Lecture 8 Gene Cloning-Cloning Vector

7/23/2019 Lecture 8 Gene Cloning-Cloning Vector

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Lecture 8:

Vectors based on the lambda

bacteriophage, cosmids and supervectors


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Revision: Plasmid Vectors

Convenient for cloning of small DNA fragmentsfor restriction mapping and for studyingregulatory regions

Relatively small insert capacity

Handling and storage of these clones is time-

consuming and difficult (the repeatedsubcultures of recombinants may result indeletions in the inserts)


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lasmid vectors can be of three main types!1. General purpose cloning vectors"

Cloning of foreign DNA fragments in general-purposecloning vectors selectively inactivates one of the

mar#ers (insertional inactivation) or derepresses asilent mar#er (positive selection) so as to differentiatethe recombinants from the native phenotype of thevector

2. Expression vectors DNA to be cloned and e$pressed is inserted

do%nstream of a strong promoter present in thevector

Revision: Plasmid Vectors


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3. Promoter probe and terminator probe

vectors &seful for isolation of regulatory se'uences such

as promoters and terminators and for studyingtheir recognition by a specific host


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Cloning Vectors Based On

Lambda Bacteriophage


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"Lambda biology

he lambda virus particle contains a linear DNAof *+", #b %ith a single stranded , e$tensionof ./ bases at both ends %hich are

complementary to each other (called cohesiveends or cos)

During infection0 the right , e$tension (cosR)0follo%ed by the entire genome0 enters the hostcell

1oth of the cosends are ligated byE. coli ligase0forming a covalently circular DNA


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cohesive ends or

cos

cohesive ends or

cos

2ector based on bacteriophage lambda allo%efficient cloning of larger fragments0 %hich is

important in constructing gene libraries


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"Development o Lambda:

! "lternatives #odes." 3ytic Cycle!

After infecting the host0 the lambda genome may

start its replication4results in the formation ofmultiple copies of the genome

/" 3ysogeny

he phage genome may enter a dormant stage(prophage) by integrating itself onto a bacterialgenome by site-specific recombination5 during thisstage it is propagated along %ith the host in thesubse'uent progeny


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Development o Lambda:

! "lternatives #odes


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The lytic cycle:

is a productive cyclethat resulting in lysis of

theE. coli cell andliberation of a numberof phage particles

Thy lysogeny cycle:

he lambda genome isable to integrate into the

bacterial chromosome0

6here it can remain'uiescent for manygenerations0

1eing replicated along%ith the hostchromosome every time

the cell divides


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he proportion of infected cells going do%n each route isinfluenced by!

i" 7nvironmental conditionii" 8enetic composition of the phage and the host

he broad ob9ectives in constructing various phage vectors are!i" the presence of cloning sites only in the dispensable fragments

ii" the capacity to accommodate foreign DNA fragments of varioussi:es

iii" the presence of multiple cloning sites

iv" an indication of incorporation of DNA fragments by a change inthe pla'ue type

v" the ability to control transcription of a cloned fragment frompromoters on the vector

vi" the possibility of gro%ing vectors and clones to high yield

vii"easy and ready recovery of cloned DNA

viii"introduction of features contributing to better biological

containment


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To types of vector have been developed:

1. !nsertion "ectors:

i" Cloning of foreign DNA e$ploits the insertionalinactivation concept

ii" ossesses at least one uni'ue restriction site into

%hich ne% DNA can be inserted

iii" 7$ample! gt.; < =A>>


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>nsertion 2ectors


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2. #eplacement "ectors:

i" A large segment of the non-essential region (stuffer)has been deleted0 and the t%o arms ligated together

ii" %o recognition sites for the R7 used for cloning"

iii" hese sites flan# a segment of DNA that is replaced

by the DNA to be cloned"

iv" 7$ample! 67?"1 < 7@13*


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Replacement 2ectors


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Cloning $ith lambda vector

." 3igation of each arm and the DNA to be cloned

/" roduce concatamers! left arm ne% DNA right arm"

B" he concatamers are then added to an in vitro pac#aging mi$0

%hich contains all the proteins needed to ma#e a lambda phageparticle

*" hese proteins form phage particles spontaneously and placeinside the particles any DNA fragment that is bet%een B to ,/ #bin length and is flan#ed by cossites

," he pac#aging mi$ cuts left arm ne% DNA right armcombinations of B ,/ #b out of the concatamers and constructslambda phages around them

" >nfection ofE. coliprocess transports the vector plus ne% DNA

into the bacteria


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!n vitro

pac$aging

A concatameris a long continuousDNA molecule that contains multiplecopies of the same DNA se'uences

lin#ed in series


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" After infection0 the cells are spread onto an agar plate

+" roduce an even layer of bacteria across the entiresurface of the agar

E" 1acteria that %ere infected die %ithin about /; min

.;" Death and lysis of the bacterium releases ne% phages

into the surrounding medium0 %here they infect ne%cells and begin another round of phage replication andlysis

.." he end result is a :one of clearing0 called a pla%ue0

%hich is visible on the la%n of bacteria that gro%s on theagar plate

./" 3igation of the t%o arms %ithout insertion of ne% DNAresults in a molecule that is too short to be pac#aged


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&'()!*(:

"ector for longer pieces of

*+,


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&osmids - are plasmids containing lambda cos

ends * to #b in si:e and are specifically designed for

cloning large DNA fragments (*;-,; #b)

Cosmids have!i" Drug resistance mar#er

ii" A plasmid origin of replication

iii" A fragment carrying the ligated cohesive ends(cos) of phage lambda

iv" Fne or more uni'ue restriction sites for cloning


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A plasmid origin ofreplication

Drug resistancemar#er

A fragment carrying

the ligated cohesiveends (cos) of phagelambda

Fne or more uni'ue

restriction sites forcloning

%tructure o a cosmid


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?ub9ect the ligation mi$ture to in vitro pac#aging

(insert should be bet%een B/ and *, #b) Concatamers of cosmid molecules0 lin#ed at their cos

sites0 act as substrates for in vitro pac#aging

he cossite is the only se'uence that a DNAmolecule needs in order to be recogni:ed as aGlambda genome by the proteins that pac#age DNAinto lambda phage particles

articles containing cosmid DNA are as infective asreal lambda phages0 but once inside the cell cannotdirect synthesis of ne% phage particles and insteadreplicates as a plasmid


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(upervectors:

,&s and /,&s


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%upervectors

AC! yeast artificial chromosomeAble to carry .;; #b or more up to / @b (.;;; $ more

DNA insert than in a plasmid) ropagated as circular plasmid inE.coli

1AC! bacterial artificial chromosomeAble to carry inserts of B;; #b

1ased on I plasmid (he prototype con9ugative plasmidassociated %ith con9ugation in a strain ofE. coli)

>mportant in genome se'uencing pro9ect


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%tructure o a &"C vector." 1amH> removes the

stuffer fragment bet%eenthe t%o telomers

/" ?na1> cuts the vector intot%o linear arms (eachcarrying a selectable

mar#er)B" >nsert is ligated bet%een

these arms

*" ransform into a yeast cell(au$otrophic mar#ers-

ensures that therecombinants containboth arms)

," ?uccessful recombinant must contain the telse'uences at each end sothat the yeast transformant can use these se'uences to build functionaltelomers


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'llustration o a chromosome


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Lecture 8 Gene Cloning-Cloning Vector

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