GENETIC ENGINEERING

ASSIGNMENT 1

Name: Saumya S

Reg. No.: 15BBT0054

Topic: Cloning Vectors

1. pUC19:
pUC19 is one of the commonly used plasmid vector for cloning,
which is usually carried out in E. Coli. It is a double stranded circular
DNA with 2686 base pairs. The copy number is high with multiple
cloning sites.
The vector is isolated from E. Coli ER2272. It is used in the
recombinant technology where the foreign DNA or the gene of
interest is introduced for further amplification of the gene.
Also it can easily be distinguished from the non-recombinants to the
recombinants based on the difference in colour of the colonies that
are grown in the media.
Components:
The isolated plasmid vector has an ampicillin resistance gene, along
with beta-galactosidase gene (lacZ). It has a multiple cloning site
polylinker that carries different restriction endonuclease acting
sites, which are called the restriction sites.
In addition to beta-galactosidase, the vector also codes for beta-
lactamase, which can degrade ampicillin and reduce the toxicity to
the host.
The origin of replication site is derived from plasmid pMB1.
The vector makes high copy number even though it is small,
specifically due to the absence of rop gene.
Function:
Transformation is the process by which the plasmid is introduced
into the bacterial cell and further amplifies to give a number of
copies.
Due to the presence of Multiple Restriction Site, a gene of interest
can be introduced into this region by treating the plasmid with the
restriction endonuclease.
The cells are then grown on the media containing ampicillin. Also
only the cells containing ampicillin resistant gene will survive.
One of the main features of this vector is that, the non-recombinant
can be distinguished from the recombinant with color. The
recombinants are found to be white with non-recombinants to be
blue.
 Fig 1: pUC19 cloning vector map

2. pJQ200:
pJQ200 is used as a plasmid vector for cloning and to carry out the
process of integration, where E. Coli serves as the host but usually
capable of a broad host range. The plasmid is a double stranded
DNA with 4900 base pairs. Also the copy number is high.
Components:
The vector is based on the p15A origin of replication.
The vector incorporates sacB from Bacillus subtilis, which is
inducible in sucrose and lethal when expressed in Gram-negative
bacteria.
It also has an antibiotic resistance marker which is gentamycin.
Function:
Suicide vector permits gene replacement and mobilization into a
wide range of gram negative bacteria. It allows positive selection for
integration.
It replicates only in enterobacteria and so functions as a suicide
vector in other Gram negative hosts.
When induced by media containing 5% sucrose, sacB is lethal in a
wide range of Gram negative bacteria, and thus permits selection
for loss of the vector.
Gentamycin is the resistance gene present in the plasmid.
Suicide genes are often utilized in biotechnology to assist in
molecular cloning. Vectors incorporate suicide genes for an
organism (such as E. Coli). The cloning project focuses on replacing
the suicide gene by the gene of interest.
Selection of vectors carrying the desired fragment is improved since
vectors retaining the suicide gene result in cell death.
 Fig 2: pJQ200 cloning vector map

