A genetically engineered DNA molecule from bacteria , phage or yeast to carry foreign DNA for the purpose of cloning and expression of the inserted DNA of interest in RDT

1. The key to success is to focus on goal and not an

2. Application
Vector
Expression
Cloning

3.  Gene cloning is molecular technique in which -
1 A fragment of DNA, containing the gene to be cloned, is inserted into a
circular DNA molecule called a vector, to produce a recombinant DNA
molecule.
2 The vector transports the gene into a host cell, which is usually a
bacterium, but it may be other living cell too.
3 Within the host cell the vector multiplies, producing numerous identical
copies of the gene, not only of itself but also of the gene that it carries.
4 When the host cell divides, copies of the recombinant DNA molecule are
passed to the progeny and further vector replication takes place.
5. After a large number of cell divisions, a colony, or clone, of identical host
cells is produced. Each cell in the clone contains one or more copies of
the recombinant DNA molecule; the gene carried by the recombinant
molecule is now said to be cloned.

5. History
• 1971-1973: New technology like recombinant
DNA technology or genetic engineering or the
process of Gene cloning developed. This
technology helped in studying the regulation of
genes, aberrations in the genes and then used
genes for production of desired proteins. •
In 1985, Kary Mullis invented the Polymerase
Chain reaction
The first cloning experiment done by Boyer and
Cohen

6. A vector is
Autonomously replicative , posses independent replicating
property and is host specific
Posses dominant selective marker for screening it may be
antibiotic resistant or any reporter gene which will indicate
transformation
Bears cloning sites needed for insertion of foreign DNA
Needs to be relatively small, ideally less than 10 kb in size, as large
molecules tend to break down during purification, more difficult
to manipulate and critical for host sustainability
, a useful cloning vector needs to be present in the cell in multiple
copies so that large quantities of the recombinant DNA molecule
can be obtained

7. Plasmid Vector
• • Plasmid vectors are double-stranded, extra-
chromosomal DNA molecules, circular, self-
replicating.
• Range -Smallest Plasmid 1.0 kb – large over
250 kb. So only a few are useful for cloning
purposes.

8. Features of Plasmids for cloning
Less than 10 kb (1-5 Kb) is desirable for a plasmid
vector
The size and copy number of a plasmid are particularly
important as far as cloning is concerned.
The copy number refers to the number of molecules of
an individual plasmid that are normally found in a
single bacterial cell.
 Stringent plasmids- larger ones & low copy number
just one or two per cell;
relaxed plasmids- present in multiple copies of 50 or
more per cell.

9. Bacterial Vector – Plasmid
It exists in three different
forms.
 A. Covalently closed circle
(CCC)-circular ds DNA is
intact (replicative)
Open Circle (OC) one strand
has nick (non replicative)
Super coiled (S). During
isolation CCC looses few
number of turns and
acquire supercoiled
configuration
All these three forms can be converted by using
the enzymes as shown in the picture

10. Types of Plasmid
The five major types of plasmid are as follows:
• Fertility or F plasmids carry only tra genes and have no characteristic beyond the ability to
promote conjugal transfer of plasmids. A well-known example is the F plasmid of E. coli.
• Resistance or R plasmids carry genes conferring on the host bacterium resistance to one or
more antibacterial agents, such as chloramphenicol, ampicillin, and mercury. R plasmids are
very important in clinical microbiology
• Col plasmids code for colicins, proteins that kill other bacteria. An example is ColE1 of E. coli.
• Degradative plasmids allow the host bacterium to metabolize unusual molecules such as
toluene and salicylic acid, an example being TOL of Pseudomonas putida.
• Virulence plasmids confer pathogenicity on the host bacterium; these include the
• Ti plasmids of Agrobacterium tumefaciens, which induce crown gall disease in the plants.
• Relaxed plasmids –many copies per cell (50copies /cell)
• Stringent Plasmid – single copy per cell./low number 91-10 copies/cell)
•

11. • To act as vector the natural plasmid is
modified like
• Insertion of polylinker site of about 30 bp
synthetic sequence by deletion of existing
sites
• Insertion of Ab resistant gene for screening
the transformation
• Some expressible gene within the cloning sites
used for transformation

12. Example pBR322
• It is genetically engineered bacteria to full fill
the requirement as vector in 1980’s.
• p means plasmid, where as BR means
developer Boliver & Rodriguez ,322
distinguishes this plasmid from others
developed in the same lab.
P Denotes plasmid
BR Name of developer
Bolavir & Rodriguez
322 Specific no. for
identification

13. ORIGIN
t was constructed by using three different naturally occurring
plasmids
RSF2124 from which amp r gene was taken
pSC101- tetr gene was taken
pMBI - origin of replication was taken.

14. Genetic Map
• Molecular size -4361 base pair and is of
relaxed type (20-30 in number/ cell).
• Marker -It has tetr and ampr as selective
marker and
• Multiple & unique cleavage sites of . more
than 20 types within the
• a. Bam HI,heI,NruI,SalI, SphI,XmaI in Tetr
region
• b. PstI, PruI,ScaIin the Ampr region

15. CloninG process
• Purified circular pBR322 is cut
within Ab Resistant gene by RE
and treated with Alakine
phosphatase(AP)
• Combined with
Genomic DNA fragment
obtained by same RE
enzyme and ligated by
T4 DNA ligase and ATP

