Cloning Vectors

Cloning Cloning vectors

vectors

Vectors • carry new DNA to desired cell

properties; • able to self-replicate in the host – unique origin of replication • can be manipulated outside the cell (in vitro) – depend on its size • always circular dsDNA – not easily digested and easily preserved • contain markers for easy selection – antibiotics resistance especially against ampicillin (amp) and tetracycline (tet) • cannot undergo conjugation – no unwanted recombination • if vector and DNA to be cloned are cut by the same restriction endonuclease, similar sticky ends are created – easy to recombine • type of vectors • cloning vector – only for cloning DNA • shuttle vectors can exist in several different species • expression vectors – the cloned gene is expressed (protein production) in the host • secretion vectors – the expressed gene is secreted out of the cell

Vectors - usual • plasmids most commonly used engineered genetically with MCS, promoter/terminator and primer sites cloning limit: up to 10 kb • lambda phages 1000 times more efficient than the plasmid vector cloning limit: up to 25 Kbp • cosmid - combination of the plasmid vector and the COS site of lambda phage cloning limit: up to 45 Kbp • bacterial artificial chromosomes (BAC) based on bacterial mini-F plasmids (fertility) cloning limit: 75-300 kb • yeast artificial chromosome (YAC) an artificial chromosome that contains telomeres, origin of replication, a yeast centromere, and a selectable marker for identification in yeast cells cloning limit: 100-1000 kb • phagemid – plasmid from phage M13 replicate either as plasmid or as phage but with phage cloning capability

Plasmids • • • • • 1. 2. 3. 4.

Double stranded closed circular DNA Size – 1 kb to > 200 kb Replicate independent of bacterial chromosome Use enzymes & proteins coded by host chromosome Impart phenotypic characters to host as resistance to antibiotics, production of antibiotics, degradation of complex organic substances, production of colicins, enterotoxins and restrictionmodification enzymes

How do you stably maintain and replicate a foreign DNA fragment? – by hitching a ride on a stable replicon Properties of plasmid cloning vector 1. Small 2. Stable in the chosen host – usually E. coli 3. High copy number 4. Easy to purifiy 5. Can accommodate foriegn DNA 6. Single “cloning” sites 7. Selectable marker – antibiotic resistance 8. Easily introduced into host (transformation or transduction

Plasmids as Cloning Vectors

Plasmid pBR322

Small, only 4361 BP, Stable in E. coli, High copy number (10003000 copies per cell) Easily isolated in the supercoiled form Foreign DNA can be inserted in good amount Restriction sites are known Single cleavage sites for several restriction enzymes Two antibiotic resistance markers Transformation easy

Plasmid Vector: pBR322 • First modern cloning vector (1976)

pBR322 •

Contains: 1. colE1 origin of replication (ORI)

Col E1 origin of replication

Direction of DNA replication

RNAse H processing

RNA II

-500 -400 -300 -200

-100 ori

100

200 300 400 500

600 rop gene

RNA I

Rop protein (63 aminoacids)

• •

• •

• • • •

Replicate in a „relaxed‟ fashion Does not require plasmid encoded functions for replication instead it relies on long lived enzymes supplied by host- DNA and RNA polymerases, & products of dnaB, dnaC, dnaD and dnaZ Can multiply in absence of host cell protein synthesis, i.e. in the presence of choranphenicol Replication of DNA is initiated by synthesis of RNA II molecule and RNA I molecule transcribed from opposite strands. RNA I binds to RNAII and prevents its folding into a cloverleaf structure necessary for formation of stable DNA:RNA hybrids Rop protein enhances binding of RNA I & RNA II Copy number can be increased by mutations that weaken interaction between RNA I & RNA II Integrity of 5‟ ss domain of RNA I is crucial for the for the interaction between RNA I & RNA II Plasmids that utilize the same replication system cannot coexist and are incompatible

pBR322 • Contains:

Non-transformed bacteria

Bacteria plus plasmid

2. Selectable Markers: • Ampicillin Resistance (β-lactamase gene) • and Tetracycline Resistance (tet gene)

Nutrient media plus antibiotic

Overnight growth Only colonies from bacteria that have plasmid

pBR322 • Contains: 3. A few good restriction sites for inserting foreign DNA BamH1

BamH1

Your favorite DNA

PstI

Eco RI

Digest with BamH1

Bam HI

and ligate

PstI

Eco RI

Your favorite DNA

Bam HI Bam HI

Gene cloning and Expression using Plasmid pBR322

pBR322 • Nice Features: √ 200 copies per E. coli cell √ Makes double stranded DNA √ All modern cloning vectors are based on pBR322

• Examples – pBR322 • One of the original plasmids used • Two selectable markers (Amp and Tet resistance) • Several unique restriction sites scattered throughout plasmid (some lie within antibiotic resistance genes = means of screening for inserts) • ColE1 ORI • Form multimers in recA+ hosts • readily lost from the cells grown in minimal media

Next Generation: pUC Plasmids •

Advantages over pBR322 1. Makes 1000‟s of copies/cell 2. Small size at 2.7 kilobase pairs (kb) = easier uptake by E. coli

pUC Plasmids • Advantages over pBR322 3. Multiple cloning site

Multiple Cloning Site (MCS or Polylinker)

pUC19 MCS

pUC Plasmids • Advantages over pBR322 4. Easier Selection –AmpR & blue/white selection

Blue/White Selection • Based on the enzymatic reaction of βgalactosidase.