3. YEp24:
Yeast Episomal plasmid 24 is a shuttle vector with URA3 marker,
used for gene over-expression in Saccharomyces cerevisiae, but
also capable of replication in E. Coli.
In S. Cerevisiae, YEp24replicates at high copy number from the
replication determinant of the yeast 2 circle plasmid.
Components:
While in E. Coli, the plasmid replicates from the pMB1 origin of
replication from pBR322.
It carries the ApR marker for selection with ampicillin. It has a
tetracycline resistance marker (TcR) from pBR322 but is separated
from its promoter by the URA3 sequence, so its expression is
variable with respect to different constructions.
Function:
Episomal yeast vectors are present as extra-chromosomal DNA and
are unstable. This can over-come by integration of vector into the
host chromosome.
In yeast, integration occurs by homologous recombination. The
yeast integration plasmids contain target sequence for integration
into chromosomal DNA, a selection marker and bacterial origin of
replication.
Before vector delivery to the yeast, it is digested with the unique
restriction endonuclease to produce linear DNA to increase the
transformation efficiency and integration.
In most of the cases integration is done in such a way that yeast
chromosomal DNA remained intact and integration may not affect
yeast growth.
But in an alternate approach, a portion of yeast chromosomal DNA
is replaced with the vector DNA through homologous recombination.
These vectors are known as transplantment integration vector 'and
they have foreign DNA, selection marker and homologous DNA to
the region of chromosomal DNA to be replaced.
 Fig 3: YEp24 cloning vector map
4. pRS414:
pRS414 is an yeast centromeric shuttle vector with 4788 base pairs,
carrying the capacity to propagate in two different host species.
Hence, DNA inserted into a shuttle vector can be tested or
manipulated in two different cell types. The plasmid also gives a
high copy number when incorporated into the host.
Components:
pRS414 is a plasmid used for creating gene fusions with lacZ. It has
a TRP1 marker with Multiple Cloning Sites derived from
pBLUESCRIPT II.
It has ColE 1 as its origin if replication. The vector carries promoter-
less lacZYA operon.
lacZ also lacks its RBS and ATG codon, so it can be used to create
gene fusions with lacZ.
The vector has a number of unique restriction sites such as EcoRI,
SmaI and BamHI, which can be identified and cleaved by the
specific restriction endonuclease for constructing a recombinant
gene of interest.
It has an autonomously replicating sequence ARS (Histone H4)
derived from S. cerevisiae and a centromere (CEN6) from the same
source.
It has an f1 origin derived from f1 and an E. coli origin from pMB1.
The vector also has an ampicillin marker derived from Tn3.
Function:
The pRS4xx series of plasmids are shuttle vectors used for gene
cloning in Saccharomyces cerevisiae, but are also capable of
replication in E. coli.
While in E. coli, they replicate from the pMB1 origin of replication
from pBR322 although the rop gene is missing.
They carry the ApR marker for selection with ampicillin and the f1
bacteriophage origin of replication for single-strand DNA production.
They carry a TRP1 selection in Saccharomyces cerevisiae, a beta-
galactosidase reporter system for visual detection of recombinants
and promoters for transcription invitro.
 Fig 4: pRS414 cloning vector map
5. pBIN19:
pBIN19 is a cloning vector plasmid with double stranded DNA of
11,777 base pairs.
It functions as a cloning vector as well as a reporter vector. Many
other cloning vectors have been derived like pBI101, pBI101.2, etc,.
It makes a high copy number when incorporated into the host
species like Agrobacterium tumefaciens, E. Coli and higher plants.
Components:
Its a vector molecule for the efficient transformation of higher
plants that has been constructed with several features that make it
efficient to use.
The binary vector like pBI19 contains a few restriction endonuclease
cloning sites in the lacZ , complementation fragment, permitting
blue/white screening for the presence of the trans gene insertion.
The origin of replication may function in both Agrobacterium and in
E. Coli. The RK2 ori present in this vector plasmid makes around 10
copies per cell.
It has a transferable (T-) DNA region, virulence region, host
specificity region and ori region.
Function:
The vector molecule has the T DNA region which carries genes
encoding plant hormones and opines.
The vir region contains the virulence genes which help in transfer of
T DNA. Crown gall disease results from transformation of the plant
genome with this part of the plasmid in a process analogue to
bacterial conjugation.
Other regions contain conjugation genes and genes concerned with
opine utilization.
The plasmid utilizes the trans acting functions of the vir region of a
co-resident Ti plasmid in Agrobacterium tumefaciens to transfer
sequences bordered by left and right T-DNA border sequences into
the nuclear genome of plants.
 The T-region contains a dominant selectable marker gene that
confers high levels of resistance to kanamycin, and a lac alpha-
complementing region from M13mpl9 that contains several unique
restriction sites for the positive selection of inserted DNA.

