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Objectives Prokaryotic and Eukaryotic vectors

Dr. M. Ravichandran Department of Microbiologi and Parasitology PPSP, Ext: 4608 Email: [email protected]

To understand the types of vector and it's properties.

Vector and Hosts

Vectors Plasmid, Cosmid, Phage, Phagemid Host E.coli, Yeast, Mammalian cells, Insect cells

Plasmid

Autonomously replicating extrachromosomal circular genetic material Properties Resistance marker: antibiotic resistance, auxotrophic marker Ori-site: enable replication in the host cell Multiple cloning site (MCS): Restriction enzyme cleavage sites Promoters: Inducible promoters, plac, ptrp, T7, SP6 Ribosomal binding site High and Low copy number Affinity tag for easy purification and detection of protein: 6 Histidine, Glutathione S transferase (GST), Maltose binding protein (MBP), Green fluorescent protein

(ROM)

Types of plasmid

Relaxed plasmid High copy number, multiple copies/cell eg. ColE1 Stringent plasmid Low copy number, limited number of copies/cell eg. F Conjugative plasmids It has a set of transfer genes. tra genes that promotes bacterial conjugation. Eg. R6K Shuttle plasmid Has origin of replication for two hosts E.coli and Yeast. It can replicate in both the hosts Plasmid incompatibility Inability of two different plasmids to co-exists in the same host cell; Eg: Two ColE plasmids can not co-exist in the same cell

Plasmid vector

Cloning vector: For cloning a gene Expression vector: Expression of recombinant protein Transcription vector: To make RNA copy of a gene PCR cloning vector: For cloning PCR products Secretion vector: For secreting recombinant proteins

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Lac Operon

Cloning vector

P(BLA)

lacZ'

EcoR I SacI AvaI XmaI KpnI SmaI BamH I XbaI SalI AccI HincII PstI SphI Hind III

P(LAC)

MCS Multiple cloning site

Amp r

2686 bp

pUC19

lacI

IPTG : Isopropyl thio galactoside -synthetic analogue of lactose -binds to repressor protein -Can not be metabolized by -galactosidase

X-gal can be hydrolyzed by -galactosidase and produces a dark blue color.

ORI

An origin of replication. The pUC family of vectors are high copy vectors. They have a ColE1 origin of replication A drug resistance marker. The pUC vectors contain a gene for ampicillin resistance (-lactamase). An inducible promoter. This family of vectors contains the lac promoter (Plac) along with the associated lac Operator region The lacI gene. This gene codes for the lac repressor protein. The lacZ' gene. This gene product is transcribed from the lac promoter and produces an amino-terminal fragment of the -galactosidase protein. Multiple cloning site(MCS). This short stretch of DNA is located just downstream from the lac promoter and after the first few codons of the lacZ' gene. It is a short stretch of nucleotides which contains a variety of restriction endonuclease sites.

P(BLA)

-complementation

Chromosomal DNA codes for carboxyterminal fragment of -galactosidase Non functional -galactosidase

E.coli host (Genetically engineered)

White colony (In the presence of IPTG and X-gal)

lacZ'

P(LAC)

r AMP

pUC19

lacI ORI

2686 bp

Chromosomal DNA codes for carboxyterminal fragment of -galactosidase + Plasmid codes for amino terminal region of -galactosidase

E.coli host (Genetically engineered) With pUC19 plasmid

Functional -galactosidase

Blue colony (In the presence of IPTG and X-gal)

Blue white colonies

Insertional inactivation

Insertional inactivation of galactosidase

lacZ'

P(BLA)

P(LAC)

MCS Multiple cloning site

Amp

r

2686 bp

ORI

pUC19

lacI

Klug & Cummings 1997)

Cloning of a foreign gene in the MCS of pUC vector will lead to inactivation of lacZ gene non functional -galactosidase E.coli carrying this plasmid will from white colonies in the presence of X-gal

Dr. M. Ravichandran

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Expression vector

Ampicillin resistance marker ColE1 origin of replication (ori)

f1 Ori MCS T7 promoter AMP R

Mechanism of T7 expression system

T7 RNA polymerase

P(LAC)

f1 Ori MCS T7 promoter AMP R

f1 origin of replication (allows single stranded vector to be produced when co-infected with M13 helper phage) lacI gene (lac repressor protein) T7 promoter (specific for phage T7 RNA polymerase) multiple cloning site (polylinker region) downstream of the T7 promoter Chromosomal DNA

lacIq

5549 bp

pHIS1

COLE1 ORI

lacIq

pHIS1

BL21 (DE3) E.coli host

T7 RNA polymerase gene is from T7 Phage, not usually present in E.coli. T7 RNA pol gene is integrated into the chromosome of E.coli using a temperate phage DE3 T7 RNA polymerase gene is under the control of a lac promoter.Thus, induction by IPTG, causes the host to produce T7 RNA polymerase The activity of T7 RNA polymerase is higer than E.coli RNA polymerase, hence the protein expression by T7 expression vector is always higher

