PMBB 0703

VECTORS FOR GENE CLONING

PART I

Lecture Set 3
(Semester I-2015)
 AN OUTLINE
PLASMIDS

PHAGEMIDS

COSMIDS

BAC

YAC

PAC

PLANT VIRAL VECTORS

 VECTOR
A DNA molecule acts as a vehicle for gene cloning.

Requirements for a vector:

Capable of replicating within the host cell

Relatively small, Molecular weight/size low as much possible
Availability of selectable markers
Contain a spectrum of restriction sites for cloning (MCS)
Multiple copies of vector desirable
 PLASMID
Extrachromosomal, self-replicating, double-stranded, covalently closed circular
DNA molecules.

Found in a wide variety of bacterial species; narrow host range and can be
maintained only in a limited set of closely related species.

Vary in size from 1 kb to more than 200 kb.

Have evolved mechanisms to maintain copy number and partition.

Dependent to a some extent on proteins encoded by their host for replication and
transcription.

Generally dispensable- known to confer traits like antibiotic resistance,

production, degradation of complex organic compounds, induction of plant tumors
etc.

Cryptic plasmids- No phenotypic traits have been ascribed.

DIFFERENT FORMS OF PLASMID DNA
Effect of intercalating agent on plasmid DNA supercoiling

Ethidium bromide*: an intercalating agent

supercoiled DNA EtBr causes untwisting

supercoiling decreases

relaxed circular form

More EtBr
introduction of excess turns
in the double helix

supercoiling in opposite direction

*Potential Carcinogen. Safer alternatives such as SYBR Safe

 Linear plasmids also exist
Linear plasmids found in a variety of bacteria
Streptomyces sp., Borrelia burgdorferi, Thiobacillus versutus
Ends are protected by two mechanisms:
I. Hair-pin plasmids: presence of repeated sequences ending in a terminal DNA
hairpin loop (Borrelia burgdorferi ). Two DNA strands are connected and form a perfectly
palindromic AT-rich terminal hairpin loop. Conserved 19 bp sequence present at ends.
II. Covalent attachment of terminal proteins or TPs at 5’ termini of each strand
as well as ITRs (Streptomyces)

Hinnebusch & Tilly, Mol Microbiol, 1993

 Linear Plasmids
Replication mechanisms of the two types of plasmids are different: linear and circular.

The two forms appear to be evolutionary distinct.

Recent studies advocate that linear variants can arise during replication of circular genomes.
This is somewhat true in case of mitochondrial and chloroplastic genomes which are plastic
in nature. Circular and linear isomers do co-exist. Mitochondria of Pichia have linear genome
with ITR loops, and circular monomeric forms are also present.

Chloroplast genomes of barley and rice are generally circular, but linear hairpin deletion
derivatives isolated from albino clones.

Conversion might occur due to:

Action of mobile elements composed of two joined telomeres. Telomere resolving enzyme
may convert circular to linear form.

Transient forms may exist and therefore distinction between circular and linear replicons may
not be absolute.

Linear plasmids with telomeres provides advantages as these regions are often recombinogeni
Lot of rearrangements and translocations occur and provide means of duplication & molecula
Evolution. Conjugation and chromosomal integrations are simplified due to availability of
linear structure.
 Plasmids exist in other organisms also

Plasmids are widespread in bacteria.

However, they are also present in eukaryotic cells as in many strains of

Saccharomyces cerevisiae such as yeast episomal plasmids (YEps).

In case of other organisms such as fungi and higher organisms there are no
reports of existence of plasmids.
 TYPES OF PLASMIDS
Conjugative Stringent

Non-conjugative Relaxed

Fertility (F)

Resistance (R)

Col

Degradative

Virulence
 CONJUGATION
Directed transfer of DNA from a donor to recipient cell

Mediated by tra and mob genes

Tra genes: Plasmid-encoded

Cluster of at least 12 genes

Responsible for synthesis of pili and

other surface components

Mob genes: Plasmid-encoded

One region encodes for a mobilising

Protein

Mob protein binds to other mob region

Formation of relaxation complex

Strand-break (nic/bom region)

 CONJUGATION
tra+ & mob + --------conjugative and mobilizable
Examples: sex factor F

tra - & mob +--------- non-conjugative, can be mobilized by another conjugative

plasmid present in same cell
Examples: ColE1 and CloDF13 are mobilised by F plasmids

tra - & mob ----------- non-conjugative & immobilizable

Non-conjugative and transfer-deficient plasmids desirable due to safety

considerations.

bom/nic mob bom/nic mob bom/nic mob

ori tra ori ori

Antibiotic Antibiotic Antibiotic

resistance gene resistance gene resistance gene
 CONTROL OF PLASMID COPY NUMBER
Copy number defined as average number of plasmids per bacterial cell: controlled by
plasmid replicon- relaxed and stringent control.

