Cosmids and Genomic Libraries

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Cosmids and Genomic Libraries
Biotechnology and Society Spring 2007 Lecture 12
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An Ideal Plasmid Vector

• A narrow range replicon with no nic site or
• A broad range replicon with a nic site
• Lack mob site
• Lack bom site
• Histochemical differentiation (blue white
  screening)
• Small size
• Amplifiable
• Multiple copy number

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Bacterial Viruses

• Frederick Twort (1915) and Felix dHerelle (1917)
  were the first to recognize bacterial viruses.

• Felix dHerelle (1917) called these viruses
  bacteriophages (bacteria eaters)

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The 1930s to 1940s

• From the 1930s, key workers discovered more
  about virus structure, biology, genetics, and
  replication.
• Salvador Luria and Max Delbruck

A picture of Max Delbruck (left) and Salvador
Luria in 1941. From www.cshl.org/History/.
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Delbruck and Luria

• The Mutual Exclusion Principle
• Alpha and Gamma phage
• Alpha has a faster time for lysis than the Gamma
  phage.
• Co-inoculation of Alpha and Gamma phage only
  resulted in Gamma phage infection.

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Bacteriophage lambda

• Discovered by André Lwoff
• Pasteur Institute in Paris France
• During WWII, converted the institute into a major
  center of Nazi resistance
• Nobel Prize, 1965, Medicine and Physiology
• How?
• Irradiated E. coli stopped growing and lysed

Andre Lwoff. From nobelprize.org/medicine/laureat
es/1965/lwoff-bio.html
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Bacteriophage lambda

• François Jacob (top) and Jacques Monod (bottom)
• Lytic cycle
• Lysogeny prophage
• Today, lambda is the most well-known virus that
  has been studied, in particular, regulation of
  genes in the genome

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Bacteriophage lambda

• By the early 1970s we knew that a good portion
  of lamda (?)was not required
• Lambda genome is 48.5 kb
• About 48 is not needed for replication
• About 20 kb used to produce the phage head and
  tail

COS
Lysis
Head
Replication
ori
Tail
Circularized lambda
Lysogeny
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http//www.chemeng.drexel.edu/web_books/EngBio/Hid
den/molec/ch4/4_1.htm
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Making a lambda vector

• Eliminate the non-essential parts of lambda
• Can now insert large pieces of DNA
• 48 of 48.5 kb 23 kb

COS
Lysis
Head
Replication
ori
Tail
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We can make it smaller lambda

• Hohn et al., empty heads and headless tails
  provided separately
• Eliminate head and tail genes

terminase
COS
Lysis
Replication
ori
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Lambda was great but

• Had to work with plaques
• Limited to hosts that can be infected by lambda,
  namely E. coli strains that has the lamb gene
• So if we were working with another bacterium such
  as Pseudomonas, how could we move our clone DNA
  into it?

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John Collins teams with the Hohns

• In 1978, shared a novel idea of inserting a cos
  site into E. coli plasmid derivatives

COS
No other lambda sequences required Cos sites on
linearized cloned DNA was clipped by a
terminase gene located at the opening of the
phage head
JC called this a cosmid
pAOP
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Broad host range replicons

• Late 70s to early 80s, studies of the symbiotic
  plasmids of a bacterium then called Rhizobium
• Large plasmids 150 to 1500 kbp
• Nitrogen fixation on legumes
• Plasmids readily replicated in E. coli, other
  bacteria
• Therefore must have mob, nic, bom

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Noel Keens Cocktail

• Wanted to have a cosmid that could be mobilized
  directly into HFB (his favorite bacterium)
• Had made pRK404, pRK414, pRK415, pDSK519
• Had nic and bom. Tet or Kan resistance
• pRK2013 has mob
• But he wasnt satisfied as he wanted to clone
  large genomic regions to find regulatory elements

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Keen and Brian Staskawicz

• Developed cosmids pLAFR3 and 5
• pLAFR1 and 3 suffered like other cosmids and
  lacked efficient ligation with target DNA and
  concatemer formation common.
• This will become clear in the next set of slides

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Making a Genomic Library with Cosmids

• Partial digest
• Ligation into site EcoRI

TetR
21.5 kb
cos
EcoRI
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Result
Sau3A partial digest of target DNA
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This Was Inefficient

• Ligation of variable pieces of either target or
  vector DNA decreased efficiency
• The cosmid pLAFR1 was improved to pLAFR3
• Addition of a polylinker in the EcoRI
• Removed EcoRI sites
• Added lacZ (peptide a region)

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pLAFR3 and 5
Advantages
Short and long arms
Directional ligation ScaI blunt end site
ScaI
polylinker
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Final Steps of a Genomic Library

• Package into heads and plug with tails
• Transduce E. coli receptor cell
• Select white colonies with tetR
• Check for plasmid
• Screen in your mutant for phenotype restoration

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Things You Should Remember

• Some plasmids are used as vectors or cloning
  vehicles but they are limited to the amount of
  DNA that can be cloned.
• A cosmid is a plasmid that has at least one COS
  (cohesive end site).
• COS comes from a bacteriophage.
• A genomic library contains at least one copy of
  every gene in an organism.