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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
Plasmid pBR322
pBR322
Contains:
1. colE1 origin of replication (ORI)
RNAse H processing
RNA II
-100 ori
100
600
rop gene
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:
2. Selectable Markers:
Ampicillin Resistance (-lactamase gene) and Tetracycline Resistance (tet gene)
Bacteria plus plasmid
Non-transformed bacteria
pBR322
Contains:
3. A few good restriction sites for inserting foreign DNA
BamH1 BamH1
PstI
Eco RI
Bam HI Bam HI
PstI
Eco RI
Bam HI
and ligate
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
pUC Plasmids
Advantages over pBR322
3. Multiple cloning site
pUC19 MCS
pUC Plasmids
Advantages over pBR322
4. Easier Selection AmpR & blue/white selection
Blue/White Selection
Based on the enzymatic reaction of galactosidase.
Blue/White Selection
Modified E. coli codes for the carboxyl portion of -galactosidase enzyme. This portion alone cannot cleave X-gal. Plasmid codes for amino portion (LacZ or peptide) of -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 complementation.
Blue/White Selection
Bacteria plus empty plasmid Bacteria with plasmid plus insert Non-transformed bacteria
Overnight
growth
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
Amp resistance gene still present (= beta lactamase), Tet resistance gene omitted
Replicon
pMB1
pMB1 p15A pSC101 Col E1
Copy number
15-20
500-700 10-12 About 5 15-20
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
Origin sequence
*DNA ligase
Some features of pUC19: 1. 2. 3. 4. 5. 6. 7. High copy number in E. coli, ~100 copies/cell, provides high yield. Selectable marker is ampR. Ampicillin in growth medium prevents growth of all other E. coli. Cluster of restriction sites called a polylinker occurs in the lacZ (galactosidase) gene. Cloned DNA disrupts reading frame and -galactosidase production. Add X-gal to medium; turns blue in presence of -galactosidase. Plaque growth: blue = no inserted DNA and white = inserted DNA. Some % of digested vectors will reanneal with no insert. Remove 5 phosphates with alkaline phosphatase to prevent recircularization (this also eliminates some blue plaques). Plasmids are transformed into E. coli by chemical incubation or electroporation (electrical shock disrupts the cell membrane). Cloned inserts >5-10 kb typically are unstable; good for <10kb.
8. 9.
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 bluewhite color selection test described in the text can be used to select for vectors with or without inserts.
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
-complementation
The portion of the lacZ gene encoding the first 146 amino acids (the -fragment) are on the plasmid The remainder of the lacZ gene is found on the chromosome of the host. If the -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 galactosidase enzyme.
MCS
plus f1 ori (single stranded DNA) T7 & SP6 Promoters (RNA production)
2pBluescript -phasmids pBluescriptSK(+/-)properties ithere are T3 and T7 phage promoters to regulate the foreign gene transcription near the MCS
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
Expression Vector
An expression vector for fusions. This is a phagemid, can be propagated either as a plasmid or as a phage
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. 2. Engineered version of bacteriophage (infects E. coli). Central region of the chromosome (linear) is cut with a restriction enzyme and digested DNA is inserted.
3.
4. 5. 6. 7.
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
Replacement Vector
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
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
N = ln(1-p)/ln(1-n)
N = # of recombinants to examine p = probability of detecting clone n = average size of insert/genome size
Vector
plasmid insertion replacement cosmid YAC
Mammals
108 109 1010 1011
105
106
107
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
X-gal: 5-bromo-4chloro-3-indolyl--Dgalactopyranoside
Cosmid vectors
termed cosmids
To a cosmid vector of 5 kb
much more than a phage- vector can accommodate.
Multiplying Process
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.
Cosmids characteristics
Circulated like phage cannot make cell lysis Containing plasmid replicon and cos site of DNA
are particularly attractive vectors for constructing libraries of eukaryotic genome fragments.