Plasmids maintain harmony between their rate of replication and that of the host by
rationing the supply of a molecule that affects the frequency of initiation of plasmid DNA
synthesis.

pMB1/ColE1: positive regulatory molecule is an RNA, known as RNAII

pSC101 and other replicons: is a cis-acting protein RepA that acts positively on origin
of replication and negatively regulates the transcription of its own gene.

Plasmids – RNAII-do not require any plasmid-encoded proteins for replication-high

copy number-rely on long lived enzymes and proteins supplied by host. When protein
synthesis is inhibited (like addition of chloramphenicol)-plasmid copy number increases.

In case of pSC101 derived replicons- plasmid replication requires continuous protein
synthesis.
 Relaxed vs Stringent Plasmids
Based on the copy number of plasmids.

Stringent: limited number per cell (generally HMW), require active protein
biosynthesis, host DNA Pol not required for their replication instead require
plasmid encoded RepA protein

Relaxed: multiple copies per cell (generally LMW), do not require de novo protein
synthesis but do require functional DNA Pol (which is a long lived enzyme in the host)

Chloramphenicol antibiotic causes plasmid amplification.

Multicopy plasmids do not require protein synthesis for replication,

while the bacterial chromosome requires.

Grow cells for sometime

add chloramphenicol

inhibition of protein synthesis

only plasmids will replicate and not the chromosome leading to amplification.
 More classes of Plasmids….
Phenotypes exhibited by plasmid genes:
Antibiotic resistance
Antibiotic production
Degradation of aromatic compounds
Heavy metal resistance
Bacteriocin production
Host-controlled restriction and modification
Grouped as:

Fertility or F plasmids: carry tra genes. Eg. F plasmid

Resistance or R plasmids: carry genes for resistance to antibiotics. Eg. RP4 (Pseudomonas
Col plasmids: carry genes for colicin productionEg. ColE1 (E. coli)
• Degradative plasmids: carry genes to metabolize unusual molecules such as toluene or
salicylic acid. Eg. TOL (Pseudomonas putida)
Virulence plasmids: carry genes for conferring pathogenicity to host bacterium.
Ti plasmids of Agrobacterium belong to this class.
 Incompatibility System
Inability of two different plasmids to coexist in the same cell in the absence of
selection pressure. Over 30 Inc groups defined for E. coli.

Compatible--------able to coexist in the same cell

Incompatible-----------unable to coexist in the same cell.

Group of plasmids which are mutually incompatible belong to the same Inc
group.

Dual incompatibility: Plasmid is incompatible against more than one Inc group.

REPLICATION CONTROL:
Plasmid Inc and exact control of copy number or of plasmid DNA replication are
intimately linked.

Ori derived from plasmid ColE1 – Best characterized copy no. and incompatibility system.
~ 20 copies per cell are maintained.

Copy no. depends upon the mechanism of initiating replication at the ColE1 origin.
 Replicon and Copy number
Replicon is a genetic unit consisting of an origin of replication and its associated control
elements.
More appropriate definition: smallest piece of plasmid DNA that is able to replicate
autonomously and maintain normal copy number.
Copy number is defined as the average
number of plasmids per bacterial cell
or per chromosome under normal
growth conditions.
Controlled by plasmid replicon.
Plasmids maintain harmony between
the rate of their replication and that of
the host by rationing the supply of a
molecule that affects the frequency of
initiation of plasmid DNA synthesis.

pMB1/ColE1-----RNAII
pSC101-------------RepA
Synthesis and activity of these regulatory molecules is modulated by ancillary trans-acting
products whose concentration is responsive to plasmid copy number or to alterations in the
physiology of the host bacterium.
 Control of Copy number
For ColE1/pMB1 replicon (high copy number plasmids)
Positive regulatory molecule is RNA. No need of plasmid-encoded proteins. Require long-
lived enzymes and proteins encoded by host such as chaperones, DNA PolI, DNA PolIII,
RNA Pol, RNaseH, DNA gyrase, Topoisomerase I.
RNAII: Positive regulatory molecule
RNA primer for DNA replication,
555 bases upstream of the origin.
RNaseH: cleaves RNAII to leave a free 3’-OH group, makes it functional i.e. at which
DNA synthesis begins.
Primer forms persistent hybrid with DNA (upstream ori)
RNAI: Regulatory molecule.
108 bases, encoded by same region of DNA as RNAII, but by the complementary
strand.
Initiation occurs within a 600 nt region containing cis-elements required for replication.
 Synthesis of RNAII precursor initiated at a promoter 550bp upstream of ori, proceeds
through origin and terminates at one of a number of closely spaced sites located 150 nt
downstream.
Countertranscript : RNAI to RNAII. RNAI & RNAII are complementary and can hybridize
to form a ds RNA helix. The 5’end of ~750 nt primary transcript folds into a complex
secondary structure that brings a G-rich loop in
RNAII into alignment with a C-rich
stretch of plasmid DNA located 20 nt
upstream of ori on the template strand.

