Gene cloning and plasmid vectors

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1. GENE CLONING AND
PLASMID VECTORS
LECTURE-3
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2. 2
Thecloningofagenebythemethoddescribedby
StanleyCohenandcoworkers(1973)requirestheuse
ofasuitablegenecloningvector.
Agenecloningvectorshouldhavecapabilityof
autonomousreplication,smallsize,originof
replication,presenceofaselectablemarkergene(s)
andpresenceofuniquerestrictionenzymesite(s).
InthefirstgenecloningexperimentplasmidpSC101
wasusedforcloningofE.colikanamycinresistant
gene.Naturalplasmidsarenotsuitablevectorsfor
genecloning.

3. 3
•SeveralplasmidvectorslikeColE1,pBR322,pUCseriesand
pGEM
R
serieshavebeendevelopedtosuitthevariousrequirements.
•ItisnoteasytocloneaDNAfragmentlargerthan10kbusinga
plasmidvectorasthetransformationfrequencydecreases
considerablywiththeincreasedsizeoftherecombinantplasmid.To
overcomethisdifficultythreeresearchgroupsindependentlyreported
theconstructionofbacteriophagelambdacloningvectorsin1974.
•Subsequently,tomeetthevariousrequirementsofcloning,the
followingvectorswereconstructedintherespectiveyearsmentioned
inthebrackets:BacteriophageM13derivatives(1977),cosmids
(1978),yeastartificialchromosomes(1987),bacteriophageP1
(1990),bacterialartificialchromosomes(1992),P1artificial
chromosomes(1994),humanartificialchromosomes(1997)and
maizeminichromosomes(2007).

