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Applied Genetics > Modern Vectors > Flashcards

Modern Vectors Flashcards

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Biology 101 flashcards
1
Q

what is a vector

A

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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2
Q

what do physical mapping systems show

A

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

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3
Q

what is an artificial chromosome

A

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

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4
Q

what is a DNA library

A

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

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5
Q

what is an F Factor

A

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

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6
Q

what are the types of DNA vectors

A 
bacteriophages
cosmids
fosmids
plasmids 
clones
artificial chromosomes:
yeast artificial chromosome
bacterial artificial chromosome
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7
Q

what is yeast artificial chromosome

A

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

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8
Q

what is yeast

A

eukaryotic cell, telomeres must be added to the artificial chromosome

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9
Q

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

A

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

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10
Q

what is bacterial artificial chromosome

A

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

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11
Q

what is a bacteriophage

A

viruses that infect bacteria

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

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12
Q

what is the structure of bacteriophages

A

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

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13
Q

what are cos sequences in bacteriophages like

A

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

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14
Q

what are bacteriophage DNA vectors used for

A

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

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15
Q

what is the structure of bacteriophage

A

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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16
Q

what are the requirements for DNA cloning - types vectors needed

A

cosmids
fosmids
plasmids

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17
Q

what are cosmids used for

A

(cos): plasmids containing DNA sequences from bacteriophage λ

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18
Q

what are fosmids used for

A

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

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19
Q

what are plasmids used for

A

used to clone small segments of DNA

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20
Q

what are plasmids

A

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

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21
Q

what do plasmids do

A

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

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22
Q

what is the origin of replication for in plasmid

A

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

23
Q

what are properties of good plasmids to clone DNA

A
  • 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
24
Q

what does pBR322 mean in plasmid map

A

who discovered it

25
Q

what is the amp in plasmid for (plasmid map)

A

amp selectable marker for ampicillin

26
Q

what is the tet in plasmid for (plasmid map)

A

tet selectable marker for tetracycline

27
Q

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

A

BamH I and EcoR I enzymes produce sticky ends

28
Q

what is MCS

A

No multiple cloning site

29
Q

how does selecting recombinant bacteria with pBR322

A

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

30
Q

what is a multiple clone site

A

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

31
Q

what is Amp R for in pBR322

A

allows selection of bacteria that have taken the plasmid

32
Q

what is LacZ for in pBR322

A

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

33
Q

what happens if insertional inactivation occurs

A

Insertional inactivation disrupts the functional enzyme

34
Q

how does the blue product form

A

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

35
Q

why does the blue product not always form

A

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

36
Q

what is IPTG

A

enhancer of production of gene in bacteria

37
Q

what is the polyadenylation sequence for in eukaryote gene

A

protection of mRNA – poly A

38
Q

what is the origin of replication

A

eukaryotic origin of replication

39
Q

what are added to plasmids

A

resistance genes

40
Q

what is needed in plasmid to work in host

A

bacterial origin or replication

41
Q

when was Recombination based cloning

Gateway® Technology invented

A

Invented in 1991 by Invitrogen

42
Q

what happens in Recombination based cloning

Gateway® Technology

A

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
43
Q

what are the advantages of Gateway® Technology

A
  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
44
Q

In Gateway® Technology where is the gene obtained from

A

Gene from:

  • PCR
  • your source
  • mRNA
  • gene synthesis
45
Q

In Gateway® Technology where is the DNA fragments from

A
  • PCR
  • cDNA library
  • restriction endonuclease digestion and ligation
46
Q

what happens in invitrogen Gateway® cloning system

A

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

47
Q

what does invitrogen Gateway® cloning system form

A

(all reversible reactions)

  • protein localization
  • protein purification
  • cell-free
  • multisite Gateway® pro
  • protein interaction
  • RNAi
  • your application
48
Q

how is a Gateway® entry clone generated

A

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

49
Q

what is added to vectors to mark them for death

A

All colonies with selected marker ccdB will die

50
Q

what can Gateway® technology be used for

A 
Native protein expression
N-terminal fusion protein expression
C-terminal fusion protein expression
Pathway reconstruction
Multiple gene expression and regulation
Protein interactions
51
Q

what is pBR322

A

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

52
Q

what is pUC18/19

A

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

53
Q

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

A

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

Applied Genetics (20 decks)

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