UNIT-II

GENETIC ENGINEERING AND BIOETHICS

12BT216

CLONING VECTORS
 Cloning Vectors
• The molecular analysis of DNA has been made possible by the cloning of
DNA. The two molecules that are required for cloning are the DNA to be
cloned and a cloning vector.
• Cloning vector - a DNA molecule that carries foreign DNA into a host cell,
replicates inside a bacterial (or yeast) cell and produces many copies of
itself and the foreign DNA
• Three features of all cloning vectors
– sequences that permit the propagation of itself in bacteria (or in yeast for
YACs)
– a cloning site to insert foreign DNA; the most versatile vectors contain a
site that can be cut by many restriction enzymes
– a method of selecting for bacteria (or yeast for YACs) containing a vector
with foreign DNA; usually accomplished by selectable markers for drug
resistance
 Types of Cloning Vectors
• Plasmid - an extrachromosomal circular DNA molecule that
autonomously replicates inside the bacterial cell; cloning limit: 100 to
10,000 base pairs or 0.1-10 kilobases (kb)

• Phagemid - derivatives of bacteriophage lambda; linear DNA

molecules, whose region can be replaced with foreign DNA without
disrupting its life cycle; cloning limit: 8-20 kb

• Cosmids - an extrachromosomal circular DNA molecule that combines

features of plasmids and phage; cloning limit - 35-50 kb

• Bacterial Artificial Chromosomes (BAC) - based on bacterial mini-F

plasmids. cloning limit: 75-300 kb

• Yeast Artificial Chromosomes (YAC) - an artificial chromosome that

contains telomeres, origin of replication, a yeast centromere, and a
selectable marker for identification in yeast cells; cloning limit: 100-
1000 kb
General Steps of Cloning with Any Vector

• prepare the vector and DNA to be cloned by digestion

with restriction enzymes to generate complementary
ends or blunt ends in case of Blunt end cloning. Or Taq
polymerase PCR using TA cloning or Topo.
• ligate the foreign DNA into the vector with the enzyme
DNA ligase
• introduce the DNA into bacterial cells (or yeast cells for
YACs) by transformation
• select cells containing foreign DNA by screening for
selectable markers (usually drug resistance)
 PLASMID
• pBR322 is a plasmid and was one of the first widely used E. coli cloning vectors. Created in
1977 in the laboratory of Herbert Boyer at the University of California, Davis , it was named
after the Mexican postdoctoral researchers who constructed it. The p stands for "plasmid,"
and BR for "Bolivar" and "Rodriguez.“

• pBR322 is 4361 base pairs in length and contains the replicon of plasmid pMB1,
the ampRgene, encoding the ampicillin resistance protein (source plasmid RSF2124) and
the tetR gene, encoding the tetracycline resistance protein (source plasmid pSC101).

• The plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these
40 sites lie within the tetRgene. There are 2 sites for restriction
enzymes HindIII and ClaI within the promoter of the tetRgene. There are 6 key restriction
sites inside the ampR gene.

• The origin of replication or ori site in this plasmid is pMB1 (a close relative of ColE1)

• The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the
count increases through the tet gene.
• Copy No=15 but can be increased 1000 fold by plasmid amplification by addition of a
protein synthesis inhibitor such as chloramphenicol (170 µg/ml) in mid-log phase and
continuing incubation for further 8 hours.
 Plasmid copy number

Plasmids vary widely in their copy number per cell depending on their origin of replication (e.g., pMB1,
ColE1, or pSC101) which determines whether they are under relaxed or stringent control; and depending on
the size of the plasmid and its associated insert.