4. Properties and construction of a vector DNA molecule
1.Capabilityofautonomousreplication
•Bacterialandviralgenomescontainonlyoneoriginof
replicationwhileeukaryotescontainmultipleorigins
2.Smallsize
•Insmallmoleculesthechancesofoccurrenceofuniquesites
forrestrictionenzymesincreases
•Efficiencyofgenetransferishighwithsmallvector
molecules
3.Presenceofselectablemarkergene(s)
•Foreasydetectionofrecombinants
•E.g.:antibioticresistantgenes,laczorresistanttotoxins,etc
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5. 4. Presence of unique restriction enzyme sites or
multiple cloning sites for inserting the target DNA
•Positionoftheserestrictionsitesshouldbesuchthatthe
insertionofasegmentofDNAinanyoftheserestrictionsites
bringaboutaphenotypicchangeinthecharacteristicofavector
moleculee.g.:lossofgeneexpressionorlossofresistancetoan
antibiotic
5.Ease of purification
6. No effect on the replicativeability of vector due to
insertion of target DNA
7. Ease of reintroduction into host cell with high
efficiency
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6. 8. Biological containment
•Vectors should be biologically contained with no possibility
of gene escape
•This can be achieved by non-conjugative and non-mobilized
plasmid vectors
9.Presence of promoters and ribosome binding
sites
10. Presence of two different origins of replication
or broad host range origin of replication
E.g.: shuttle vectors that contain two different origins of
replication
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7. •1973 Plasmid
•1974 Bacteriophagelambda
•1977 Plasmid pBR322
BacteriophageM13(M13mp1)
•1978 Cosmid
•1987 Yeast artificial chromosome-YAC
•1990 BacteriophageP1
•1992 Bacterial artificial chromosome-
BAC
•1994 P1artificial chromosome-PAC
•1997 Human artificial chromosome-HAC
•2007 Maize mini-chromosomes
Cloning Vectors Used in Genetic Engineering
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8. 1.Generation of the DNA fragment
2. Construction of the recombinant DNA
molecule by joining of DNA fragment with a
vector
3. Introduction of the recombinant vector into
host cell, multiplication of recombinant DNA
molecule along with host cell
Steps involved in gene cloning
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9. Plasmid characteristics
1.Capabilityofautonomousreplication
2.Smallsize
3.Presenceofselectablemarkergene(s)
4.Presenceofuniquerestrictionenzymesites
5.Non-conjugativeandnon-mobilizable
6.Repliconunderrelaxedcontrol
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10. Natural plasmid vectors for E. coli
pSC101
•First used for in vitro cloning of eukaryotic DNA
•9kbp in size
•Low copy number(1-2copies)
•Has advantage of a single Eco RI site at which DNA can be
inserted
•Has selectable marker for tetracycline resistance
•Derived from the conjugative plasmid R6-5
Disadvantages are
•Large size
•Stringent replicativecontrol
•Low copy number
•Low insert capability
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11. pSC101
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12. pSF2124( RSF2124)
•Produced by transfer of the ampicillinresistance gene
•Has ability for colicinbiosynthesis
•Has high copy number
•Has single sites for Bam H1and EcoR1
•Not currently used as vector as it does not provide easy
selection by insertionalinactivation
•Mobilizableplasmid
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13. Col E1Plasmid
•Small, circular colicingenicplasmid
•Codes for 57kDa protein toxin
•Size is 6,466bp
•Has ceagene for colicinproduction
•immfor immunity against colicin
•killfor killer or lysisprotein
•mob for mobilization
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14. Col E1Plasmid
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15. Artificially constructed plasmid vectors
for E. coli
pBR322
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16. •pBR322isaplasmidandwasthefirstwidely-usedE.
colicloningvector
•Createdin1977,itwasnamedafteritsMexican
creators,pstandingforplasmid,andBRforBolivar
andRodriguez
•pBR322is4361basepairsinlengthandcontainsa
repliconregion(sourceplasmidpMB1)
•Theamp
R
gene,encodingtheampicillin
resistanceprotein(sourceplasmidRSF2124)
•Thetet
R
gene,encodingthetetracyclineresistance
protein(sourceplasmidpSC101)
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17. •Hasuniquerestrictionsitesformorethan40
restrictionenzymes
•11ofthese40sitesliewithinthetet
R
gene
•Has2sitesforrestrictionenzymes
HindIIIandClaIwithinthepromoterofthetet
R
gene
•Sixkeyrestrictionsitesinsidetheamp
R
gene
•Theoriginofreplicationororisiteinthisplasmid
ispMB1(acloserelativeofColE1)
•TheoriencodestwoRNAs(RNAIandRNAII)and
oneprotein(calledRomorRop)
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18. DERIVATIVES OF pBR322
pBR324
pBR325
pBR327
pBR328
pBR329
pAT153
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19. pUCseries
•pUC19is one of a series of
plasmidcloning vectors
created by Messing and co-
workers in the University of
California.
.
•The p in its name stands for
plasmid and UC represents
the University in which it was
created. It is a circular double
stranded DNA and has 2686
base pairs
•Series include pUC18 pUC
19,pUC12and pUC13, etc
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20. •Ithasoneamp
R
gene(ampicillinresistancegene)
•HasanN-terminalfragmentofβ-galactosidase(lacZ)geneofE.
coli
•Themultiplecloningsite(MCS)regionissplitintothelacZgene
(codons6–7oflacZarereplacedbyMCS),where
variousrestrictionsitesformanyrestrictionendonucleasesare
present
•Theorisiteorreplicon,repisderivedfrompMB1vector
•pUCvectorissmallbuthasahighcopynumber.Thehighcopy
numberofpUCplasmidsisaresultofthelackoftheropgene
andasinglepointmutationinrepofpMB1
•ThelacZgenecodesforβ-galactosidase
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21. pGEM
R
series
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22. •The pGEM-3Z Vector is intended for use as a standard cloning
vector, as well as for the highly efficient synthesis of RNA in
vitro
•The vector carries thelacZα-peptide and the multiple cloning
region arrangement from pUC18 allowing selection of
recombinants by blue/white screening
•In addition, the vector contains both the SP6 and T7RNA
polymerase promoters flanking the multiple cloning region
•The pGEM-3Z and pGEM-4Z Vectors are essentially identical
except for the orientation of the SP6 and T7promoters
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23. 23

24. •The pET-28a-c(+) vectors are most powerful
for cloning and expression of recombinant
proteins in E. coli.
•The pET-28a-c(+) vectors carry an N-terminal
His•Tag®/thrombin/T7•Tag® configuration
plus an optional C-terminal His•Tagsequence.
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