Blue/White Selection – Modified E. coli codes for the carboxyl portion of β-galactosidase enzyme. This portion alone cannot cleave X-gal. – Plasmid codes for amino portion (“LacZ” or αpeptide) of β-gal. – However, when the two peptides are expressed together in a cell, X-gal is cleaved, and an indigo product stains the bacterial colony blue. This process is called αcomplementation.

Blue/White Selection What is white selection?

Blue/White Selection Bacteria plus empty plasmid

Bacteria with plasmid plus insert

Non-transformed bacteria

Nutrient media plus antibiotic plus X-Gal

Overnight

Colonies with insert - white Colonies w/o insert - blue

growth

Only colonies from bacteria that have plasmid

– pUC18 • Derivative of pBR322 • Advantages over pBR322: – Smaller – so can accommodate larger DNA fragments during cloning (5-10kbp) – Higher copy # per cell (500 per cell = 5-10x more than pBR322) – Multiple cloning sites clustered in same location = “polylinker”

• Interruptable gene encoding for enzyme beta galactosidase (lacZ) – – – – –

Polylinker resides in the middle Enzyme activity can be used as marker for gene insertion Disrupted gene = nonfunctional Intact gene = functional Media containing XGAL chromagenic substrate used (blue colonies = intact; white colonies = disrupted)

• Amp resistance gene still present (= beta lactamase), Tet resistance gene omitted

Replicons in currently used plasmid vectors Plasmid

Replicon

Copy number

pBR322 and its derivatives

pMB1

15-20

pUC vectors

pMB1

500-700

pACYC and its derivatives

p15A

10-12

pSC101 and its derivtives

pSC101

About 5

Col E1

Col E1

15-20

Hosts for cloning vectors • Ideal hosts: rapid growth, capable of growth in cheap culture medium, not harmful or pathogenic, transformable by DNA, stable. • Prokaryotic hosts: E. coli, Bacillus subtilis. • Eukaryotic hosts: Saccharomyces cerevisiae. – DNA virus SV40, a virus causing tumors in primates, can be used as a cloning vector into human culture lines. – Retroviruses, vaccinia virus are useful too. – Baculovirus, an insect DNA virus can be used to transfer DNA to insect cell lines

Must have: 1) Ori 2) A dominant selectable Marker 3) Cleavage sites for cloning 4) (high copy no.) 5) Lack rop gene

The plasmid cloning vector pUC19. This plasmid has an origin of replication (ori), an ampR selectable marker, and a polylinker located within part of the -galactosidase gene lacZ+.

Fig. 7.4, pUC19

Polylinker: restriction sites

lacZ+ gene

Ampicillin resistance gene

Origin sequence

Fig. 7.5 *Cut with same restriction enzyme

*DNA ligase

Some features of pUC19: 1.

High copy number in E. coli, ~100 copies/cell, provides high yield.

2.

Selectable marker is ampR. Ampicillin in growth medium prevents growth of all other E. coli.

3.

Cluster of restriction sites called a polylinker occurs in the lacZ (galactosidase) gene.

4.

Cloned DNA disrupts reading frame and -galactosidase production.

5.

Add X-gal to medium; turns blue in presence of -galactosidase.

6.

Plaque growth: blue = no inserted DNA and white = inserted DNA.

7.

Some % of digested vectors will reanneal with no insert. Remove 5’ phosphates with alkaline phosphatase to prevent recircularization (this also eliminates some blue plaques).

8.

Plasmids are transformed into E. coli by chemical incubation or electroporation (electrical shock disrupts the cell membrane).

9.

Cloned inserts >5-10 kb typically are unstable; good for <10kb.

Insertion of a piece of DNA into the plasmid cloning vector pUC19 to produce a recombinant DNA molecule. The vector pUC19 contains several unique restriction enzyme sites localized in a polylinker that are convenient for constructing recombinant DNA molecules. The insertion of a DNA fragment into the polylinker disrupts part of the -galactosidase (lacZ+) gene, leading to nonfunctional  -galactosidase in E. coli. The blue–white color selection test described in the text can be used to select for vectors with or without inserts.

a-complementation – relies on modular structure of galactosidase - basic idea is often used with cloning vectors – called insertional inactivation

a-complementation LacZ+ - blue colony LacZ- - while colony -of you interrupt the lacZ gene, the colony is white

Brock Biology of Microorganisms, vol. 9, Chapter 10

How does acomplementation work? It all comes down to galactosidase Certain strains supply the -Gal  fragment When a is supplied in trans, this allows -Gal to function

α -complementation • The portion of the lacZ gene encoding the first 146 amino acids (the α -fragment) are on the plasmid • The remainder of the lacZ gene is found on the chromosome of the host. • If the α -fragment of the lacZ gene on the plasmid is intact (that is, you have a nonrecombinant plasmid), these two fragments of the lacZ gene (one on the plasmid and the other on the chromosome) complement each other and will produce a functional β galactosidase enzyme.

