Gene Technology - 2020. 04 - Vectors 1

Gene Technology - 2020. 104 - Vectors

Vectors

• Plasmids

– prokaryotic

– eukaryotic

• Phage

– M13

– lambda

• Cosmids

• Artificial chromosomes

2

• Cloning vectors

• Expression vectors

04 - VectorsGene Technology - 2020.

Plasmids• First plasmid described was found in Japan in Shigella species

during an outbreak of dysentery in the early 1940's

• Three plasmid types are the most studied: F factor (fertility factor), R plasmids (antibiotic resistance) and Col (colicinogenic)

• F factor: important for pilus formation - allows genes to be transferred by conjugation from one bacterium carrying the factor to another bacterium lacking the factor.

• R plasmids - medically importantPenicillin was introduced for general use in early 1940's1946 - 14% of Staphylococcus aureus were penicillin resistant1969 - 59% PenR

1970's - almost 100% of S. aureus were PenR (US data)


Plasmids


Major plasmid types


4 groups of wild type plasmids based on transfer properties:

– Nontransmissible - can neither initiate contact with recipient nor transfer DNA

– Conjugative - can initiate contact with recipient bacterium– Mobilizable - can prepare its DNA for transfer– Self-transmissible - is both conjugative and mobilizable

• Donation - a conjugative plasmid (such as F) can provide conjugative function to a mobilizable plasmid (such as ColE1) such that both plasmids can be transferred.

• Plasmid conduction - a self-transmissible plasmid (such as F) can recombine with a non-mobilizable plasmid and transfer the cointegrate.

604 - Vectors

Plasmids used in molecular cloning are nontransmissible

Gene Technology - 2020.

Plasmid replication• theta replication (either uni- or bidirectional) or

rolling circle

• replicon - DNA molecules that can replicate autonomously (chromosomes, plasmids, phages)

• replicon must have on origin of replication (ori)

• there are different types of ori

• functions of the ori region

– Host range determination - narrow or broad host ranges

– Copy number determination


Rolling circle replication

• https://www.youtube.com/watch?v=FhcZLqvs5yg

Gene Technology - 2020. 04 - Vectors 8

Regulation of the copy number at the ColE1 origin of replication

9

Origin incompatibility*: one host cell cancontain only one type of plasmid from thesame incompatibility group (except in thepresence of different selection markers)


Transformation of E coli with plasmid• Requires CIRCULAR DNA

• Efficiency relatively low (106 – 1010 cfu/mg DNA)

10

Mandel & Higa, 1970

D. Hanahan, 198304 - VectorsGene Technology - 2020.

can be frozen,

+glycerol,

-80 C

http://szilagyl.web.elte.hu/SH-2018/Classical%20papers/mandel1970.pdf

http://szilagyl.web.elte.hu/SH-2018/Classical%20papers/hanahan1983.pdf

Electroporation• Salt free!!! cells (50-100 ml) and DNA (can be linear too)

• Cooling in ice

• Electroporation cuvette (distance of electrodes 1 to 2 mm)

• Short impulse (10-6 – 10-3 s) of high voltage (1.0 – 1.5 kV)


pBR322: first successful cloning vector

12

(Bolivar et al. 1977)

(4363 bp)

rep/ori pMB1 plasmid

Apr (bla) Tn3 transposon

Tcr pSC101 plasmid


Restriction map of pBR322

Selection I

• Selection for the uptake of plasmid

– Antibiotic resistance

• Ampicillin

• Tertacycline

• Chloramphenicol

• Streptomycine

• Kanamycine


Antibiotic resistance genes

ampicillin cell wall synthesis -lactamase(karbenocillin)

tetracycline aa-tRNA binding 40 kD membrane protein

chloramfenicol peptidyl-transferase chl-acetyltransferase (CAT)

streptomycin initiation , missreading st.-phosphotransferase

kanamycin missreading kan.-acetyltransferase(neomycin)


Most important antibiotics

15

Ampicillin Tetracycline Chloramphenicol

Streptomycin Kanamycin04 - VectorsGene Technology - 2020.