Fig 5: pBIN19 cloning vector map

6. pGreen:
pGreen is a cloning binary vector with 3228 base pairs. Binary Ti
vectors are the plasmid vectors of choice in Agrobacterium-
mediated plant transformation protocols.
The pGreen series of binary Ti vectors are configured for ease-of-use
and to meet the demands of a wide range of transformation
procedures for many plant species.
Components:
The pGreen plasmid contains a mutant version of GFP linked to
another gene called beta-galactosidase.
This combination of genes was chosen because the protein
produced from this combination turns bacteria yellow-green, even in
normal light. If you expose the colonies to a UV light, they also
fluoresce.
The plasmid also contains the antibiotic resistance gene to allow
growth in the presence of ampicillin.
It also has a pSa replication origin of pSoup.
Function:
The plasmid system allows any arrangement of selectable marker
and reporter gene at the right and left T-DNA borders without
compromising the choice of restriction sites for cloning. Since the
pGreen cloning sites are based on the well-known pBluescript
general vector plasmids, its size and copy number in Escherichia
coli offers increased efficiencies in routine in vitro recombination
procedures.
The removal of RepA and Mob functions has enabled the size of
pGreen to be kept to a minimum.
Versions of pGreen have been used to transform several plant
species with the same efficiencies as other binary Ti vectors.
 pSoup is a helper plasmid that provides the replicase function for
the pSa replication origin of pGreen.
pSoup is tetracyclin resistant and a complementary incompatibility
group such that it can co-exist with pGreen in the Agrobacterium
cell.
pSoup: the original help plasmid for pGreen. pGreen will not
replicate in Agrobacterium if it is not present.

Fig 6: pGreen cloning vector map

7. pCaSpeR-hs:
pCaSpeR-hs is one of the most commonly used Drosophila
transformation vectors. They have a great utility in the
contemporary world but multiple cloning sequences have a limited
number of unique restriction sites.
Also the large size of the transgenesis vectors requires sequence
manipulations such as site-directed mutagenesis or deletion
dropouts to be done in small plasmid vectors and the modified DNA
to be moved to the transgenesis vectors.
The lack of matching shuttle vectors further constrains the usable
cloning sites and can make moving large genomic fragments
between a cloning vector and a transgenesis vector problematic.
Components:
The plasmid is a P element vector with a white selectable marker for
expressing open reading frames under heat shock control.
Two segments of the hsp70 gene were inserted between the EcoRI
and PstI sites of pCaSpeR.
The hsp70 promotor, extending from the XbaI site to the XmnI site,
was inserted upstream from the hsp70 3' region.
This 3' region was excised from pHT4 as a 525 bp EcoRI - PstI
fragment, extending from the SalI site at the stop codon to approx.
350 bp downstream from the poly A addition site.
Function:
pCaSpeR is a type of insect expression vector with ampicillin as its
resistance marker derived from bacteria.
 The plasmid vector pCaSpeR-hs expresses transposase. We use
small plasmid vectors pBluescript for initial cloning of PCR products
and manipulation of gene segments.
A polylinker containing 7 unique sites can be used to insert an open
reading frame for expression.
The vector contains divergently transcribed white, CAT and lacZ
reporter genes.

