Modern Vectors Flashcards
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
what do physical mapping systems show
size of molecules
e.g. plasmids up to 15 kilo base pairs
bacteriophages P1 90 kb
what is an artificial chromosome
functional chromosome created by genetic engineering, with a centromere and a telomere and it is transmissible in cell division
what is a DNA library
collection of fragmented DNA stored and propagated in a population of micro-organisms via molecular cloning (studying genomes)
what is an F Factor
fertility factor in bacteria, also called episome and involved in DNA transduction
what are the types of DNA vectors
bacteriophages cosmids fosmids plasmids clones artificial chromosomes: yeast artificial chromosome bacterial artificial chromosome
what is yeast artificial chromosome
DNA construct, based on replication and preservation criteria in yeast cells
YAC
what is yeast
eukaryotic cell, telomeres must be added to the artificial chromosome
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
what is bacterial artificial chromosome
DNA construct, based on a fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria
BAC
what is a bacteriophage
viruses that infect bacteria
sequence is known, and the size of the dsDNA sequence is ~ 50 kb
what is the structure of bacteriophages
linear double-stranded molecules with single-stranded complementary ends (cos region)
They have cohesive termini
what are cos sequences in bacteriophages like
cos sequences are ssDNA, and the cohesive ends have specific affinity for each other
what are bacteriophage DNA vectors used for
clone larger segments of DNA ( clones ~ 20 kb phages used to clone large segments)
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
what are the requirements for DNA cloning - types vectors needed
cosmids
fosmids
plasmids
what are cosmids used for
(cos): plasmids containing DNA sequences from bacteriophage λ
what are fosmids used for
are similar to cosmids but they are based on the bacterial F (fertility) plasmid containing F factor
what are plasmids used for
used to clone small segments of DNA
what are plasmids
Small circular dsDNA that autonomously replicate independently of the chromosome of the host cell
“molecular parasites”
what do plasmids do
carry one or more genes, some confer resistance to certain antibiotics
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
- fast and flexible
- highly efficient
- does maintain orientation and reading frame without using restriction enzymes or ligation
- can clone up to 4 segments at once
- makes expression-ready clones.
- segments bigger than 5 Kb can be cloned
- 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
