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Plasmid as a Genetic Material

Dr. Aris T Wahyudi

Bogor Agricultural University2009

Plasmid

Chromosome

Escherichia coli

Cell

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What is a Plasmid?

•DNA extra chromosome, small circular

•Autonomous replication

•Variable number of copies and size

•Non essential for the basic operation of the cell

•Contain antibiotic resistance gene or the others

•Plasmids that confer conjugation (F plasmids)

•As cloning vectors (multicopy)

Figure . Physical map of the complete genomesequence of Mesorhizobium loti

Plasmid

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Mega Plasmid

Figure . Physical map of the complete genomesequence of Shinorhizobium meliloti

Genomes: Sizes and Numbers of GenesGenome Group Size (kb) Number of genesEukaryotic nucleusSaccharomyces cerevisiaeCaenorhabditis elegansArabidopsis thalianaHomo sapiens

YeastNematodePlantHuman

13,500 (L)100,000 (L)120,000 (L)3,000,000 (L)

6,00013,50025,000100,000

ProkaryoteEscherichia coliHemophilus influenzaeMethanococcus jannaschii

BacteriumBacteriumBacterium

4,700 (C)1,830 (C)1,660 (C)

4,0001,7031,738

VirusesT4HCMV (herpes group)

Bacterial virusHuman virus

172 (L/C)229 (L)

300200

Eukaryotic organellesS. cerevisiae mitochondriaH. sapiens mitochondria

YeastHuman

78 (C)17 (C)

3437

Marchantia polymorphaChloroplast Liverwort 121 (C) 136PlasmidsF plasmidpSymApSymBpMLapMLb

E. coliSinorhizobiumSinorhizobiumMesorhizobiumMesorhizobium

100 kb (C)1.35 MB (C)1.68 MB (C)351.341 kb (C)206.315 kb (C)

29????

----------------------------------------------------------------------------------------------------------------------NOTE: C = circular; L = linear; L/C = linear in free virus, circular in cell

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Naming PlasmidsA. The genes they carry

pBR322 : constructed by Bolivar and Rodriguez fromThe ColE1 plasmid and is derivative number 322.

pJL1703 : constructed by Jose Loper and Lindow S.pATW38: constructed by Aris T Wahyudi

B. Number and letter/person(s) namep for plasmid, proceedes capital letters.

Plasmid Structure

Most plasmids are circular, a few known plasmids are linear.

Linear plasmids have been found in Streptomyces and Borrelia.

Covalently closed circlar (CCC), supercoiledRelaxed, no supercoiling

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Plasmid Purification

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Properties of Plasmid

-Plasmids have the ability to replicate independently.DNA that can replicate autonomously in the cell: “Replicon”-Replicon in type of cell, DNA must have at least one originof replication (ori site).-The plasmid origin of replication is often named: oriV.-Replication:

~Theta Replication-opening the two strands of DNA at the ori region,creating a structure that look like “Theta”.RNA primer begins replication: one/two direction.-Unidirectional Replication: single replication forkmoves around the molecule until it returns to theorigin. Two doughter DNAs separate.-Bidirectional Replication: Two replication forksmove out from the ori region, one in either direction,and replication is complete when two forks meetsomewhere on the other side of the molecule.

~Rolling Circle

-Rolling circle plasmids. This type of plasmid is widespreadfound in Gram negative and positive, and archaea.

-Replication occurs in two stages:1. Double-stranded circular plasmid replicates to form

double-stranded circular DNAsingle-stranded circular DNA

2. Complementary strand is synthesized on the single-stranded DNA to make another double-stranded DNA

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Mechanisms:[1] Rep protein recognizes double-stranded origin (DSO) on the DNA

and single-stranded break.[2] DSO is palindromic sequence which might form a cruciform structure

by base pairing between the inverted repeat.[3] After Rep protein has made a break in the DSO, it remains covalently

attached through one of its tyrosines to the Phosphate at 5’ end.[4] DNA Pol III uses the free 3’ OH- end at the break as a primer to re-

plicate around the circle, displacing one of the strands.[5] DNA pol III has completed it circle, the Rep protein make another

nick and releasing the displaced strand.[6] The ends of the displaced single strand are joined by Rep protein.

Phosphate is transferred from tyrosine of Rep protein to 3’OH ofthe displaced strand.

[7] The newly synthesized strand is ligated by host DNA ligase, creatingDouble stranded and single strand------Two double stranded plasmid

Fig. Some common schemes of plasmid replication

Unidirectional

Bidirectional

Rolling-Circle

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Host Range

~Host range is usually determined by ori region.pBR322, pET, pUC (ori from ColE1) “Narrow host range”only replicate in E. coli and Salmonella and Klebsiella.

