Lecture 13 Cloning for synthetic biology pt 3

Necessary Features of Shuttle Vectors:

Origin or rep for species 1 AND 2

selectable marker for species 1 AND 2

MCS

Cosmid:

Small multicopy plasmid that carries lambda Cos sites and can carry around 45 kb of cloned DNA

BAC, PAC, and YAC

BAC: bacterial artificial chromosome

PAC: P1 artificial chromosome

YAC: yeast artificial chromosome

Shuttle Vector:

A vector that can survive in and be moved between more than one type of host cell

features of lambda replacement vectors

Remove non-essential region (middle) of phage and clone sequence of interest

Use lambda to package and deliver

Mutant head proteins → non-functional until mixed

features of cosmid vectors

Hybrid between traditional plasmid and lambda phage, can clone in almost anything, can be linearized

Possesses sequences to be packaged into virus particles → produces gene of interest

Can package larger gene sequences than lambda

how cos ends affect the conformation of lambda DNA

Lamba is typically linear and ds, except for ends which are ss

Cos ends are "cohesive end sites" → 5' overhangs with complementary sequences

Can pair and form cos site, circularizes the molecule into plasmid

Describe the features of YACs

Circular plasmid that functions as a chromosome in yeast

When it is moved into yeast, it will linearize. Gene will be on artificial chromosome & expressed

Describe the induction/repression of the lacUV promoter

inducer: IPTG

Repressor: LacI

Uses LacZ promoter region

Describe the induction/repression of tet promoters

Inducer: Tetracycline

Repressor: TetR

Uses lambda left promoter, PL

tetON: tetracycline turns gene ON by inducing (repression removed)

tetOFF: tetra turns gene OFF tTa unable to bind TRE

Name three methods of getting vectors into mammalian cell

Cationic lipid-mediated delivery

Calcium phosphate coprecipitation (electroportation)

viral transduction

two different oris typically used in TRANSIENTLY expressed mammalian vectors and the accessory proteins needed for each origin

SV40 ori requires SV40 large T-antigen protein

OriP/EBV ori requires Epstein Barr nuclear antigen (EBVA1) in the cytoplasm

transient transfections

DNA NOT integrated into genome, remains in nucleus, no selection required, is NOT PASSED down to daughter cells

stable transfections

DNA integrates into the genome, genetic material is CARRED stably from gen to gen requires selection (i.e. drugs)

transfection relative to mammalian expression vectors

intro of vectors, WITHOUT VIRUSES, into mammalian cells

transduction relative to mammalian expression vectors

intro of vectors into mammalian cells USING VIRAL-mediated delivery

mode of action of common selection agents for mammalian EXPRESSION vectors

G418/Geneticin and Hygromycin B = blocks ribosome from elongation protein chain

Puromycin: induces premature chain termination during protein synthesis

Blasticidin: inhibits protein translation termination

ALL INHIBIT PROT SYNTH

gene (genomic) library,

Collection of cloned DNA segments, contains at least one copy of every gene from a particular organism (source = genomic DNA, includes introns)

cDNA library

Collection expressed genes (mRNA) lacking introns, uses reverse transcriptase