Genomic Host

5. SEL INANG DAN VEKTOR

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Gene (s) cloning

Vector

utilizes

Host’s cells

requires

Procaryotic Eucaryotic

• E. coli• B. subtilis

• S. cerevisiae• Sel tanaman• Sel hewan

Inserted cloned gene

• Transformatiom• Transfection• Packaging in

vitro• Microinjection• Biolistic

• An origin of replication

• Selectable marker(s)

• Single restriction site

• Multiple cloning site

• Expression signals

Inserted to the host

Plasmids Phages Cosmids Phagemids Yeast

artificial chromsomes

Viruses

Circular DNA smolecule

Specifically set up

M13

Insertionvectors

• λgt10• Charon

16A

Replacementvectors

• EMBL4• Charon 40

n/or

including

Specific method

Could haveMust have

thorugh

Can be

Add type

Such as

dua tipeyang

seperti seperti

Main types

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Learning Outcome (LO)LO 42: menjelaskan sel inang yang digunakan dalam kloning gen

LO 43: menjelaskan vektor yang digunakan dalam kloning gen

LO 44: menjelaskan sel inang prokariotik

LO 45: menjelaskan sel inang eukariotik

LO 46: menjelaskan vektor plasmid yang digunakan dalam sel iang E. coli

LO 47: menjelaskan vektor bakteriofaga yang digunakan dalam sel inang E. coli


LO 49: menjelaskan vektor yang digunakan dalam sel inang eukariotik

LO 50: menjelaskan kromosom artifisial

LO 51: menjelaskan transformasi

LO 52: menjelaskan transfeksi

LO 53: menjelaskan metode mikroinjeksi dan biolistik3

Three steps to do for isolating gene from recombinanDNA sequences : The molecules must be physically separated fully Recombiant sequences must be mulptiplied for

futher analysis The targetted fragment must be selected using a

method based on its specific bases. Gene cloning steps are always related with selection

and utilization of : vector (carrier) Host-cells (to multiply the vectors).

Host’s cell and vector

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Host types

LO 42: menjelaskan sel inang yang digunakan dalam kloning gen

Host types depend on the cloning purposes: Isolating gene for structure analysis: simple type Expressing genetic information of the higher

eucaryotics like plant: use the more specific one

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LO 42: menjelaskan sel inang yang digunakan dalam kloning gen

Host types

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Ideal hosts must be: Easy to be handled for propagation Has many strains Could accept various vectors

Escherichia coli is an ideal host Others like :

Bacillus Pseudomonas Streptomyces

Procaryotic hosts

LO 44: menjelaskan sel inang prokariotik7

However, E. coli has some diasadvantages : Particular eucaryotic gene might not function in E. coli Procaryotic host cells might not produced full function

eucaryotic protein .

Eucaryotic hosts:Yeast (S. cerevisiae)Aspergillus nidulansNeurospora crassaAlgae (Chlamydomonas reinhardtii )Plant cellsAnimal cells

5.1.2 Eucaryotic hosts

LO 45: menjelaskan sel inang eukariotik8

LO 43: menjelaskan vektor yang digunakan dalam kloning gen

Type vector

Plasmid Bakteriofaga (faga) Others Vectors

Vector

Small size Has a point for replicatiion (ori) Selection points Has more than one restriction

site

Important charaters

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In nature they can be found in bacteria and yeasts. DNA molecule is circular and extrachromosomal. May cause antibiotic resistance. The antibiotic resistance genes are coded by the DNA

plasmid (pDNA) and are used for constructing vedtor Nomenclature:

P = plasmid, preceeded by the founder (s) Numerical number to classify specific isolate. pBR322 founded by Francisco Bolivaret al., isolate

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Plasmids

LO 46: menjelaskan vektor plasmid yang digunakan dalam sel iang E. coli10

Note: Antibiotic abbreviations are as follows: Ap, ampicillin; Cm, chloramphenicol;Km, kanamycin; Sm, streptomycin; Sn, sulphonamide; Tc, tetracycline. E1imm andDF13imm represent immunity to the homologous but not to the heterologouscolicin. Thus plasmid ColE1 is resistant to the effects of its own colicin (E1), but notto colicin DF13. Copy number is the number of plasmids per chromosomeequivalent. r, resistance.


Features of pBR322 plasmid Light in molecular weight Have antibiotics resistance genes Has the point of origin of replication (ori) Some have single sites for specific

restriction enzyme


Plasmid pBR322

Fig. 5.1. Map of plasmid pBR322. Important regions indicated arethe genes for ampicillin and tetracycline resistance (Apr and Tcr) andthe origin of replication (ori). Some unique restriction sites are given.The hatched region shows the two fragments that were removedfrom pBR322 to generate pAT153.