~Plasmid RK2, RF1010, RC plasmid from Gram +“Broad host range”.ori from RK2 replicate most type of Gram –ori from RSF1010 replicate most type of Gram +pUB110 isolated from S. aureus, replicate in Gram +

Copy Number

[“Average number of the particular plasmid per cell”]

High Copy Number : ColE1Low Copy Number : Plasmid F

Regulation of Copy Number:ctRNA and protein (Counter transcribed RNA)

Antisense RNActRNA onlyProtein only--------binds to repeat sequences in the

plasmid called “iteron” inhibiting replication

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1.2.

3.

4.

5.

6.

7.

RNA I inhibit plasmid replication by interfering with RNA IIIn the absence of RNA I, RNA II forms an RNA-DNA hybridat the Replication origin.RNA I inhibit replication by interfering with primer formationby RNA II by forming double-stranded RNA with it.These two RNA are complement.Initial pairing of RNA I & RNA II is very weak, called “Kissingcomplex”. Protein Rop helps to stabilize the kissing complex.Formation of double-stranded RNA prevents the RNA II fromforming the secondary structure required for it to hybridizeto the DNA before being processed by RNAse H to form matureprimer.Rop protein helps RNA I to pair with RNA II, therefore helpinhibit plasmid replication.More RNA I will be made when the concentration of the plasmidis high. High conc of RNA I interferes with the processing ofmost of the RNA II Replication is inhibited.

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ColE1-Derived Pasmid(Regulation by a ctRNA and a protein)

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ColE1(6646 bp)

R1 and ColiB-P9 Pasmid(Regulation of Rep protein by antisense RNA)

1.

2.3.

4.

5.

6.

RepA is required for initiation of replication, transcribed fromtwo promoters:PcobB-----transcribes both repA and copB genes

yield protein RepA and CopB.PrepA-----in the copB gene, make RepA protein.PrepA is repressed by CopB protein.Once the plasmid has attained its copy number, the regulationof synthesis of RepA, and therefore the replication of plasmid,is regulated by an antisense RNA called CopA.CopA RNA is made from the same region encoding repA gene,but from the other strand. The two RNA will be complementaryto make double stranded RNA.The RNA-se III cleave double stranded RNA, will cleave theCopA-RepA mRNA.Translation of RepA is coupled to the translation of leader po-lypeptide. Cleavage of the mRNA by RNAse III in the leaderregion will interfere with the translation of this leader polypep-tide and blocking the translation. Also block translation of thedownstream RepA.

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7.

8.

CopA RNA activate cleavage of themRNA for the RepA proteinupstream of the RepA coding seq,the plasmid copy number iscontrolled by amount of CopARNA.

Higher conc of plasmid, more CopARNA will be made and less RepAprotein will be synthesizedmaintain copy number

The Iteron Plasmid(Regulation by Coupling)

1.

2.

3.

4.

5.

Iteron: The oriV contain several repeats of a certain set of DNA basespSC101, F, R6K, P1, RK2.

pSC101 iteron sequence: 17-22 bp~~3-7 copy in the ori required region.

RepA protein is required for initiation of replication. Iteron: R1, R2, R3

RepA is plasmid-encoded protein required for replication of pSC101.

RepA ~ activator replication. Host encodes other protein: DnaA,B,C,G

Iteron plasmid replication is regulated by two mechanisms:

[A] RepA represses it own synthesis by binding to its own promoter

region and blocking transcription of its own gene.

The higher conc of plasmid, more RepA will be made and more it

will repress its own synthesis. “Transcriptional Autoregulation”.

[B] Caupling: When the conc of plasmid is high enough, they become

coupled to each other through the RepA protein, thereby inhibiting

the replication of both caupled plasmid.

“Control of replication”.

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Incompatibility

1.

2.

3.

4.

Many bacteria isolated from nature, often contain more than onetype of plasmid.

These plasmids stably coexist in the bacterial cell and remain thereeven after many cell generation.

However, not all type of plasmids can stably coexist in the cell. Sometypes will interfere with each other’s replication or partitioning sothat if two such plasmid are introduced into the same cell, one orother plasmid will lost. Phenomenon:”Plasmid Incompatibility”.

Plasmids that can not stably coexist: “Incompatibility Group”.Plasmid that can stably coexist: “Different Incompatibility Group”.

5.

6.

Plasmid RP4

RK2

Plasmid RSF1010

RP4

“Incompatibility Group”

“Different Incompatibility”, RSF1010can be stably maintained with RP4

EndogenousPlasmid

Physical and genetic map of R. sphaeroides 2.4.1 genome

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Incompatibility Due to Replication Control

1.

2.

3.

4.

5.

Two plasmids that share the same mechanism of replication controlwill be “Incompatible”.

The replication control system will not recognize the two as differentand so either may be randomly selected for replication.