5549 bp

6 Histidine tag- useful for purification of protein by Metal affinity chromatography COLE1 ORI Recombinant protein can be expressed only in Ecoli host rTEV protease cleavage site which can produce T7 RNA polymerase (BL21DE3)

Other T7 expression vector

High-level expression from the bacteriophage T7 promoter N-terminal polyhistidine (6xHis) tag for rapid purification by metal affinity chromatography N-terminal Xpress epitope for protein detection with the Anti-Xpress Antibody Enterokinase cleavage site for removal of fusion tag f1 origin for ssDNA rescue to allow easy sequencing and mutagenesis

Fusion protein expression

Gene-X

Used for the detection of recombinant protein using anti-epitope antibody 6His Used for purification of this protein using Nickel column 6His Epitope

Transcription and Translation

EK

Enterokinase clevage site

Protein X

Epitope

EK

Protein-X

Pure recombinant protein

Transcription vector

SP6 Promoter MCS T7 Promoter COLE1 ORI

PCR cloning vector

Taq polymerase adds "A" at the 3' end of PCR product PCR products generated by Taq polymerase can be ligated to the `T' overhang of the PCR cloning vector Other properties Blue/White screening (lacZ') f1 origin of replicationSingle stranded DNA preparation Ampicillin and Kanamycin resistance marker In-vitro transcription using T7 RNA polymerase M13 reverse and T7 primers can be used for sequencing

2462 bp

pSP72

AMP(R)

DNA cloned downstream from the SP6/T7 promoter can serve as a template for RNA synthesis In-vitro transcription can be done using SP6 and T7 RNA polymerase, ribonucleotide triphosphates, rUTP, rATP, rGTP and rCTP and buffer In-vitro translation Sense and Antisense RNA can be produced using this vector RNA probes

Dr. M. Ravichandran

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Type of vectors Cloning vector (pUC19)

Properties Resistance marker -Ampicillin Origin of replication Multiple cloning site(MCS) lacZ lacI R, O, M Ampicillin resistance Strong promoter (T7) 6 HIS- affinity tag Enzyme cleavage site F1 origin of replication R, O, M Ampicillin resistance T7 promoter SP6 promoter R, O, M Ampicillin & Kanamycin resistance "T" overhang F1 origin of replication T7 promoter M13 reverse priming site

Use Type of vectors Cloning a gene Screening by -complementation and insertional inactivation ..Blue/White colony screening Secretion vector pET21 Properties R, O, M Ampicillin resistance F1 origin of replication T7 promoter Use Recombinant protein production, secretion and purification using nickel column

PelB signal peptide

Recombinant protein production and purification using nickel column 6 HIS- affinity tag Enzyme cleavage site Cosmid pWE15 R, O, M Cos site (Does not have the lambda phage gene except COS site) Can be package with head and tail MCS in the c1 repressor gene Can be package with head and tail Single stranded DNA Infect E coli having pilus (F') Phage and plasmid properties Contains M13 initiator and terminator Upto 40 kb inserts can be cloned Used for making gene libraries

Expression vector pHis, pRSET

Transcription vector- (pSP72)

Sense and antisense RNA probes by Invitrotranscription Invitro-translation Cloning PCR product Invitro transcription -complementation and insertional inactivation Blue/White colony screening

Lambda insertion vector lgt10 Filamentous phage Phagemid

Upto8-23 kb inserts can be cloned Used for making gene libraries useful for sequencing, in-vitro mutagenesis Upto 3 kb inserts can be cloned Used for making gene libraries Converting into plasmids with out subcloning

PCR cloning vector (pTOPO 2.1)

Bacteriophages

Lambda phage: Head and tail 49kb in size, double stranded Cos site Upto 8-23 kb inserts can be cloned in lambda phage

Package with head and tail Only the foreign DNA inserted in c1 repressor will form clear plaque

in-vitro Packaging of -DNA

-DNA ligated with foreign DNA

Cosmid

Plasmid with lambda phage cos site Upto 40 kb inserts can be cloned in cosmid Foreign DNA cloned, packaged and infected into E.coli but in E.coli, it propagates like a plasmid pWE15 carries neomycin marker and SV40 promoter for neomycin gene expression in eukaryotic cells

+

Assembly proteins Pre-head proteins Tail Protein D

+ ATP

Produced separately by lysogenised E.coli (s)

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in-vitro Packaging of Cosmid

Cos site Cos site

Filamentous phage: M13, f1 phage

6407 bases, single stranded DNA Infect E coli having pilus (F') Replicate form is double stranded DNA, this form can be used for genetic manipulation useful for sequencing, in-vitro mutagenesis Upto 3 kb inserts can be cloned in M13 phage

G8 protein

Cosmid ligated with foreign DNA

+

Assembly proteins Pre-head proteins Tail Protein D

+ ATP

ssDNA 6407bases

G3 protein

Produced separately by lysogenised E.coli (s)