RNAII is processed by RNAse H

(cleaves RNA within a sequence of
five A residues at the origin) as it
forms DNA-RNA hybrid at the replication
Origin.

RNAII functions as primer for

DNA Pol I to initiate replication by
synthesis of leading strand.

Extension of stable DNA-RNA hybrid

exposes sites on complementary DNA
strand at which discontinuous synthesis
of lagging strand is initiated.
Primer Processing Model
Replication control is mediated by RNA I which forms hybrid (ds RNA helix with RNAII).

This interferes with the processing of RNAII by RNAse H and hence replication does not ensue.

Dynamic interaction between RNAII

and short-lived RNAI (T1/2=1-2 min).
Folding of RNAII is vulnerable to
interference by RNAI when the nascent
RNAII transcript is between 80 and 360nt.

Hybrid involves 5’end of RNAII and

entire length of RNAI.

Steady state concentration of cytoplasmic

RNAI is determined by gene dosage and
this controls copy number.

If copy no. increases above normal, conc.

of RNAI increases and plasmid DNA
replication is inhibited.

This coupling of plasmid copy number

and RNAI conc. only works if half-life
of RNAI is short. Solar and Espinosa, Mol Microbiol, 2000
 Control of Copy number
Degradation of RNAI proceeds in two stages.
The five 5’-terminal nucleotides are removed by endonucleolytic cleavage by RNAse E. The
truncated molecule may still bind to RNAII but is now susceptible to degradation by a
ribonuclease whose activity is responsive to growth rate. This regulation is responsible in
providing a mechanism to maintain a constant number of plasmids even when growth rate of
cells is fitful.

ROM/ROP PROTEINS EMPOWER NEGATIVE REGULATORY ACTIVITY OF RNAI

Rom-RNA Modulator
Rop-Repressor of primer
Both are plasmid-encoded proteins.
Improve efficiency of binding of RNAI to RNAII. Rom binds to RNAI and RNAII with similar
affinities and drives unstable intermediates formed between the two complementary RNAs into
more stable structure.
Rom recognizes sequence and structural elements in both RNAI and RNAII. Rom binds to the
stem of interacting RNAs, stabilizing the ‘kissing complex’ and initiating formation of perfect
RNAI-RNAII hybrid.
Mutation or deletion of Rop/Rom genes results in increase in plasmid copy number.
Eg. Deletions results in more than 500 copies (normally 15-20 copies per cell for pBR322).
Insertional mutagenesis results in lethal runaway plasmid replication.
 Control of Copy number
For pSC101 (stringent/low copy number plasmid)
Ori contains an array of of 17-22 bp long
directly repeated sequences known as ‘iterons’.
Characteristic of each plasmid.
Specifically bind cognate Rep protein.
Iterons belonging to same plasmid show a high
degree of sequence conservation.

These iterons, in conjunction with nearby cis-acting par locus, “handcuff” plasmid DNA
molecules by sequestering the plasmid- encoded RepA protein which is a regulatory
molecule, resulting in inhibition of replication.

RepA is the only plasmid-encoded protein required for replication. Sequestration of RepA
proteins results in inhibition of binding of host-encoded proteins to origin and prevents
replication.
RepA protein represses its own synthesis by binding to its own promoter and blocking
transcription of its own gene.
Copy number HIGH-----synthesis of RepA is repressed. After cell divisions, the copy
number and concentration will drop and this initiates replication.
Increases in active RepA promotes initiation by overcoming handcuffing, but handcuffing
dominates when copy number reaches a threshold.
Mutations in RepA protein are known to increase copy number of plasmids.
Chattoraj, Mol Microbiol, 2000
 Control of Copy number
For pSC101 (stringent/low copy number plasmid)
RepA (initiator protein) is the only plasmid-encoded protein required for replication.
Rep exists in both monomer and dimer forms.
Monomers bind to iterons specifically and serve as initiators. Two globular domains of
monomer bind to two halves of iterons.
Purified protein found predominantly in dimer from that is inactive in iteron binding.
Dimers can be converted into monomers by a conformational change mediated by chaperones.
Dimers can bind to operators of rep genes of some plasmids.
Sequestration of RepA proteins results in inhibition of binding of host-encoded proteins to
origin and prevents replication.
Iterons are negative regulators as increase in iteron concentration with increase in copy
number of the plasmid eventually turns replication off.
Handcuffing is the most reasonable model for negative control, but it blocks initiation is not
clear.
RepA protein represses its own synthesis by binding to its own promoter and blocking
transcription of its own gene.