Latest Generation: pGEM and pBluescript

MCS

 Features similar to pUC

 plus f1 ori (single stranded DNA) T7 & SP6 Promoters (RNA production)

Latest Generation: pGEM and pBluescript

 Features similar to pUC

 plus f1 ori (single stranded DNA) T7 & T3 Promoters (RNA production)

（2）pBluescript -phasmids pBluescriptSK(+/-)properties： （i）there are T3 and T7 phage promoters to regulate the foreign gene transcription near the MCS

T3 RNA polymase bind at T3 promoter, Foreign DNA （－）strand transcripted

T7 RNA polymase bind at T7 promoter, Foreign DNA （+）strand transcript

(ii)Other are same with PU118 or PU119.

Applications for cloning vectors Great to make lots of your favorite DNA For sequencing making libraries initial step for subcloning & expressing your favorite DNA

Preparation of plasmid DNA • Traditional method • Conventional method

Cloning Genes-General Cloning Scheme • Vector and foreign gene to be inserted are purified/modified separately before ligating the two together • Ligated products are introduced into “competent” bacterial cells by transformation techniques. Individual colonies are analyzed separately.

• Vectors able to survive under antibiotic selection are amplified in bacterial hosts by autonomous replication • Plasmid DNA containing the gene of interest is purified from large scale cultures

• Subsequent steps in the experimental design are undertaken: – – – –

Subcloning Mutagenesis Sequencing Transfection of eukaryotic cell lines (calcium phosphate precipitation, lipofection, electroporation, dextran sulfate, microinjection,…..) – Fragment isolation for transgenic mice production (microinjection) – PCR

Expression Vector

• An expression vector for fusions. This is a phagemid, can be propagated either as a plasmid or as a phage

Vectors for larger DNA fragments  vectors - Can insert fragments of DNA up to 25 kb. - Can introduce into cells at a very high efficiency BAC vectors (bacterial artificial chromosomes) - Contain sequences from the E. coli F plasmid – present at one copy per cell. - Can clone up to 200 kb per BAC clone. YAC vectors (Yeast artificial chromosomes) - Contains sequences required to replicate and maintain chromosome in budding yeast (like , end up as a linear molecules) - a yeast origin of replication, a centromere, and a telomere at each end. - Can clone >2,000 kb (2 Mb).

The DNA into which a foreign piece of DNA is cloned is called a ‘VECTOR’

There are several classes of vectors in use: 1. Plasmids: Accept up to ~10 kb ‘foreign’ DNA 2. Phage : 5-20 kb fragments (its own genome is only 50 kb!) Commonly used in making genomic libraries. (very high efficiency of transfection) 3. Cosmids: 35-45 kb – similar to plasmids (high efficiency for transformations) 4. YACs (Yeast Artificial Chromosomes): 300-2000 kb! (essential for cloning very large fragments)

Phage  cloning vectors: 1.

Engineered version of bacteriophage  (infects E. coli).

2.

Central region of the  chromosome (linear) is cut with a restriction enzyme and digested DNA is inserted.

3.

DNA is packaged in phage heads to form virus particles.

4.

Phages with both ends of the  chormosome and a 37-52 kb insert replicate by infecting E. coli.

5.

Phages replicate using E. coli and the lytic cycle

6.

Produces large quantities of 37-52 kb cloned DNA.

7.

Like plasmid vectors, large number of restriction sites available; useful for larger DNA fragments than pUC19.

Fig. 3.13

Fig. 7.6

Phage  Cloning Vectors • plasmids • viruses • bacteriophage • lambda () • filamentous (ssDNA) • combination

• large phage that infects E. coli • phage attaches to bacteria and injects DNA • complex genetics and life cycle with two phases: • lytic • lysogeny

 as a Cloning Vector • infectious  can be assembled in vitro • foreign DNA can be incorporated into the  genome • non-essential genes removed • phage assembly can occur with 40-52 kb of DNA (wild-type   50kb)

Insertion Vector

• accommodates up to 710 kb foreign DNA (depending on vector)

Replacement Vector

• 13 kb ‘stuffer fragment’ (lysogeny genes) discarded • accommodates 11-20 kb foreign DNA

Bacteriophages as Cloning Vectors Modified lambda phages as cloning vectors

12kb-20kb

<12 kb, can not be packaged efficiently >20 kb, too large to fit into the phagehead

Phage replication

Phage replication

12kb-20kb

Library Construction in  1 Prepare foreign DNA 2 Prepare vector DNA 3 Mix vector, foreign DNA and ligase 4 In vitro packaging 5 Infect host E. coli 6 Screen plaques 7 Plaque purification 8 Subclone fragment into plasmid