Phagemid

Hybrid phage vector with plasmid functions Contains M13 initiator and terminator region Cloned DNA can be excised in-vivo as a part of a plasmid when co-infected with M13 phage No need for subcloning

In-vivo excision

Individual lambda phage or an amplified library are allowed to infect E. coli cells which are co-infected with filamentous helper phage. Inside the cell, trans-acting proteins from the helper phage recognize initiator (I) and terminator (T) domains within the ZAP Express vector arms. Both of these signals are recognized by the helper phage gene II protein and a new DNA strand is synthesized, displacing the existing strand. The displaced strand is circularized and packaged as a filamentous phage by the helper phage proteins, and secreted from the cell. pBK plasmids are recovered by infecting an F' strain and growing in the presence of kanamycin.(F-cells carry the receptor for the single-stranded bacteriophage on their pili)

http://www.stratagene.com/vectors/cloning/zap_express.htm

Phagemid

Initiator (M13) ColEI Lambda DNA

AmpR MCS

Eukaryotic expression vector

Advantages

Terminator (M13)

lacZ

Clone the DNA of interest in MCS In-vitro packaging Infect F' E coli Co-infect with M13 phage

Replicative form is dsDNA (plasmid)

Post translational modification eg. Glycosylation Disulfide bond formation Proper protein folding Endotoxin free recombinant protein Example: Yeast vector Features of Eukaryotic vector

Multiple cloning site Selection marker (Resistance /complementing) Origin of replication (both eukaryotic and prokaryotic) Promoter: Simian virus 40 promoter Cytomegalovirus promoter (CMV) Poly A tail Introns Genetic manipulation is normally done in E.coli and transferred to eukaryotic cells

M13 phage with cloned DNA (ssDNA) Infects F' E.coli

Gene cloned in plasmid

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Yeast vector

Integrative Vectors They consist of a ColE1 replicon and an appropriate complementing marker; replication of this type of vector occurs in E.coli but not in yeast. The plasmid get integrated into the chromosome by homologous recombination. Autonomous Vector These are again subdivided into plasmid derived and chromosomally derived vectors: Plasmid derived vectors

These vectors depend on cryptic plasmids present in yeast called 2-micron circle. This plasmid is 6.3 kb in size with 50-100 copies per genome. Origin of replication for Yeast and E.coli (Shuttle vector)

Pichia- Integrative vector

Chromosomally derived vectors: YAC

http://www.invitrogen.com

Yeast artificial chromosome (YAC)

Chromosome structure

A YAC can be considered as a functional artificial chromosome (self replicating element), since it includes three specific DNA sequences that enable it to propagate from one cell to its offspring: TEL: The telomere which is located at each chromosome end, protects the linear DNA from degradation by nucleases. CEN: The centromere which is the attachment site for mitotic spindle fibers, "pulls" one copy of each duplicated chromosome into each new daughter cell.

Telomere

Centromere

Telomere

ORI: Replication origin sequences which are specific DNA sequences that allow the DNA replication machinery to assemble on the DNA and move at the replication forks. It also contains few other specific sequences like: A and B: selectable markers that allow the easy isolation of yeast cells that have taken up the artificial chromosome. Recognition site for the two restriction enzymes EcoRI and BamHI.

Is it possible to make chromosome artificially?

Various vectors and their insert size

Up to 1000 kb

Dr. M. Ravichandran

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Vector and Host Plasmid

Summary

Properties Relaxed plasmid Stringent plasmid Conjugative plasmids Shuttle plasmid Plasmid incompatibility Cloning vector

-complementation,Insertional inactivation

Bacteriophages gt10 in-vitro Packaging of -DNA Cosmid in-vitro Packaging of Cosmid Filamentous phage Phagemid Eukaryotic expression vector Advantages Features of Eukaryotic vector

Yeast vector

Intergrative Vectors Autonomous Vector Plasmid derived vectors Chromosomally derived vectors: YAC Yeast artificial chromosome

Expression vector Mechanism of T7 expression system Fusion protein expression Transcription vector PCR cloning vector

Suggested readings

Questions

Dr. M. Ravichandran

Department of Microbiologi and Parasitology PPSP, Ext: 4608 Email: [email protected]

Lecture notes:

Principles of Gene Manipulation- An introduction to Genetic Engineering, RW.OLD & S.B primrose Genetics: TA Brown Gene cloning : TA Brown Molecular Biology: David Freifelder Molecular cloning: A laboratory manual, Sambrook, Fritsch, Maniatis, Vol 1-3 Molecular Biology catalogs

Stratagene: Phagemid, Cosmid Promega- Restriction enzymes cloning vectors Invitrogen: pRSET, pTOPO2.1 Novagen: BL21(DE3)

http://10.2.5.75/lecturers/mravi/gtb204_2002_Index.htm

Dr. M. Ravichandran

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