Fig 7: pCaSpeR cloning vector map

8. pUChsneo:
pUChsneo is a double stranded plasmid with 5073 base pairs. It
mostly functions as a transposomal element.
The plasmid is usually incorporated into D. Melanogaster larvae and
dorsophila. This particular vector is derived from pUC8 and hsp70-
neo.
Components:
The plasmid has the entire pUC8 plasmid sequence as well as the
hsp70 promoter.
It has the neo gene contained between the P-inverted repeats. The
pUChsneo vector uses the bacterial neomycin resistance gene to
render the larvae resistant to G418 introduced.
The promoter which is shown to function in nearly all embryonic and
larvae tissues, ensures that the neo gene will be expressed in all
larval cells, likely to come in contact with the drugs.
In contrast to other P vectors, the design of pUChsneo places the
bacterial plasmid sequences within the P terminal repeats.
The entire pUC8 sequence, including the polylinker, ampicillin
resistance gene, and beta-galactosidase sequences, is included with
the neo gene between the P terminal repeats derived from the p6.1
clone.
Function:
The transposon that integrates in the Drosophila genome includes
the pUC8 sequence, allowing the recovery of the inserted DNA by
the plasmid rescue method.
 Genomic DNA of the transformed fly is digested with a restriction
enzyme that does not cut the transposon and diluted and ligated
under conditions that allow circularization of the fragments.
The ligation mixture is then used directly to transform E. Coli hosts.
This technique is useful in isolating the sequences flanking the
insertion site and in analyzing the inserted transposon for
rearrangements or mutations.
The lacZ fragment and the polylinker of the pUC8 vector facilitate
the cloning and identification of insertions.
But the presence of HindIII sites in the P sequences and of a PstI site
in the hsp70 promoter, these two enzymes cannot be used for
cloning even though their sites are present in the polylinker.

Fig 8: pUChsneo cloning vector map

9. pPA1:
pPA1 is vector constructed for it is to be incorporated into the host
mammalian cells. It has the ORF of size 870 base pairs.
cDNA description: Full length Clone DNA of Homo sapiens
pyrophosphatase.
It has a vector which is pGEM-T vector and a plasmid which is pGEM-
PPA1.
It consists of sequencing primers which are SP6 and T7 or M13-47.
Components:
It has the ampicillin antibiotic in E. Coli. The pGEM-T is 3kb in length,
and contains the amplicin resistance gene, conferring selection of
the plasmid in E. coli, and the ori site which is the bacterial origin of
replication.
The plasmid has multiple cloning sites as shown below. The coding
sequence was inserted by TA cloning.
Many E. coli strains are suitable for the propagation of this vector
including JM109, DH5 and TOP10.
The coding sequence can be easily obtained by digesting the vector
with proper restriction enzyme(s). The coding sequence can also be
amplified by PCR with M13 primers, or primer pair SP6 and T7.
 Fig 9: Mammalian cloning vector map

10.HACs:
A human artificial chromosome (HAC) is a microchromosome that
can act as a new chromosome in a population of human cells.
That is, instead of 46 chromosomes, the cell could have 47 with the
47th being very small, roughly 6-10 megabases (Mb) in size instead
of 50-250 Mb for natural chromosomes, and able to carry new genes
introduced by human researchers.
Ideally, researchers could integrate different genes that perform a
variety of functions, including disease defense.
The genetic material introduced by these vectors not only leads to
different expression levels, but the inserts also disrupt the original
genome.
HACs differ in this regard, as they are entirely separate
chromosomes. This separation from existing genetic material
assumes that no insertional mutants would arise.
This stability and accuracy makes HACs preferable to other methods
such as viral vectors, YACs and BACs.
 HACs allow for delivery of more DNA (including promoters and copy-
number variation) than is possible with viral vectors.
Components:
Create a small minichromosome by altering a natural human
chromosome. This is accomplished by truncating the natural
chromosome, followed by the introduction of unique genetic
material via the Cre-Lox system of recombination.
Function:
Researchers formed a human artificial chromosome by truncating
chromosome 14. Genetic material was then introduced as
mentioned above, using the Cre-Lox recombination system.
This particular study focused on changes in expression levels by
leaving portions of the existing genomic DNA.
By leaving existing telomeric and sub-telomeric sequences, they
were able to amplify expression levels of genes coding for
erythropoietin production over 1000-fold.
This work also has large gene therapy implications, as
erythropoietin controls red blood cell formation.
HACs have been used to create transgenic animals for use as
animal models of human disease and for production of therapeutic
products.

Fig 10: Human Artificial Chromosome