Vector DNA • purchase pre-cut and dephosphorylated

Genomic DNA Options • preliminary Southern blots • optimal enzyme (s) •  size fractionation • adaptors

EcoRI BamHI AATTCGAACCCCTTCG GCTTGGGGAAGCCTAG

GATCCNNNNNN---GNNNNNN----

Library Construction in  1 Prepare foreign DNA 2 Prepare vector DNA 3 Mix vector, foreign DNA and ligase 4 In vitro packaging 5 Infect host E. coli 6 Screen plaques 7 Plaque purification 8 Subclone fragment into plasmid COSLLLLLLLLG AATTCFFFFFFFG AATTCRRRRRRRRR ||||||||| ||||||||| |||||||||| LLLLLLLLCTTAA GFFFFFFFCTTAA GRRRRRRRRRCOS

Library Construction 1 2 3 4 5

Prepare foreign DNA Prepare vector DNA Ligation reaction In vitro packaging Infect host E. coli (plate on ‘lawn’) 6 Screen plaques 7 Plaque purification 8 Subclone fragment into plasmid Plaque: clear zone on bacterial lawn cause by lytic phage

 



Plaque Lift

Library Construction 1 2 3 4 5

Prepare foreign DNA Prepare vector DNA Ligation reaction In vitro packaging Infect host E. coli (plate on ‘lawn’) 6 Screen plaques 7 Plaque purification 8 Subclone fragment into plasmid

• punch out plaque(s) with Pasteur pipette • elute phage particles from agar • re-plate and re-screen as needed

Plaque hybridization

Library Construction 1 2 3 4 5

Prepare foreign DNA Prepare vector DNA Ligation reaction In vitro packaging Infect host E. coli (plate on ‘lawn’) 6 Screen plaques 7 Plaque purification 8 Subclone fragment into plasmid

• amplify cloned phage • purify phage DNA • excise insert • ligate into plasmid

To Screen Again or To Re-Make the Library? • no guarantee that clone of interest is in library • statistical estimates • # of independent inserts • identical clones from previous successful screen

N = ln(1-p)/ln(1-n) N = # of recombinants to examine p = probability of detecting clone n = average size of insert/genome size

Bacteria Protists Plants Insects Insert (kb)

Vector plasmid  insertion  replacement cosmid YAC

0-5* Fish 0-10 Amphibians 11-20 35-45 Reptiles 100-2000 Birds

*transformation efficiency  with  size (10-20 kb is generally upper limit)

105

106

107

Mammals 108

109

base pairs per haploid genome

1010

1011

Cosmids: plasmid vectors containing foreign DNA plus only the cos (cohesive end) site from the lambda genome • Sizes of a cloning gene the vectors can carry: YAC>Cosmid>Lambda>Plasmi ds

Vectors for DNA Sequencing: Bacteriophage M13 • Phagemid: a hybrids between a filamentous phage, like M13 and a plasmid Vectors with cloned DNA

Vectors without cloned DNA

X-gal: 5-bromo-4chloro-3-indolyl--Dgalactopyranoside

Other specialized vectors • Expression vectors: to obtain synthesis of the protein coded for by the foreign gene cloned into the vector. • Secretion vectors: protein product is not only expressed but secreted (excreted) from the cell. • Shuttle vectors: move DNA between unrelated organisms, can replicate in two different organisms

Cosmid vectors

Plasmids contain a fragment of λ DNA including the cos site

termed cosmids

be used as gene-cloning vectors be used in vitro packaging system.

the cos sites are substrates for the packaging-dependent cleavage

insertion of 32–47 kb of foreign DNA

To a cosmid vector of 5 kb

Multiplying Process

much more than a phage-λ vector can accommodate.

the particle is packaging in vitro, used to infect a suitable host. The recombinant cosmid DNA is injected and circularizes like phage DNA  cosmid DNA replicates as a normal plasmid without the expression of any phage functions.

Transformed cells are selected on the basis of a vector drugresistance marker.

in vitro packaging system Principles of Gene Manipulation

Simple scheme for cloningHebei in a University cosmidofvector. Economics

Cosmids characteristics

Circulated like λphage cannot make cell lysis Containing plasmid replicon and cos site of λDNA

provide an efficient means of cloning large pieces of foreign DNA.

are particularly attractive vectors for constructing libraries of eukaryotic genome fragments.

Existed problems

Recombinant cosmid may contain two fragments

Solution

ligated to form a single insert.

Dephosphorylating the foreign fragments to prevent their ligation together.

Recombinants may have a duplicated vector

are unstable and break down in the host by recombination

resulting in the propagation of a nonrecombinant cosmid vector.

Principles of Gene Manipulation Hebei University of Economics

Solutions

Principles of Gene Manipulation Hebei University of Economics

Modern cosmids ( pWE and sCos series) contain features such as:

(i) multiple cloning sites

(ii) Phage promoters flanking the cloning site;

(iii) Unique NotI, SacII or Sfil sites (rare cutters) flanking the cloning site to permit removal of the insert from the vector as single fragments. required.