Existed problems
Solution
Solutions
Modern cosmids ( pWE and sCos series) contain features such as:
(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
Origin of replication
40kb-50kb
Colonies No plaques
Cosmid cloning vectors: 1. 2. 3. 4. 5. 6. 7. Features of both plasmid and phage cloning vectors. Do not occur naturally; circular. Origin (ori) sequence for E. coli. Selectable marker, e.g. ampR. Restriction sites. Phage cos site permits packaging into phages and introduction to E. coli cells. Useful for 37-52 kb.
Fig. 7.7
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.
Phage
Bacteria
Plasmid
Bacteria
Up to 50kb
Infection Very efficient Multiply Colonies
~ 12-20 kb
Infection Very efficient Multiply and kill Plaques
~ 1-8 kb
Transformation Less efficient Multiply Colonies
Application
Genomic library
Genomic library
Cloning
Shuttle vectors: 1. 2. Capable of replicating in two or more types of hosts.. 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.
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. 3. 4. 5. 6. Yeast centromere sequence. Selectable marker (amino acid dependence, etc.) on each arm. Autonomously replicating sequence (ARS) for replication. Restriction sites (for DNA ligation). Useful for cloning very large DNA fragments up to 500 kb; useful for very large DNA fragments.
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)
contains a packaging site (pac) Contain two loxP sites. recognized by the phage recombinase
digested
long arms
unite
Usage be used to construct genomic libraries ( mouse, human, fission yeast and Drosophila DNA) .
cloning sites
P1
The oriS and repE genes mediate the unidirectional( replication of the F factor,
Compositions
from
from P1
two cloning sites (HindIII and BamHI) several G+C restriction enzyme sites
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.
Two widely used BAC vectors, pBeloBAC11 and pECBAC1, are derivatives of pBAC108L
Varieties of BAC vectors Two widely used BAC vectors: pBeloBAC11 and pECBAC1, are derivatives of pBAC108L pECBAC1 has only the EcoRI site in the lacZ gene.
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
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.
P1 phage
PAC BAC
70~100 kb
130~150 kb 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.
2.
Useful for cloning up to 200 kb, but can be handled like regular bacterial plasmid vectors.
Useful for sequencing large stretches of chromosomal DNA; frequently used in genome sequencing projects.
3.
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.
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
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 Origin Composition plasmid bacteria Double strand DNA ori region phage double-stranded DNA virus 1 cos site 2 origins of viral and complementary strands cosmid Plasmid+cos site of plasmid replicon and cos site of DNA phasmid Plasmid+phage Plasmid origins and phage origins of replication
Feature
low molecular weight, High cloning capacity, High genetic stability, clone single and double strand DNA Need no package
Cloning Sequencing expression libraries
Usage
Clone/ expression
Shortcoming Example
pUC118 /119 pBluescriptSK(+/-) BACs and PACs cos sites substrates for the packagingdependent cleavage combines many different features Some contain pac gene
attention
Packaging in vivo
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).
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
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
if
to prepare RNA probes of the cloned gene to obtain large amounts of gene product.
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).
Methods
Method for preparing RNA probes from a cloned DNA using a phage SP6 promoter and SP6 RNA polymerase.
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 detectable synthesis is not sufficient must be maximized.
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
Many different vectors have been constructed for regulated expression of gene inserts
compatible Constitutive
Strategy for regulatingPrinciples of Gene Manipulation genes cloned into the expression of Hebei University of Economics pET vector
Tag gene
cleave gene
After purification, the tag protein can be cleaved off with the specific protease to leave a normal or nearly normal protein.
Strategy 1
To use a polyhistidine fusion for purification
steps
Strategy 2
Purification of a gene product fused to the biotin carboxylase carrier (tag).
One of the problems associated with the overproduction of proteins in E. coli is the production of the insoluble aggregates or inclusion bodies
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.
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.
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)
Ba m HI
EcoRI
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 MFa 1S PGK1 P Eco RI Bam HI Pst I
Example 1
Example 2
The polylinkers are located in the lacZ gene and inserts in the polylinker prevent -complementation.
Blue/white screening can be used to distinguish clones with inserts from those with no insert.
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.