particular gene if the cloned fragments in the plasmid are 4kb?

4x106 (bp) ln(1-0.99)

n= =103 N= = 4603 clones
4X103(bp) ln(1-1/103)
It should be noted that to achieve 100% (1.00) probability an infinite number of clones would have to be used!
Plasmid DNA library

genome

Limitation: the size of DNA fragments

that can be cloned is small (up to 10 kb).

Usually plasmids are used for cDNA

libraries
Phage DNA libraries
Sau3A BamHI

• Almost 50 % of λ genome is not necessary for

replication (the central part of the viral
genome is not essential for lytic growth).

• These regions can be replaced for large

fragments of DNA

• The λ vector arms and ≈20-kb genomic

fragments are mixed, ligated, and packaged in
vitro to produce recombinant λ phage virions.

• The recombinant λ phage virions are plated on

a lawn of E. coli cells to obtain individual
plaques.
Molecular Cell Biology. 4th edition.
Phage DNA libraries

cos cos

terminase

In vitro λ packaging (Gigapack®)

Phage DNA libraries
• Particularly, lambda phage represents a feasible choice for constructing complex
genome libraries because of its simple structure and flexible DNA.

• With the help of restriction enzymes, extracted genome DNA can be prepared as
numerous fragments and inserted into engineered lambda vectors.

• After in vitro packaging the ligated

recombinant DNA into lambda phage
particles and amplification in host
bacterial, a comprehensive library of
phage clones can be obtained.

• Each plate carries a different DNA

fragment which collectively constitute
the (almost) whole genome sequence of
the study organism.
Phasmid DNA libraries

• Phasmids combine elements of both

phage and plasmids.

• Cosmids are phasmids that contain cos

regions of λ phage, required for packaging
of DNA fragments into λ particles in vitro.

• Cosmids allow the clonation of DNA

inserts up to 50 kb.
BAC DNA libraries

• Bacterial Artificial Chromosomes (BACs) are vectors based on F plasmid origin of

replication, so they have a copy number of only 1 in E. coli.

• Only one copy is an adventage since if the DNA insert exists in many copies, particularly
if they are large, recombination between repeated sequences in the clones can
rearrange the sequences.

• BACs can accomodate very large inserts, on the order of 300 kb of DNA.

• This was expected since it was known that F’ factor can be very large and are quite
estable, especially in a RecA- host.

• An F’ factor is a naturally occurring plasmid in which the F plasmid has incorporated a

large region of the E. coli chromosome.
BAC DNA libraries
The plasmid pBeloBAC11:

• Is a derivative of a mini-F plasmid

• Encodes repE gene for self-replication

• Contains CmR, a selectable marker

• Regulates its copy number inside a cell: sop

genes functions for active partitioning to
ensure that each daughter cell gets a copy
of the plasmid.

• Includes a lacZ’ gene into the multiple

cloning site and a lambda cos site for
packaging into phage lambda particles if
desired.
PAC and YAC DNA libraries
• PACs (P1-derived artificial chromosomes) are based on P1 E. coli phage.

• As BACs, PACs vectors permit the stable maintenance of large DNA fragments, in an E.
coli host strain.

• At the same time, both BAC and PAC molecules can easily be separated from the E. coli
genomic DNA, using a standard plasmid isolation protocol.

• YACs (Yeast artificial chromosomes), which provide a larger cloning capacity (about
1,000 kb), are unstable, prone to rearrangements, and difficult to separate from the
endogenous yeast chromosomal DNA.
PAC and YAC DNA libraries
P1 DNA digested + plasmid replicon

(85-100 kb)

Cloning into a P1 Artificial chromosome (PAC) Cloning into a Yeast Artificial chromosome (YAC)
APPLICATIONS OF
DNA LIBRARIES
Whole genome shotgun sequencing

• Genomic DNA is fragmented into random pieces and cloned as a bacterial library.
• DNA from individual bacterial clones is sequenced.
• The sequence is assembled by using overlapping DNA regions.
• Gaps can be filled by primer walking.
Identification of genes by
complementation
A mutation on the unknown gene X gives specific phenotypic characteristics.

This genetic method for

identifying clones is based on
their ability to complement
mutations in the chromosome.

Only cells transformed by a plasmid

cloning vector with the geneX will
multiply to form a colony in the
appropriate medium.

Ex. GeneX = thyA, only complemented cells are able to grow without thymine
Cloning by complementation:
Anabolic genes

Shuttle vector
Cloning catabolic genes

Because of its simplicity, agar plate screening

is most commonly used in the identification
of novel enzymes with diverse functions
Recent Advances in Function-based Metagenomic Screening
Cloning catabolic genes

Identification and characterization of alkaline serine protease from goat skin

surface metagenome (PMID: 21906326)
Identification of transcriptional
regulators
The gene X is regulated by an unknown transcriptional regulator.

This genetic method for identifying

X promoter::reporter gene
transcriptional regulators is based on
the construction of a transcriptional
fusion between the promoter of the
gene X and a reporter gene.

Only cells transformed by a plasmid

cloning vector with the transcriptional
regulator will affect the expression of
the reporter gene.
Isolation of regulator genes

Px::gfp
Isolation of regulator genes
Cloning virulence genes

DIGESTION ISOLATION

LIGATION
INOCULATION GENE INVOLVED IN
TRANSFORM VIRULENCE
AVIRULENT STRAIN

DNA LIBRARY
Identification of homologue genes
• Identification of an unknown homologue gene based on its sequence in other organisms

• The first step is the design of degenerate oligonucleotide probes based on the conserved
known homologues

ProtX
E. coli
Shigella
Providencia

Conserved region

Design of probes
Screening in DNA library
Identification of homologue genes

conserved region in other organisms

Identification of homologue genes

The method of plate hybridizations is like the Southern blot hybridizations, except that
the DNA transferred to filters is from phage plaques or bacterial colonies rather than
being bands on gels. The colonies or plaques on a plate are transferred to a membrane
filter (two filters may be used per plate to discard false positives).
Identification of homologue genes

The DNA on the filters is denatured and then hybridized to the labelled probes to identify
the colonies or plaques that contain DNA complementary to the probes, which can be
recovered from the master plate for sequencing.
Cloning toxic genes
Cloning toxic genes
Cloning toxic genes
Cloning toxic genes

IPTG binds to the LacI repressor and releases the tetrameric repressor from the lac operator
T7 lysozyme is a natural inhibitor of T7 RNA polymerase
Cloning toxic genes
Cloning toxic genes

PcnB is required for the rapid degradation of RNAI, the antisense

RNA that controls the copy number of ColE1-related plasmids.