Some plasmids, such as the pUC series and derivatives, have mutations which allow them to reach very high
copy numbers within the bacterial cell. Plasmids based on pBR322 and cosmids are generally present in
lower copy numbers. Very large plasmids and cosmids are often maintained at very low copy numbers per
cell.
The pUC 18 or 19 Cloning Vector

Lac Z α

RE Sites in blue occur only once in the plasmid

After Induction by Lactose or IPTG
 Alpha-complementation
• Alpha-complementation is the most common form of insertional inactivation. In alpha-
complementation, the vector molecule contains the regulatory and coding regions for the first 146
amino acids of the ß-galactosidase (lacZ) gene. A polycloning/ Multiple Cloning Site (MCS) site has been
engineered into the coding sequence without disrupting the activity of the gene product (the amino-
terminus of ß-galactosidase). During ligation, vector molecules are cut at a particular sequence in the
polycloning site and incubated with pieces of insert DNA cut with the same restriction enzyme.

• In some instances, insert DNA is successfully ligated into a vector molecule causing disruption of
the lacZ coding sequences on the vector. However, some vectors will not be cut by the restriction
endonuclease or will have their ends religated without incorporation of an insert.

• After ligation, the resulting DNA is used to transform a competent E. coli strain in which only the region
of the lacZ gene coding for the carboxy-terminal portion of ß-galactosidase is present. In cells that
contain a vector without an insert, the amino-terminal ß-galactosidase subunit will interact with the
carboxy-terminal ß-galactosidase subunit to form an active ß-galactosidase enzyme (the inducer IPTG is
often necessary for transcription).

• ß-galactosidase catalyzes the transformation of the clear substrate X-GAL into a blue precipitate, giving
colonies with an active ß-galactosidase protein a distinctive blue color. In contrast, clones containing a
polycloning site into which a piece of exogenous DNA has been inserted (i.e., recombinant clones) will
not produce a functional amino-terminal subunit, and will consequently appear white in color. It is the
recombinant clones that are of interest, and hence they are preferentially selected from agar plates for
further analysis
Restriction & Ligation
Insertion of Restriction Fragment into
Vector
Multi Cloning Site
Transformation and Selection

White White Blue Dead

Screening
 BLUE/WHITE SCREENING
• Colony Selection: finding the rare bacterium
with recombinant DNA
• Only E. coli cells with resistant plasmids grow on
antibiotic medium
• Only plasmids with functional lacZ gene can
grow on Xgal
lacZ(+) => blue colonies
lacZ functional => polylinker intact => nothing
inserted, no clone
lacZ(-) => white colonies polylinker disrupted =>
successful insertion & recombination!
 α -complementation
• The portion of the lacZ gene encoding the first
146 amino acids (the α -fragment) is on the
plasmid

• The remainder of the lacZ gene is found on the

chromosome of the host.

• If the α -fragment of the lacZ gene on the plasmid

is intact (that is, you have a non-recombinant
plasmid), these two fragments of the lacZ gene
(one on the plasmid and the other on the
chromosome) complement each other and will
produce a functional β -galactosidase enzyme.
 SCREENING RECOMBINANTS

• In the example shown above, the b-galactosidase gene is

inactivated. The substrate "X-gal" turns blue if the gene is
intact, ie. makes active enzyme. White colonies in X-gal
imply the presence of recombinant DNA in the plasmid.
• Plasmids are used as vectors for cloning small pieces of DNA.

• The limitation of this vector is the size of DNA that can be introduced into the cell by
transformation.

• This presents problems when you are trying to create a genomic library of a large genome such as
with plants.

• A genomic library contains all of the DNA found in the cell of any organism.

• If you digest genomic DNA to completion with a restriction enzyme, ligate those fragments into a
plasmid vector and transform bacterial cells, only a portion of those fragments will be represented in
the final transformation products.

• If a gene of interest is located on a large fragment then you will not be able to isolate that gene from
a plasmid library.

• But what can be done to increase the probability of obtaining a clone which contains the entire
gene.

• First you need to use a vector that can accept large fragments of DNA. Examples of these are
bacteriophage and cosmid vectors and more recently yeast artificial chromosomes.