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Has an ori Has resistance gene for

ampisilin Has lacZ gene (fragment -

peptide of -galactosidase); a specific gene for screening recombinant-gene using an X-gal substrate

In the lacZ gene, it haspolylinker or multiple cloning sites (MCS)


Plasmid pUC18

Feature of pUC18 plasmid

Fig. 5.2. Map of plasmid pUC18.

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Note: There are hundreds of variants available from many different suppliers. A good sourceof information is the supplier’s catalogue or website. Apr–ampicillin resistance; Tcr–tetracycline resistance; Neor–neomycin resistance (selection using kanamycin in bacteria,G418 in mammalian cells); MCS – multiple cloning site; SP6/T7 are promoters for in vitrotranscription; lacZ – -galactosidase gene; CMV – human cytomegalovirus. The terms pGEM,pCI and pCMV-Script are trademarks of the suppliers.


It is able to inject DNA molecule to bacteria Both phages, dan M13,are commonly used in cloning.

Bacteriophages (Faga)


Fig. 5.3. Structure of bacteriophages and M13. Fag memiliki kapsid atau kepala yang membungkus genom DNA beruntai ganda. Daerah ekor diperlukan untuk adsorpsi ke sel inang. M13 memiliki struktur yang lebih sederhana, dengan genom DNA beruntai tunggal yang dibungkus dalam mantel (coat) protein.

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Bakteriofaga This type of phage is used for vector cloning as not all genomes of

this organisms is important for its function.

Fig. 5.4. Peta genom faga . Beberapa gen ditunjukkan.Daerah fungsional ditunjukkan oleh garis horizontal.Daerah non-esensial (blok hitam) dapat dimanipulasidalam konstruksi vektor.

The genome length is = 48.5 kb, consits of 46 genes.

Both ends are linear with single strand which consist of (12 bases) and is known as cos site.

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(a) insertion vector is shown Fig 5.7. has single restriction site (RS). To obtain recombinant DNA the DNA fragment is inserted to this site. The fragment size which will be inserted is determined bby the difference size between vector size and maximum fragment size.

(b) A replacement vector . Has two restriction sites (RS) which is separated by an area called as fragmen stuffer. The phage genome is then replaced by DNA fragment (ii). This might then possibly to do a cloning using larger DNA molecule.

Bacteriophage divided into two main classes:(i) Insertion vector ( gt10 and Charon 16A)(ii) Substituon vector ( EMBL4 and Charon 40)


Fig. 5.7. Insertionand replacement phage vectors

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LO 48: menjelaskan vektor bakteriofaga yang digunakan dalam sel inang E. coli18

Genome M13:circular single strand DNA 6407 bases.

Injected only E. coli that has F-pili. When the DNA enters the cell, the

single strand DNA will then be changed to double strands DNA which is known is replicative (RF).

Is produced and excluded from the cell as M13 particles.


Bacvteriophage M13

Fig. 5.10. Map of the filamentous phage vector M13mp18. The double-stranded replicativeform is shown. The polylinker region (MCS) is the same as that found in plasmid pUC18 (Fig.5.2). Genes in the REP region encode proteins that are important for DNA replication. The CAPand MOR regions contain genes that specify functions associated with capsid formation andphage morphogenesis, respectively. The vector M13mp19 is identical except for theorientation of the polylinker region.

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Cosmid: a combination of plasmid cos fag site. 4-6 kb in size, therefore it can accomodate DNA

fragment at 47 kb. Has a plasmid character like E. coli. Cloning capacity is tice larger than vector ,

(replacement vector). Phagemid (phasmid): vector hybrid plasmid/faga f1

Like: ZAP pEMBL9 pBluescript

Cosmid and Phagemid (phasmid)


A range of vectors for use in yeast cells has been developed, withthe choice of vector depending on the particular application. Yeast episomal plasmids (YEps) are based on the naturally

occurring yeast 2 m plasmid, and may replicate autonomouslyor integrate into a chromosomal location.

Yeast integrative plasmids (YIps) are designed to integrate intothe chromosome in a similar way to the YEp plasmids,

Yeast replicative plasmids (YRps) and remain as independentplasmids and do not integrate.

Yeast centromere plasmids (YCps) contain sequences fromaround the centromeric region of chromosomes and thesebehave essentially as minichromosomes.

Vector which is commonly used in eucaryotic cell

LO 49: menjelaskan vektor yang digunakan dalam sel inang eukariotik24

The aims of genetic engineering in higher eukaryotes are twofold:(i) to introduce recombinant DNA into plant and animal cells in

tissue culture, for basic research on gene expression or forthe production of useful proteins

(ii) to alter the genetic makeup of the organism and produce atransgenic, in which all the cells will carry the geneticmodification.