Fig 4.14A. Cell contain 2 types of plasmid that belong to differentincompatibility group, use different replication control system.

Plasmid exist in equal number before cell division, but after divisionthe two doughter cell are not likely to get the same number of eachplasmid.

However the new cell, each plasmid will replicate to reachits copy number.

Fig 4.14B. The cell has two plasmid in the same Inc group, both ofwhich use the same replication control system.

First----plasmid exist in equal number. Cell division, cell will notreceive the same number of the two plasmid.

Next, the doughter may not receive the same number of plasmid.

Next Division, one of the two plasmid to be cured.

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Incompatibility Due to partitioning

1.

2.

Two plasmids can also be incompatible if they share the same parfunction. When coexisting plasmids share the same par function, oneor the other will always be distributed into the doughter cell duringdivision.

One doughter cell will get the other plasmid type, producing cellcured of one or the other plasmid.

Determining the Incompatibility

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Plasmid Cloning Vector

1.

2.

3.

4.

5.

Generally does not kill the host cell

Relatively easy to purify

Relatively small

Contain origin of replication (oriV)

Carry selectable marker (antibiotics, ..)

How to Make Plasmid Cloning Vector?

1.

2.

3.

4.

Finding the plasmid ori origin

Introducing selectable genes

Introducing unique restriction sites

Insertional inactivationDirectional cloning

Finding the ori origin

1. Digest plasmid with restriction enzyme

Piecess containing ori are ligated another

piece of DNA of that selectable marker (AP)

2. Transform to bacteria (E. coli)

3. Selection on media Agar+antibiotic (Ap)

4. Cutting the fragment ori---smaller--clone

Introducing selectable marker

1.

2.

3.

Gene which confer resistance to an antibioticmake convinient selectable marker

introduce into cloning vector

Selection by plating on media containingantibiotic. Antibiotic resistance genes are oftentaken from: transposon or other plasmids.

Have been introduced in clonning vector: CmRfrom Tn9; TcR from pSC1010; ApR from Tn3;KmR from Tn5.

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Introducing unique restriction sites

1.

2.

Cloning vector should have unique restriction.

Unique site of RE: cut the vector at only oneMCS: by : site-specific mutagenesis

partial digest of the plasmid.

Introducing unique restriction sites

1.

2.

Insertional inactivation: the unique sites arelocated in selectable marker insertion offoreign DNA in the site will inactivateselectable genes.

Directional cloning: small region on theplasmid for cloning of foreign DNA/gene(MCS).

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Figure . Map of Plasmid pBR322

HincIIPstISphIHindIII

EcoRISacIKpnISmaIXmaIBamHISalIAccI

EcoO109 2674AatII 2617

SspI 2501

XmnI 2294

ScaI 2177

PvuI 2066

AvaII 2059

FspI 1919

AvaII 1837BglI 1813

GsuI 1784Cfr101 1779

PpaI 1766

HgiEII 1387

PvuII 628

PvuI 276PvuII 306

NarI 235BglI 245FspI 256

(EcoRI/PvuII) 1HgiEII 181

NdeI 183

lacZ

lacI

AflIII 806

ORI

MmeI 996

MmeI 1180AlwNI 1217

Eco57I 1333

Ap pUC192686 bp

Fig . Map of plasmid pUC19

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ORF Size (bp) Protein Homology Protein Homology

1 828 Rep protein Pseudomonas fulva

Mob protein2 1110 Listeria monocytogen

Table Homologous searches

SacI

NcoI

HincII

HincII

SacI

SmaI*

SacI

BamHI XhoI*SphI

ORF2

pMGT(3741 bp)

Fig. Construction of plasmid pUMG

NcoI

HincII

SphIBamHI

pMGT3.7 kb

ligation

lacZ

BamHI

lacIAp pUC192.7 kb

ORI

NcoI

HincII

HincII

NcoIBamHI

ORF1

NcoIFig. Map of a cryptic plasmid pMGT

lacZBamHI

ORIlacI

Ap HincII

NcoI

Digested withBamHI

Digested withBamHI

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Shuttle Vector

Ori

pUMG

6.4 kb

mob

Ori

mob

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Procedure 1-1.5 ml kultur

Larutan II ~ mix slowly 4-6 X

Larutan III ~ mix slowly 4-6 X

Supernatan +fenol/kloroform/isoamilalkohol

Lap atas ambil, + 100% alkoholInkubasi pada –20 C, 20-30

Cuci DNA pelet dng 70% alkohol

Keringkan dan larutkan dalamTE atau akuades steril

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Larutan isotonis

SDS-lisis sel,denaturasi proteinNaOH-denaturasi DNA

Renaturasi,netralisasi

Pengendapprtein/lemak

Presipitasi DNAplasmid

Pencuci DNA

Plasmid Isolation

Pelet + Larutan I ~ campur