Selection II

• Selection for the presence of insert in theplasmid

– pBR322: insertional inactivation, replica plating

– pUC: „blue-white” selection


Replica plating

17

replica


http://szilagyl.web.elte.hu/SH-2018/Classical%20papers/Lederberg-replica%20plating.pdf

pUC18/19 (Vieira & Messing, 1982)

pMB1 (ColE1) replikon

„MCS” (polylinker) (57 bp, 10 sites)

18/19; reverse orientation

(sequence on slide #21)

lacZ’

CAP binding site

Plac, operator, RBS,

-gal -fragment: 60 aa, MCS: instead of codon no. 6 and 7


lac OPERON


complementation: „blue – white” selection


M13 phage• Single stranded, filamentous

phage (length: 800-2000 nm, width: 6-8 nm)

• Does not lyse cell

• 10, non overlapping genes

• Double stranded form in the cell (RF)

• Rolling circle replication generates single stranded


Life cycle of M13 phage


Cloning vector from M13

MCS + lacZ’ from pUC18/19 (+ lacI) inserted into intergenicregion


Cloning into M13 vectors


pBluescript (phagemid)• 21 restriciton sites (in two

orientations, KS/SK)

• Blue-white selection

• Promoters for RNA pol and sequencing

• f1 ori in two orientations (+/-)

– single stranded rescue

– needs helper phage*!

• Restiction sites with alternating 3’ and 5’ overhang

– Nested deletions

25

Helper phages produce all proteins necessery for virion formation but

contain defective ori, and package their own ssDNA at low level


Nested deletion


Expression plasmids


Promoters in expression plasmids


Positive selection vectors

• pTR262– PR (-phage) promoter – tetracycline resistance; cI gene

– insert in cI gene

• pZErO (Invitrogen)

– insert in ccdB killer gene

– cytotoxic F plasmid gene (DNA gyrase toxine)

– gyrA462 strain is resistant (to clone vector)

29

cI Pr Tc


Bacteriophage linear doublestranded genome (48,5 kb, 50 genes)

icosahedral head + flexible tail

adsorption (maltose transporter LamB),

in bacteria circularization (cos sites),

temperate phage-lysogen (prophage) vs. lytic lifecycle – regulated by lambdarepressor (cI)

- and „rolling-circle” replication,

30

Electron micrograph of rolling

circle replication

phage


http://szilagyl.web.elte.hu/SH-2020Classical%20papers/03-Bacteriophage.pdf

Assembly of bacteriophage


packaging extract –

extracellular (in vitro)

assembly

phage vectors

Size limit:

there is a lower and upper limit of an infective genome:

38 kb < recombinant > 52 kb (78%-105%)

40 % of the genome is not necessary for lytic growth

Removal of unwanted restrictionsites


problems to be solved:

substitution vectors40% of the genome removed

contains „stuffer” fragment

stuffer fragment removed by digestion

left arm (A-J virion genes; 20 kb)right arm (pL, pR, regulation, ori; 10 kb)

ligation of inserts results concatamer

only recombinant makes infective phage

33

Difficult to remove stuffer fragment - positive selection:

WT phage do not infect E. coli lysogenic for bacteriophage P2; Sensitive to Ps Interference (spi).

Stuffer fragment carries genes for the spi phenotype

Nonrecombinants (carrying the stuffer fragment) will not grow on an E. coli host carrying a P2 lysogen

Cloning into phage


in vitro packaging

Gigapack(Agilent)

terminase, head and tail proteins

infection: 107-109 pfu/mg

insertion vectors

10 kb removed, infective

retained capability for lysogeny

expresses cI – forms turbid„cloudy” plaques

cloning into cI gene – clear plaques

positive selection: infecting Hfl(High frequency lysogeny) cells„empty” phages don’t form plaques

good for creation cDNA library

screening by plaque hybridization

35

• COSMIDSplasmid - -phage hybrid vectors(~8 kb)2 cos-site (250 bp)

pMB1 repliconin vitro packing, infection, plasmid

replicationinsert: 30-45 kb

genomic libraries, genome mapping(YAC subclones)

FOSMID: F plasmid replicon (1-2 copy)


P1 phage vectors

• P1 (temperate) bacteriophage (110 kb)• insert: 75-100 kb• in vitro packaging, • „headful” mechanism; pac site (162 bp), packase enzyme,

phage head and tail• infection, site specific recombination (linear phage → circular

plasmid)• cre/loxP system• plasmid replication• P1 plasmid (1 copy) and lytic replicon (multiple copies)


The cre-lox system

38

Cre recombinase (Cyclization Recombination) of P1 phageTwo domains: large C, small Nthe C domain is similar to Integrase frombacteriophage λ. This is also the catalytic site of theenzyme.

Lox P siteLox P (locus of X-over P1) is a site on theBateriophage P1 consisting of 34 bp. There exists an asymmetric 8 bp sequence in between with 2 sets of palindromic, 13 bp sequences flanking it.

Orientation and location of the loxP sites determines how the genetic material will be rearranged

13bp

8bp 13bp

ATAACTTCGTATA - GCATACAT -TATACGAAGTTAT


The cre-lox system• Inversion: If the loxP sites are on the same DNA strand and are in opposite

orientations, recombination results in an inversion and the region of DNA between the loxP sites is reversed.