Vectors used for plant and animal cells may be introduced intocells directly by techniques viruses or other infectious agents such as Agrobacteria.

Some examples of the types of system that have been used in thedevelopment of vectors for plant and animal cells are given inTable 5.5.


The development of vectors for cloning very large pieces of DNAwas essential to enable large genome sequencing projects.

YACs has enabled DNAfragments in the megabaserange to be cloned

YACs have centromeric andtelomeric regions, and therecombinant DNA is thereforemaintained essentially as ayeast chromosome.


Yeast artificial chromosomes (YACs

Fig. 5.11. Map of the yeast artificial chromosome vector pYAC2

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BACs are based on the F plasmid, which is much larger than thestandard plasmid cloning vectors, and therefore offers thepotential of cloning larger fragments.

BACs can accept inserts of around 300 kb, and many of theinstability problems of YACs can be avoided by using thebacterial version.

Much of the sequencing of the human genome has beenaccomplished using a library of BAC recombinants.


Bacterial artificial chromosomes (BACs)

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Manipulation of vector and insert DNAs to producerecombinant molecules is carried out in the test-tube,and we are then faced with the task of getting therecombinant DNA into the host cell for propagation.

The efficiency of this step is often a crucial factor indetermining the success of a given cloning experiment,particularly when a large number of recombinants isrequired.

The methods available depend on the type ofhost/vector system, and range from very simpleprocedures to much more complicated and esotericones.

To the host’s cell

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Transformation: the uptake of plasmid DNA The cells need to be made competent.

by soaking the cells in an ice cold solution of calcium chloride Transformation of competent cells is carried out

by mixing the plasmid DNA with the cells incubating on ice for 20–30 min giving a brief heat shock (2 min at 42 °C)

The transformed cells are usually incubated in a nutrient broth at37 °C for 60–90 min to enable the plasmids to become establishedand permit phenotypic expression of their traits.

The cells can then be plated out onto selective media forpropagation of cells harbouring the plasmid

transformation frequencies of around 106 or 107 transformantsper microgram are more often achieved in practice.

Transformation

LO 51: menjelaskan transformasi30

LO 51: menjelaskan transformasi31

Transfection: the uptake of phage DNA. introduction of phage

DNA into E. coli cells require a packaging in vitro

To enable packaging in vitro, the components of the capsid, and the endonuclease, must be available.

Transfection

LO 52: menjelaskan transfeksi

Fig. 5.12. Phage DNA and packaging(a) A concatemeric DNA molecule composed of wild-type phage DNA (+). The individual genomes are joined at the cos sites. (b) Recombinant genomes ( rec) are shown being packaged in vitro.

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An alternative to transformation procedures is to introduce DNAinto the cell by some sort of physical method.

One way of doing this is to use a very fine needle and inject theDNA directly into the nucleus.

This technique has been usedsuccessfully with both plant andanimal cells.

The cell is held on a glass tubeby mild suction and the needleused to pierce the membrane.

The technique requires amechanical micromanipulatorand a microscope, and plenty ofpractice!

Microinjection

LO 53: menjelaskan metode mikroinjeksi dan biolistik

Fig. 5.13. Microinjection of a protoplast-derived potato cell

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The technique involves literally shooting DNA into cells

Biolistic

LO 53: menjelaskan metode mikroinjeksi dan biolistik

• The DNA is coated onto microprojectiles, which are accelerated by the macroprojectile on firing the gun.

• At the stop plate the macroprojectile is retained in the chamber and the microprojectiles carry on to the target tissue.

Fig. 5.14. Biolistic apparatus

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Kloning Gen

Vektor

menggunakan

Sel inang

memerlukan

Prokariot Eukariot

• E. coli• B. subtilis

• S. cerevisiae• Sel tanaman• Sel hewan

Memasukan DNA terklon

• Transformasi• Transfeksi• Packaging in

vitro• Mikroinjeksi• Biolistic

• An origin of replication

• Selectable marker(s)

• Single restriction site

• Multiple cloning site

• Expression signals

dimasukkan dalam sel inang

Plasmids Phages Cosmids Phagemids Yeast

artificial chromsomes

Viruses

molekul DNA sirkuler

dibuat secara spesifik

M13

Insertionvectors

• λgt10• Charon

16A

Replacementvectors

• EMBL4• Charon 40

dan

meliputi

seperti seperti

memerlukan metode untuk

seperti

dapat mempunyai

harus mempunyai

dengan cara

dan dapat

tipe lain

berupaseperti

dua tipeyang

seperti seperti

dua tipe

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Genomic Host

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