Cosmids

• cosmid vectors are plasmids with ‘cos’ sequences • cos sequence permits in vitro packaging • infection produces colonies

Cosmids as the DNA carriers

Origin of replication

40kb-50kb

Colonies No plaques

Cosmid cloning vectors: 1.

Features of both plasmid and phage cloning vectors.

2.

Do not occur naturally; circular.

3.

Origin (ori) sequence for E. coli.

4.

Selectable marker, e.g. ampR.

5.

Restriction sites.

6.

Phage  cos site permits packaging into  phages and introduction to E. coli cells.

7.

Useful for 37-52 kb.

Fig. 7.7

Yeast Artificial Chromosome

Example of a yeast artificial chromosome (YAC) cloning vector. A YAC vector contains a yeast telomere (TEL) at each end, a yeast centromere sequence (CEN), a yeast selectable marker for each arm (here, TRP1 and URA3), a sequence that allows autonomous replication in yeast (ARS), and restriction sites for cloning.

Yeast Artificial Chromosomes (YACS) • replicates as linear chromosome in yeast • can incorporate 100 kb - >2 Mb of foreign DNA • vector contains: • bacterial ori and ampr • yeast centromere and ARS • ciliate telomere • yeast selectable marker

Cosmids, Phages, and plasmids as DNA carriers Cosmid

Phage

Plasmid

Host

Bacteria

Bacteria

Bacteria

Insert size

Up to 50kb

~ 12-20 kb

~ 1-8 kb

Entry into Cells

Infection

Infection

Transformation

Efficiency

Very efficient

Very efficient

Less efficient

Outcome

Multiply

Multiply and kill

Multiply

Appearance of infected cells

Colonies

Application

Genomic library

Plaques

Genomic library

Colonies

Cloning

Shuttle vectors: 1.

Capable of replicating in two or more types of hosts..

2.

Replicate autonomously, or integrate into the host genome and replicate when the host replicates.

3.

Commonly used for transporting genes from one organism to another (i.e., transforming animal and plant cells).

Example: *Insert firefly luciferase gene into plasmid and transform Agrobacterium.

*Grow Agrobacterium in large quantities and infect tobacco plant. http://cwx.prenhall.com/bookbind/ pubbooks/horton3/medialib/media_ portfolio/23.html

Cosmids as the DNA carriers Cosmids behave both as plasmids and as phages They contains the Cos ends from the  phage DNA Allow the DNA to be packaged into  phage heads Cosmids

They also contain a origin of replication from the plasmid so they replicate like plasmid in bacteria Host: E. coli Vector size: usually about 5-7 kb. Insert size: up to 50kb

Cosmids

Other Vectors • Yeast artificial chromosomes (YAC)

YAC vectors are only about 10 KB, but can carry 200-800 KB DNA sequences YAC has been developed and used for the human genome project

Yeast Artificial Chromosomes (YACs): Vectors that enable artificial chromosomes to be created and cloned into yeast. Features: 1. Yeast telomere at each end. 2.

Yeast centromere sequence.

3.

Selectable marker (amino acid dependence, etc.) on each arm.

4.

Autonomously replicating sequence (ARS) for replication.

5.

Restriction sites (for DNA ligation).

6.

Useful for cloning very large DNA fragments up to 500 kb; useful for very large DNA fragments.

5.2.3 BACs and PACs as alternatives to cosmids

5.2.3.1 a P1 vector system

5.2.3.2 BAC system (bacterial artificial chromosome)

5.2.3.3 PAC (artificial chromosome)

Principles of Gene Manipulation Hebei University of Economics

5.2.3.1 The phage P1 vector system has a capacity to clone DNA as large as 100 kb .

about twice capacity of cosmid less than that of yeast artificial chromosome (YAC) .

Phage P1  a temperate bacteriophage used for genetic analysis of E. coli , mediate generalized transduction.

Compositions (2)

necessary for in vitro packaging

contains a packaging site (pac) Contain two loxP sites. recognized by the phage recombinase

The P1 vector Ad10

digested

long arms

The phage P1 vector system. cleaved at the pac site by pacase

unite

DNA-replicates using plasmid replicon

Cre recombinase circularizes DNA at the loxP sites.

Plasmid copy number is increased by induction of P1 lytic replicon.

P1 clones feature Clones are maintained in E. coli as low-copy-number plasmids

A high copy number can be induced acting on the P1 lytic replicon

Usage be used to construct genomic libraries ( mouse, human, fission yeast and Drosophila DNA) .

5.2.3.2 BAC system (bacterial artificial chromosome)

Bacterial cloning system for mapping and analysis of complex genomes

based on the single-copy sex factor F of E. coli.

unique cleavage sites for excision of inserts.

cloning sites

cleavage sites for λ terminase

P1 λ cleavage sites P1 cre protein

To generate RNA probes Chloramphenicol resistance marker

maintain the copy number at a level of one or two per genome.