16. Advantage & Disadvantage
 Advantage
 It is an ideal cloniong vector due to following advantage
 Plasmid is smaller than natural plasmid so it is more resistant to damage
& shearing and increase the efficiency of uptake during transformation
 Since this small foreign DNA up to 6Kb can be inserted
 Marker gene allow selection of transformant
 With the use of chloromphenicol no. of plasmid can be increased up to
1000-2000 since it has relaxed origin of replication
 It can act as raw materials for construction of several other useful vector
 Presence of single copy of restriction sites for many different enzymes
allow easy insertion.
 Disadvantage
 Besides many advantage it has disadvantage
 1, Instability-in continuous culture , it get lost.so can not consider for
commercial purpose.
 Limited res sites and many lies outside the cloning sites
 Limited Ab R marker gene
 Limites host for cloning i.e can replicate in the G –ve bacteria.
 Time consuming procedure as it requires many steps in screening the
transformants
 Carrying capacity is of 6-10 Kb

17. • To overcome pUC19 was developed with following features
• Size -2686 bp
• Marker gene - ampr
• regulatory segment of beta glycosidase ( lac Z) of the lac operon,
• lac I – repressor gene for regulation of expression of beta glycosidase
• a short sequence with multiple cloning sites (EcoRI, SacI,KpnI,XmaI, SmaI,
BamHI,XbaI, SalI, PstI, HindIII etc )used for the insertion of cloned DNA
Cloning with pBR322 has many limitation . for instance
Although insertional inactivation of an antibiotic resistance gene provides
an effective means of recombinant identification, the method is made
inconvenient by the need to carry out two screenings, one with the
antibiotic that selects for transformants, followed by the second screen,
after replica-plating, with the antibiotic that distinguishes recombinants

18. pUC19
2686 bp lon

19. SCREENING OF
TRANSFORMED COLONY

20. • Transformation is genetic recombination process
when one bacterial stain is converted into another
bacterial strain due to integration of another gene
transferred . It was first observed in 1928 by
Frederick Griffith, when he co -cultured the R
(rough/virulent strain ) starin with heat killed S
train of pneumococcus bacteria

21. Griffith Experiments of Transformation
Rat is killed due to transfer of s type DNA in to
live R stain resulting transformation of R stain
into S train

22. RDT[Recombinant DNA technology ]
• A technology to produce DNA with integrated heterologous
DNA from other source and is commonly generated for the
cloning and expression of integrated cloned DNA/ gene
purposes in RDT

23. Screening of Transformed Bacterial
colony or Transformants
• Transformed colony is called transformants
• During Cloning A genomic DNA fragment of interest is
inserted within the dominant Marker gene used for
screening
• For example in pBR322 Gene is integrated in the TET r
region which is present within the Multiple cloning
sites
• When inserted Tet R is transformed in to Tet S as the
reading frame is disturbed.
• When such bacteria is cultured in culture media
containing tetracycline . The transformed colony is not
able to grow while non transformed used to grow

24. Transformation & Screening
Transformed with
CaCl2 and high
temp.

25. Screening of pBR 322 transformants
Since both non transormants and transformant
are Amp resistant , the tend to grow in culture
media containing only Ampiciline
Non Transformed -Amp r and Tet r
Culture of Bacteria after integration (transformed) in
media containing both tetracycline & ampiciline
Transformed –
Amp r & Tet s
Non
transformed
-Amp r & tet r
Colonies grown only in amp
medium

26. Screening of Transformants in pUC19
It utilized blue white colony screening methods .
 The cloned DNA inserted into the MCS will inactivate the
expression of Lac Z gene .
IPTZ (Isopropyl ß D thiogalactopyranoside is an inducer of lac
operon which binds with Lac I product (repressor) and prevent
to bind at promoter operator region allow expression of ß
galactosidase .
The product of Lac Z combines with the protein encoded by
chromosomal DNA to form active hybrid ß galactosidase.
If X gal ( 5 bromo 4 chloro 3 indolyl-ß galactopyranoside) is
present in the medium , it will be hydrolysd by the enzyme and
blue product bearing colonies will formed .
If Plasmid contains foreign DNA the Lac Z is inactivated and so X
gal is not hydrolysd since there is no ß galacotosidase
expression .
Thus transformant produces white and notransformants
produces blue colonies.

27. Lac Operon

28. In presence of IPTZ (Isopropyl ß D thio
galactopyranoside) repressor , product
of Lac I is inactivate r and induce the
expression of Enzyme
Galactocidase formed
In presence of IPTZ (Isopropyl ß D thio galacto
pyranoside) repressor , product of Lac I is
inactivated but lac Z can not express since it is
hampered .
Galactosidase not formed
Such colonies can be screened by using X gal ( 5 bromo 4 chloro 3
indolyl-ß galactopyranoside– a substrate for galactosidase

29. Screening of Transformant s
No transformation
transformation

30. Transformants
No Lac Z expression in
presence of Inducer IPTZ
X gal remain as such
Non Transformants
Lac Z expression in presence
of X gal and IPTZ producing
Blue colonies

31. Screening with suicidal gene
suicidal gene within the correct reading frame of Lac Z gene is inserted
which lie in the cloning sites .
The transformants will grow
non transformants will be killed as it produces a protein (enzyme) that
prevent rejoining of double strand breaks in the chromosomal DNA and
accumulation of double strands break will kill the bacteria

32.  Medium containing no IPTG and intact plasmid – no killing since no gene product to
promote growth , Lac operon off
 Medium with IPTG and plasmid intact –killed as suicidal gene expressed
 Medium with IPTZ and plasmid with insert –not killed since ß galactosidase and suicidal
genes are affected by the insertion of cloned DNA ,so this colony belongs to transformants

33. Conclusion
Plasmid is universal vector which are enginered as per require
in order to clone the DNA, like reduction in the length(about 3
kb ) as compare to natural ,bearing resitant gene and multiple
cloning sequence
Stanford and UCSF researchers fused a segment of DNA
containing a gene from the African clawed frog Xenopus with
DNA from the bacterium E. coli and placed the resulting DNA
back into an E. coli cell. This was the first time an animal gene
was cloned - that is, isolated and propagated like this.