• Deletion: If the sites face in the same direction, the sequence between the loxPsites is excised as a circular piece of DNA (and is not maintained).

• Translocation: If the sites are on separate DNA molecules, a translocation event is generated at the loxP sites.


Artificial chromosomes

• PAC• (P1-derived Artificial Chromosome)• electroporation• pCYPAC2, pPAC4 (19 kb)•

• BAC • (Bacterial Artificial Chromosome)• insert: >300 kb• electroporation• pBAC108L, pBACe3.6 •

•

• YAC (Yeast artificial chomosomes)[yeast vectors]


Bacterial artificial chromosome

Developed in 1992 (pBAC108L)

Essential components:

regulatory sequences from F factor:

ori S, repE for undirectional replication

parA, parB mantain copy# one or two

selection marker (cmR)

cloning sites (HindIII and BamHI)

cosN and LoxP – linearization

rare cutters for excision of inserts

T7 and SP6 promoters – RNA probegeneration


http://szilagyl.web.elte.hu/SH-2020/Classical%20papers/pBAC108L.pdf

Yeast plasmids• Yeast Integrating plasmids (YIp): These plasmids lack an ORI and must be

integrated directly into the host chromosome via homologous recombination.

• Yeast Replicating plasmids (YRp): Contain an Autonomously Replicating Sequence (ARS) derived from the yeast chromosome. These vectors can replicate independently of the yeast chromosome; however, they tend to be unstable and may be lost during budding.

• Yeast Centromeric plasmids (YCp): Low copy vectors and incorporate part of an ARS along with part of a centromere sequence (CEN). These vectors replicate as small independent chromosomes and are thus typically found as a single copy. Unlike the ARS vectors, CEN vectors are stable without integration.

• Yeast Episomal plasmids (YEp): Similar to bacterial plasmids and are considered “high copy”. A fragment from the 2 micron circle (a natural yeast plasmid) allows for 50+ copies to stably propogate per cell.


https://blog.addgene.org/plasmids-101-yeast-vectors

Genomic librariesA genomic library is a collection of the total genomic DNA from a single organism.

The DNA is stored in a population of identical vectors, each containing a different insert of DNA.

Construction of a library:

By Aluquette - Own work, CC BY-SA 3.0,

https://commons.wikimedia.org/w/index.php?curid=

25760209


How to choose vector for genomic library

• Carbon and Clarke formula:

N is the necessary number of recombinantsP is the desired probability that any fragment in the genome will occur at

least once in the libraryi is the insert sizeg is the genome size

• g = 3x 109 (human genome)

• i = 2x 104 ( phage)

• P = 0.99


Types of vectors

Vector type Insert size (thousands of bases)

Plasmids up to 15

Phage lambda (λ) up to 25

Cosmids up to 45

Bacteriophage P1 70 to 100

P1 artificial chromosomes (PACs) 130 to 150

Bacterial artificial chromosomes (BACs) 120 to 300

Yeast artificial chromosomes (YACs) 250 to 2000


https://en.wikipedia.org/wiki/Vector_(molecular_biology)

https://en.wikipedia.org/wiki/Insert_(molecular_biology)

https://en.wikipedia.org/w/index.php?title=Basepairs&action=edit&redlink=1

https://en.wikipedia.org/wiki/Plasmid

https://en.wikipedia.org/wiki/Lambda_phage

https://en.wikipedia.org/wiki/Cosmid

https://en.wikipedia.org/wiki/P1_phage

https://en.wikipedia.org/wiki/P1-derived_artificial_chromosome

https://en.wikipedia.org/wiki/Bacterial_artificial_chromosome

https://en.wikipedia.org/wiki/Yeast_artificial_chromosome

cDNA libraryContains mRNA sequences from a given organism or tissue

Does not contain introns and regulatory elements

Vector usually phage

Screeningcolony/plaque hybridizationPCR


Laboratory strains of E. coli used forcloning

https://blog.addgene.org/plasmids-101-common-lab-e-coli-strains


These strains are all

based on E. coli K-12

and considered BSL-1

(biosafety level 1: little

to no threat of infection

in healthy adults


Common gene mutations found in E. coli strains

https://blog.addgene.org/plasmids-101-common-lab-e-coli-strainsGene Technology - 2020. 04 - Vectors 48


Common gene mutations found in E. coli strains

https://blog.addgene.org/plasmids-101-common-lab-e-coli-strainsGene Technology - 2020. 04 - Vectors 49


Documents

Gene Technology - 2020. 04 - Vectors 1