• Bacteriophage lambda vectors were developed because several observations were made that
suggested that they could complete their life cycles even if foreign DNA was inserted into a portion
• Lambda was first discovered at the Pasteur
Institute by Andre Lwoff when he observed
strains of E. Coli.
• He showed that the cells of these bacterial
strains carried bacteriophage in a dormant
form (prophage).
• Phage can alternate between lysogenic (non-
productive) and lytic (productive) growth
cycles.
 The Life cycle of lambda.

• Adsorption - the phage particle binds at a maltose receptor site of the bacterial cell;
growing the cell in the presence of the sugar increase the number of receptor sites
• Penetration - DNA is injected into the cell; at this point it can enter one of two
pathways;
– Lysogenic pathway - the phage DNA becomes integrated into the genome and
is replicated along with the bacterial DNA; it remains integrated until it enters the
lytic pathway
– Lytic pathway - large scale production of bacteriophage particles that eventually
leads to the lysis of the cell; base pairing at the cos site leads to a circular
molecule.

• Early transcription - transcription proceeds from thepL and pR promoters, through the N and cro genes and stops at terminators tL and tR1; a
low level of transcription through the O and P genes occurs and terminates at tR2; the N product is an antitermination factor that is important for
the next stage of transcription
• Delayed early transcription - the N product binds to RNA polymerase and transcription proceeds past thetL, tR1 and tR2 terminators; genes to
the left of N, involved in recombination, to the right of cro, involved in replication, are expressed at this point; another protein expressed from
the Q gene is used for antitermination of later transcription
• Replication - early replication is through a theta form initiated from a single origin of replication site; later replication is via rolling circle
replication; this produces long concatamers of the phage DNA that are cleaved at the cosL and cosR sites
• Late transcription - the protein product of the crogene builds up to a critical level and then binds to theoL and oR to stop early transcription;
another protein, a product of the Q gene, has built up and activates transcription at the p'R promoter by antitermination; transcription terminates
with in the b region; this transcription results in the production of the proteins required for the head and tail of the mature phage particle and
those required for bacterial cell lysis.

• Assembly - a prophage head is produced; a unit length DNA is placed into the head
by the action of the Nu1 and A proteins; the DNA is locked into place by
the D protein and ter function of the A protein clips the DNA at
the cosL and cosR sites; the concatamer is released, the tail is added and the
• Packaging of the DNA into the head does not
require a complete length of wild type
lambda. It has been determined that a lambda
molecule that is between 78% and 105% of
wild type length can be packaged. This is from
37 to 53 kb in length.
 l Bacteriophage based Cloning Vectors
• Double stranded DNA molecule
• 12-base-pair sticky ends (cos sites) at 5'
• It is used as a cloning vector, accommodating fragments of DNA up
to 15 kilobase pairs long. For larger pieces, the cosmid or YAC’s are
used.
• Will accept foreign DNA and still complete their life cycle.
• Distinguish cells that have foreign and non foreign DNA.
• Should replicate in host (E.coli)
• Gene of interest can be identified and grown in large amounts.
• Non essential genes can be removed and replaced by foreign gene.
 Cont.
• Should carry one or more selectable markers that
identify the parent and recombinant vectors
• Should have restriction sites in non-essential regions
of DNA into which foreign DNA can be inserted
• Should be easy to make and maintain library
http://www.gmu.edu/departments/biology/385-Ch04c-rDNA/
 Lambda vectors
• 1) Insertion vectors
cos cos

EcoRI

• 2) Replacement vectors

cos cos

20Kb
EcoRI EcoRI

• http://www.sh.lsuhsc.edu/gradcore/IDSP117/16
 Insertion Vector
-Can not fit as much new foreign DNA in
-Central 1/3 is the “stuffer” fragment.
-Segments of the lambda DNA are removed and a stuffer fragment
is put in, this keeps the vector at a correct size

