Modern Vectors Flashcards by Yasmin Yau

what is a vector

DNA molecule that functions as a “molecular carrier” that brings the DNA of interest into the host cell and facilitates its replication

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what do physical mapping systems show

size of molecules
e.g. plasmids up to 15 kilo base pairs
bacteriophages P1 90 kb

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what is an artificial chromosome

functional chromosome created by genetic engineering, with a centromere and a telomere and it is transmissible in cell division

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what is a DNA library

collection of fragmented DNA stored and propagated in a population of micro-organisms via molecular cloning (studying genomes)

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what is an F Factor

fertility factor in bacteria, also called episome and involved in DNA transduction

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what are the types of DNA vectors

bacteriophages
cosmids
fosmids
plasmids 
clones
artificial chromosomes:
yeast artificial chromosome
bacterial artificial chromosome

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what is yeast artificial chromosome

DNA construct, based on replication and preservation criteria in yeast cells
YAC

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what is yeast

eukaryotic cell, telomeres must be added to the artificial chromosome

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what is the potential problem with using yeast as a artificial chromosome

DNA inserts can be rearranged, inverted or deleted by host cell, can lead to mutations

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what is bacterial artificial chromosome

DNA construct, based on a fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria
BAC

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what is a bacteriophage

viruses that infect bacteria

sequence is known, and the size of the dsDNA sequence is ~ 50 kb

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what is the structure of bacteriophages

linear double-stranded molecules with single-stranded complementary ends (cos region)
They have cohesive termini

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what are cos sequences in bacteriophages like

cos sequences are ssDNA, and the cohesive ends have specific affinity for each other

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what are bacteriophage DNA vectors used for

clone larger segments of DNA ( clones ~ 20 kb phages used to clone large segments)

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what is the structure of bacteriophage

right and left arm that are essential for the survival of the phage
Left arm and right arm make the outside of the virus
Left arm = tail
Right arm = head
Nucleic acid is non-essential for plasmid

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what are the requirements for DNA cloning - types vectors needed

cosmids
fosmids
plasmids

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what are cosmids used for

(cos): plasmids containing DNA sequences from bacteriophage λ

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what are fosmids used for

are similar to cosmids but they are based on the bacterial F (fertility) plasmid containing F factor

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what are plasmids used for

used to clone small segments of DNA

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what are plasmids

Small circular dsDNA that autonomously replicate independently of the chromosome of the host cell
“molecular parasites”

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what do plasmids do

carry one or more genes, some confer resistance to certain antibiotics

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what is the origin of replication for in plasmid

origin of replication (ORI) which is a region of DNA that allows multiplication of the plasmid within the host

what are properties of good plasmids to clone DNA

must be small in size
DNA sequence must be known
must produce a high number of copies
must have a selectable marker
must possess a second selectable gene
must have a large number of unique restriction sites

what does pBR322 mean in plasmid map

who discovered it

what is the amp in plasmid for (plasmid map)

amp selectable marker for ampicillin

what is the tet in plasmid for (plasmid map)

tet selectable marker for tetracycline

what is BamH I and EcoR I for (plasmid map)

BamH I and EcoR I enzymes produce sticky ends

what is MCS

No multiple cloning site

how does selecting recombinant bacteria with pBR322

all colonies are grown on master plate - complete medium, press plate onto velveteen sterilized velveteen with imprint of all colonies transferred to: 1. complete medium and incubated = all colonies grow 2. minimal medium and incubated = mutants don't grow

what is a multiple clone site

Short sequence of DNA with restriction sites, usually unique in a vector

what is Amp R for in pBR322

allows selection of bacteria that have taken the plasmid

what is LacZ for in pBR322

subunit with its promoter. Lactose operon provides the means to select plasmids that have taken the insert. In this particular plasmid subunit is produced by the plasmid

what happens if insertional inactivation occurs

Insertional inactivation disrupts the functional enzyme

how does the blue product form

LacZ gene has an insertion which will transform colonies LacZ gene has a deletion of amino acid sequence Inside the chromosome of the bacteria the plasmid produces the LacZ fragment, this binds to the inactive beta-galactosidase and activates it, causing the X-gal substrate to form blue product

why does the blue product not always form

If don’t have this interaction because you have an introduction of a segment of DNA will not lead to the blue product production

what is IPTG

enhancer of production of gene in bacteria

what is the polyadenylation sequence for in eukaryote gene

protection of mRNA – poly A

what is the origin of replication

eukaryotic origin of replication

what are added to plasmids

resistance genes

what is needed in plasmid to work in host

bacterial origin or replication

when was Recombination based cloning | Gateway® Technology invented

Invented in 1991 by Invitrogen

what happens in Recombination based cloning | Gateway® Technology

Once phage infect cells two phases - lytic phase, virus reproduce and break down cells - lysogenic, virus inside of bacteria chromosome, till virus ready to replicate, is integrated DNA into E.coli chromosome

what are the advantages of Gateway® Technology

1. fast and flexible 2. highly efficient 3. does maintain orientation and reading frame without using restriction enzymes or ligation 4. can clone up to 4 segments at once 5. makes expression-ready clones. 6. segments bigger than 5 Kb can be cloned 7. allows interdisciplinary collaborations

In Gateway® Technology where is the gene obtained from

Gene from: - PCR - your source - mRNA - gene synthesis

In Gateway® Technology where is the DNA fragments from

- PCR - cDNA library - restriction endonuclease digestion and ligation

what happens in invitrogen Gateway® cloning system

Gene of interest put into entry clone – silent clone can recombine and use for many things

what does invitrogen Gateway® cloning system form

(all reversible reactions) - protein localization - protein purification - cell-free - multisite Gateway® pro - protein interaction - RNAi - your application

how is a Gateway® entry clone generated

Short recombination sites that are recombined with attB1 and attB2 1 hr incubation with BP clonase® 1 hr incubation with Gateway® LR clonase®

what is added to vectors to mark them for death

All colonies with selected marker ccdB will die

what can Gateway® technology be used for

``` Native protein expression N-terminal fusion protein expression C-terminal fusion protein expression Pathway reconstruction Multiple gene expression and regulation Protein interactions ```

what is pBR322

the first plasmid and the selection of recombinant bacteria requires imprinting of colonies

what is pUC18/19

has a multiple clone site and the selection of recombinant bacteria is through insertional inactivation of the Lac gene

what is an entry clone from Gateway® technology like and function

silent and it can be used to shuttle the DNA of interest into multiple expression clones to study the gene of interest at different molecular levels