Structure of a BAC vector derived from a mini-F plasmid. pBAC108L Principles of Gene Manipulation Hebei University of Economics

The oriS and repE genes mediate the unidirectional(单向） replication of the F factor,

Compositions

from λ

from P1

includes cos N and lox P sites,

two cloning sites (HindIII and BamHI) several G+C restriction enzyme sites

(SfiI, NotI to facilitate the gene excision)

The cloning site is flanked by T7 and SP6 promoters

feature BAC can be transformed into E. coli very efficiently, and avoiding the packaging extracts

required with the P1 system. with a high degree of stability BACs usages  be capable of maintaining human and plant genomic fragments of greater than 300 kb for over 100 generations. been used to construct genome libraries with an average insert size of 125 kb

defect The first BAC vector, pBAC108L, lacked a selectable marker for recombinants.

clones with inserts be identified by colony hybridization.

Two widely used BAC vectors, pBeloBAC11 and pECBAC1, are derivatives of pBAC108L

Varieties of BAC vectors pECBAC1 has only the EcoRI site in the lacZ gene.

Two widely used BAC vectors: pBeloBAC11 and pECBAC1, are derivatives of pBAC108L

pBeloBAC11 has two EcoRI sites, one in the lacZ gene and one in the CMR gene,

in which the original cloning site is replaced with a lacZ gene carrying a multiple cloning site

Further improvements to BACs have many.

5.2.3.3 Artificial chromosome (PAC)

developed a P1-derived artificial chromosome (PAC)

combining features of the P1 and the F-factor systems.

PAC vectors are able to handle inserts in the 100– 300 kb range.

A self-replicating DNA molecule that transfers a DNA segment between host cells.

  

A cloning site (multi-clone site) A replicator (origin of replication) A selectable marker

Vector

Targeting Fragment Length

Plasmid

0~10 kb

Replacement λ vector

9~23 kb

Insertion λ vector

0~10 kb

Cosmid

33~44 kb

P1 phage

70~100 kb

PAC

130~150 kb

BAC

300 kb

YAC

0.2~2.0 Mb

Bacterial Artificial Chromosomes (BACs): Vectors that enable artificial chromosomes to be created and cloned into E. coli. Features:

1.

Useful for cloning up to 200 kb, but can be handled like regular bacterial plasmid vectors.

2.

Useful for sequencing large stretches of chromosomal DNA; frequently used in genome sequencing projects.

3.

Like other vectors, BACs contain: 1.

Origin (ori) sequence derived from an E. coli plasmid called the F factor.

2.

Multiple cloning sites (restriction sites).

3.

Selectable markers (antibiotic resistance).

PACs and BACs • PACs - P1-derived Artificial • BACs - Bacterial Artificial Chromosomes Chromosomes • E. coli bacteriophage P1 is • These chimeric DNA similar to phage lambda in that molecules use a naturallyit can exist in E. coli in a occurring low-copy number prophage state. bacterial plasmid origin of • Exists in the E. coli cell as a replication, such as that of plasmid, NOT integrated into the F-plasmid in E. coli. E. coli chromosome. • Can be cloned as a plasmid • P1 cloning vehicles have been constructed that permit cloning in a bacterial host, and its of large DNA fragments- few natural stability generally hundred kb of DNA permits cloning of large • Cloning and propogation of the pieces of insert DNA, i.e. up chimeric DNA as a P1 plasmid to a few hundred kb of DNA. inside E. coli cells

RETROVIRAL VECTORS • Retroviral vectors are used to introduce new or altered genes into the genomes of human and animal cells. • Retroviruses are RNA viruses. • The viral RNA is converted into DNA by the viral reverse transcriptase and then is efficiently integrated into the host genome • Any foreign or mutated host gene introduced into the retroviral genome will be integrated into the host chromosome and can reside there practically indefinitely. • Retroviral vectors are widely used to study oncogenes and other human genes.

Types of expression systems • Bacterial: plasmids, phages • Yeast: expression vectors: plasmids, yeast artifical chromosomes (YACs) • Insect cells: baculovirus, plasmids • Mammalian: – viral expression vectors (gene therapy): • • • •

SV40 vaccinia virus adenovirus retrovirus

– Stable cell lines (CHO, HEK293)

EXPRESSION VECTORS • Allows a cloned segment of DNA to be translated into protein inside a bacterial or eukaryotic cell. • Vectors will contain the ff: • (a) in vivo promoter • (b) Ampicillin selection • (c) Sequencing primers

EXPRESSION VECTORS • Produces large amounts of a specific protein • Permits studies of the structure and function of proteins • Can be useful when proteins are rare cellular components or difficult to isolate

Common problems with bacterial expression systems • Low expression levels: ▪ change promoter ▪ change plasmid ▪ change cell type ▪ add rare tRNAs for rare codons on second plasmid • Severe protein degradation: – use proteasome inhibitors and other protease inhibitors – try induction at lower temperature • Missing post-translational modification: co-express with kinases etc. • Glycosylation will not be carried out: – use yeast or mammalian expression system • Misfolded protein (inclusion bodies): – co-express with GroEL, a chaperone – try refolding buffers