Copy number HIGH-----synthesis of RepA is repressed. After cell divisions, the copy number
and concentration will drop and this initiates replication.

Increases in active RepA promotes initiation by overcoming handcuffing, but handcuffing

dominates when copy number reaches a threshold.

Mutations in RepA protein are known to increase copy number of plasmids.

 Control of Copy number
Replication cycle of plasmid P1

Replication activated by increase in RepA

and inhibited by handcuffing.

Rep saturation of ori iterons not only affects

initiation but also represses the rep
promoter
that maps within ori iterons
(autorepression).

Replication activates the promoter by

cleaning the iterons bound to Rep.

Newly made Rep molecules associate readily

to from dimers that are inactive.

Pre-existing active Rep gets titrated by

Negative regulation by iterons is by handcuffing, although
daughter ori, which allows handcuffing and
initiator limitation is involved. Availability of active
promoter repression.
monomeric Rep is crucial for initiation and protein acts as a
positive regulator by rescuing situations that reduce copy
Handcuffing is effectively reversed by number.
Chattoraj, Mol Microbiol, 2000
 LOW COPY NUMBER PLASMID VECTORS

Low copy plasmid vectors are built around replicons such as R1 that keep plasmid DNA
synthesis under a very tight rein

Designed to solve problems of toxicity that arose with cloning certain type of sequences
for eg. genes coding for membrane proteins, certain promoters and regulatory sequences

Sometimes toxicity so high that it is impossible to isolate transformed strains using high
copy number vectors.

To solve these problems, multi-purpose low copy number vectors have been developed
that carry tightly regulated prokaryotic promoters with a low level of basal expression for
example in pET vectors.

In high copy number plasmids, the toxicity can be controlled by propagating recombinant
plasmids in a E. coli strain carrying a mutant allele of pcnB gene. The WT allele of this
gene is responsible for unstable RNAI as it polyadenylates RNAI.
 Run Away Replication
Temperature-sensitive control of copy number:

Plasmid replication affected by temperature changes. Also, known as “run away

replication”.

Runaway vectors replicate in normal fashion upto 34oC-copy number increases with
temperature and above 39oC replication becomes uncontrolled.

Eg. IncFII (Low copy number plasmid). RepA synthesis is under control of
bacteriophage pR or pL promoter. The activity of this promoter is controlled by
temperature-sensitive lambda repressor cI857.
The amount of protein constitutes 50% of the total protein.
Problem: Severe metabolic strain resulting in decreased growth rate and sometimes
cell death.
Solution: Optimize time of induction.
 Antibiotic resistance
One of the useful characteristics for designing cloning vectors
Also known as positive selection markers
 More selection markers

Reversal of auxotrophy:
hisB+----histidine

inactivation is positively selected:

sacB--encodes levansucrase, activity lethal to cells on medium with 7% sucrose.

ccdB-- toxin is lethal to cells, if host contains DNA gyrase mutation (gyrA) the cells can
grow. Very useful in Gateway cloning system.
Strategy for cloning in plasmids
 α-Complementation/Blue-white screening

Vector: amino terminal of lac Z gene product (beta-galactosidase)

Host: carboxy terminal of lac Z which is capable of intra-allelic
complementation with amino peptide.

X-gal: 5-bromo-4-chloro-3-indolyl-β-D-galactoside
IPTG: Isopropylthiogalactoside
 PLASMIDS USEFUL IN MOLECULAR BIOLOGY
pBR322: widely used, designed cloning vector
among early vectors, best and most widely used
contains ampr and tetr genes as selectable markers
>40 unique enzymes sites
restriction sites present in marker genes
pBR322 widely used for cloning
Model system for the study of prokaryotic
transcription & translation.

Improved vectors designed from pBR322

Addition of restriction sites
insertion of new selectable markers
Use of polylinkers or MCS as seen in
pUC vectors
many restriction sites for efficient cloning
lacZ inserted for blue/white screening
 Suggested Reading
1. Principles of Gene Manipulation & Genomics: Primrose and Twyman

2. Gene Cloning: T A Brown

3. From Genes to clones: E L Winnacker

4. Genes IX: B. Lewin

5. Molecular Cloning, A laboratory manual : Sambrook and Russell