•
steps in cloning with l :
– Isolate vector DNA and gene of
interest
– Cut both with restriction
enzyme(EcoRI)
– Connect two fragments of foreign
DNA with DNA ligase. (recombinant
DNA)
– Package DNA by adding cell
extracts containing head and tail
proteins
– Transfer recombinant molecules
into host cell
– Grow/select clones: check
recombinant phage for the
presence of desired foreign DNA
sequence by observing its genetic
properties.
Brock Biology of Microorganisms, 9th Edition (2000)
Prentice Hall, Madigan, Martinko, Parker
 Limitations
• Size of DNA to be introduced into the host cell

• Problem: when making genomic library of large size

(plants and mammals) only a portion of those
fragments will be represented. If gene of interest is
located in a large fragment, then you won’t be able
to isolate that gene from the library.
• Solution :use a vector that can accept large
fragments of DNA
Cosmids
• Cross between phage and plasmid
• Circular ds DNA
• Contain cos sites and can be packaged into
phage heads
• Carry more DNA than plasmid and can be
maintained and manipulated as plasmids

Cloning using cosmids

• Clone into vector as would with plasmid.

• Introduce DNA to cell as you would with

phage.

• Propagate as you would a plasmid.

• Cosmids have about 5kb DNA and therefore

can get ~ 33-48kb foreign DNA into a phage
head.
cosmids
Cosmids are plasmid vectors that contain one or two “cos sites”.

(The cos site and the length between 2 cos sites is the only requirement for DNA to be
packaged into a phage particle).

How do we clone DNA into a cosmid vector ?

1. We use a polylinker.
2. We package this DNA into phage particles like phage DNA.
3. We propagate the cosmid as a plasmid (a plasmid with a selection marker. (Amp –resistance)
4. We purify the cosmids as if they were plasmids.

As the head of a phage can accept between 40 and 55 kb of DNA and as most cosmids are
about 5 kb in length, between 33 and 48 kb of DNA can cloned in these vectors.
This cosmid is cut at a BglII site next to the cos site.

Donor genomic DNA is cut using Sau3A, which gives sticky ends compatible with BglII.
Fragment are treated with alkaline phosphatase.

A tandem array of donor and vector DNA results from mixing.

DNA is packaged as for the phage.
http://www.fmv.ulg.ac.be/genmol/MODGEN/ChapterIII/Fig14_9.htm
from:
http://www.bi.umist.ac.uk/users/mjfssps/2GEN/Cosmid2.htm
 Vector types:
1. Plasmids- small circular DNA molecules which
can replicate their DNA independently of their
bacterial chromosome. They are found
naturally in bacteria and replicate inside the
bacterial cell. They can insert pieces up to Vector Insert size (kb)
10kb(kilobases) or 100 to 10,000 base pairs.
Examples: pBR322 and pUC18
2. Bacteriophage l- They are double stranded Plasmid <10 kb
linear DNA vector. They replicate in E. Coli
in the lytic or lysogenic mode. They can insert
fragments up to 15kb. Examples are lgt10 Bacteriophage 9-15 kb
and lZAP l
3. Cosmids- are hybrid vectors of l phage and Cosmids 33-50 kb
plasmids. They can replicate their DNA in the
cell with a plasmid and be packaged like a
phage. They can insert up to 50kb. YACS 100-1000 kb
4. Yeast artificial chromosomes (YAC)- primarily
used in genome sequencing projects. They
host large inserts up to 1000kb.
M13 , a single stranded filamentous phage
• Phage DNA is packaged in the core of a helical particle
• The length of particle is dependant on length of DNA
• In all M13 preps the following occur
• polyphage- more than one genome length
• minphage- 0.2-0.5 genome length
• maxiphage- genetically defective but more than one genome length
• Molecular biologists use this to create cloning vectors
• Can insert long stretches of DNA into non essential regions
• No sharp cut off in length that can be packaged
• Some decrease in efficiency of packaging with increasing length
• 10% longer not affected, 50% longer replicate more slowly