REPORTER GENE VECTORS • A gene that encodes a protein whose activity can be easily assayed in a cell in which it is not normally expressed • These genes are linked to regulatory sequences whose function is being tested • Changes in transcriptional activity from the regulatory sequences are detected by changes in the level of reporter gene expression

SHUTTLE VECTORS • Shuttle vectors can replicate in two different organisms, e.g. bacteria and yeast, or mammalian cells and bacteria. • They have the appropriate origins of replication. • Hence one can clone a gene in bacteria, maybe modify it or mutate it in bacteria, and test its function by introducing it into yeast or animal cells.

compare Vector

plasmid

phage

cosmid

phasmid

Origin

bacteria

Plasmid+cos site of λ

Plasmid+phage

Composition

Double strand DNA ori region

double-stranded DNA virus 1 cos site 2 origins of viral and complementary strands

plasmid replicon and cos site of λDNA

Plasmid origins and phage origins of replication

Feature

segregative instability Incompatibility

1single-stranded vectors 2 transfect 3 RF and singlestranded 4 plaques / infected colonies

1 Packing in vitro 2 Function as plasmid in host

low molecular weight, High cloning capacity, High genetic stability, clone single and double strand DNA Need no package

Usage

Clone/ expression

sequencing Mutagenesis probe

cloning in vitro packaging libraries of eukaryotic genome

Cloning Sequencing expression libraries

Shortcoming

Cloning gene smaller

Cloning gene smaller

Self-link duplicated vector

Example

pUC18/pUC/9/pB R322

M13, f1 and fd

attention

Par gene

Packaging in vivo

pUC118 /119 pBluescriptSK(+/-) BACs and PACs cos sites substrates for the packagingdependent cleavage

combines many different features Some contain pac gene

5.2.4 Choice of vector （two point to consider) The size of insert ( the only importance feature). The absence of chimeras and deletions (more important).

Maximum DNA insert with different cloning vectors.

advantages of the BAC and PAC over YACs

lower levels of chimerism Construct library easily

manipulate and isolate inserted DNA easily.

BAC clones is far more faithful than YAC or cosmid clone. BAC clones be suited for sequence analysis,

5.3 Specialist-purpose vectors 5.3.1 Vectors to make single-stranded DNA 5.3.2 vectors for expression 5.3.3 Vectors for making RNA probes 5.3.4 Vectors for maximizing protein synthesis

5.3.5 Vectors to facilitate protein purification 5.3.6 Vectors to promote solubilization of expressed proteins

5.3.7 Vectors to promote protein export

5.3.1 Vectors used to sequence single-stranded DNA

The methods to sequence a new gene or a novel genetic construct.

an M13 vector single-stranded DNA was obtained

a pUC-based phagemid vector（pUC118)

contains M13 ori and pUC (Col E1) ori.

Single-stranded DNA can be produced by infecting cultures with a helper phage

The helper phage M13KO7 The helper phage (can replicate on its own).

+ a phagemid bearing a wild-type origin of replication

single-stranded phagemid is packaged preferentially secreted into the culture medium.

DNA purified from the phagemids can be used directly for sequencing

5.3.2 Expression vectors

if

to prepare RNA probes of the cloned gene to obtain large amounts of gene product.

In either case, transcription of the cloned gene is required.

Expression vectors are required

Promoters selection to use the promoter of the cloned gene

This method is more usual

to utilize a promoter specific to the vector.

been optimized for binding of the E. coli RNA polymerase be regulated easily by changing the culture conditions.

Promoter structure common promoter consists of the −35 region (5'-TTGACA-) and the −10 region, or Pribnow box (5'-TATAAT).

The distance between the −35 and −10 regions is also important.

other bases flanking these regions can affect promoter activity

5.3.3 Vectors for making RNA probes

Methods

Method for preparing RNA probes from a cloned DNA using a phage SP6 promoter and SP6 RNA polymerase.

incubate four ribonucleoside triphosphates

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Principles of Gene Manipulation Hebei University of Economics

5.3.3.1 why using a phage promoter? (three reasons)

the promoters are very strong, so large amounts of RNA be made in vitro.

the phage promoter not recognized by the E. coli RNA polymerase and no transcription occur inside the cell. E. coli RNA

the RNA polymerases encoded by phages such as SP6, T7 and T3 are much simpler molecules to handle than the E. coli enzyme, RNA

5.3.3.2 make probes corresponding to both strands? to use a cloning vector, in which the insert is placed between two different opposing phage promoters.

each promoters is recognized by different RNA polymerase, the transcription direction is determined by the used polymerase.

5.3.4 Vectors for maximizing protein synthesis

to study its property be commercial value,

detectable synthesis is not sufficient  must be maximized.

Factors affecting the expression of cloned genes.

The effects of over expression on the host cell

Many gene products can be toxic to the host cell

high-level synthesis exerts a metabolic drain on the cell.

Examples : surface structural protein proteins that regulate basic cellular metabolism

Over expression leads to slower growth and enriched variants, with lower or no expression of the cloned gene

To minimize the problems associated with high-level expression, it is usual to use a vector

the cloned gene is under the control of a regulated promoter.

Many different vectors have been constructed for regulated expression of gene inserts

most of those contain λPL , T7, trc (tac) or BAD.

The pET vectors-----

a family of expression vectors

utilize phage T7 promoters to regulate synthesis of gene products

compatible Constitutive

Strategy for regulatingPrinciples the expression of genes cloned into of Gene Manipulation Hebei University of Economics pET vector

5.3.5 Vectors to facilitate protein purification

the expressed protein be fused to another protein, called a tag.

to facilitate protein purification 5.3.5.1 Tags varieties：

glutathione- S-transferase, the MalE (maltose-binding) protein multiple histidine residues,

5.3.5.2 Rules to construct the tag vectors

Tag gene

cleave gene

Target gene

The coding sequence for an amino acid

After purification, the tag protein can be cleaved off with the specific protease to leave a normal or nearly normal protein.

（cleaved by a specific protease）

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Principles of Gene Manipulation Hebei University of Economics

Strategy 1 To use a polyhistidine fusion for purification

proteolytic cleavage site

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Structure a vector (pBAD/His） Hebeiof University of Economics

steps

the gene of interest is inserted into a vector

induce the synthesis of the fusion protein

the cells lyse

the viscosity of the lysate is reduced by nuclease treatment.

The lysate is applied to a column (containing immobilized divalent nickel)

selectively binds the polyhistidine tag.

Elution---wash away any contaminating proteins

the fusion protein is eluted from the column

treated with enterokinase to release the cloned gene product.

Strategy 2 Purification of a gene product fused to the biotin carboxylase carrier (tag).

Principles of Gene Manipulation Hebei University of Economics

Principles of Gene Manipulation Hebei University of Economics

5.3.6 Vectors to promote solubilization of expressed proteins

One of the problems associated with the overproduction of proteins in E. coli is the production of the insoluble aggregates or „inclusion bodies‟

Inclusions of Trp polypeptide–proinsulin fusion protein in E. coli.

How to prevent the formation of inclusion body 1 regulate the culture temperature and growth rate

Lowering the growth temperature to increases the yield of correctly folded, soluble protein

Media compositions and pH values to reduce the growth rate, decrease the formation of inclusion body.

2 three ‘genetic’ methods)

the host cell is engineered to overproduce a chaperon in addition to the protein of interest.

Even with excess chaperon there is no guarantee of proper folding. making minor changes to the amino acid sequence of the target protein.

many proteins （produced as insoluble aggregates in their native state） are synthesized in soluble form as fusion proteins

Example the interest gene is cloned into an MCS and the gene product is a thioredoxin fusion protein, with an enterokinase cleavage site at the fusion point.

After synthesis, the fusion protein is released from the host cells by osmotic shock and purified.

The desired protein is released by enterokinase cleavage.

5.3.7 Vectors to promote protein export Many natural signal sequences support the efficient translocation of heterologous peptides across the inner membrane

cytoplasmic protein

Secreted proteins

Secreted proteins may be released into the periplasm or integrated into or transported across the outer membrane.

How to ? Strategy to make a protein secreted

to modify proteins, so they are transported through the outer membrane and secreted into the growth medium.

to express recombinant proteins on the surface of the bacterial cell, using one of the carrier proteins （surface display)

Sac I Not I Xba I

Ba m HI

Kpn I Apa I Xho I Sal I Amp r 1Lac Za Xba I ilv2 Bam HI ADH1T P lac pMGI6 8610bp Kpn I GLA ilv2

EcoRI Aat II Eco RI HindIII

MFa 1S PGK1 P Eco RI

Bam HI Pst I

5.4 vectors with combinations of features

Many of the vectors have combinations of the features

Example 1

LITMUS vectors are used for the generation of RNA probes.

Example 2

the PinPoint series of expression vectors

5.4.1 LITMUS vectors---used to generate RNA probe.

LITMUS vectors exhibit the following features:(5)

The polylinkers are located in the lacZα gene and inserts in the polylinker prevent α-complementation.

Blue/white screening can be used to distinguish clones with inserts from those with no insert.

polylinkers contain 32 unique restriction sites.

carry 2 ori regions （pUC and M13） Normally vector replicates as a plasmid,

On infection with helper phage (M13KO7), single-stranded molecules are produced and packaged in phage protein.

The single-stranded molecules have all the features necessary for DNA sequencing

The vectors are small (< 3 kb) and have a high copy number.

5.4.2 the PinPoint series of expression vectors

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These vectors have the following features （6）

Expression is under the control of either the T7 or the tac promoter.

Some carry a DNA sequence specifying synthesis of a signal peptide.

an MCS adjacent to a factor-Xa cleavage site.