Chapter 3 Cutting DNA molecules

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Chapter 3 Cutting DNA molecules

• 1.DNA is easily broken by physical methods
• 1) Ultrasound 300bp
• 2) High speed stirring (1500r/min) 8kb
• 3) DNA is cut randomly in this way, usually
  there is a single-stranded region at each end of
  the DNA fragment, which makes the DNA ligation
  difficult.

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• 2.DNA could be cut specifically by endonucleases
• 1) Phenomenon 1960s, there existed host
  restriction( restriction endonucleases) and
  modification( modification enzymes) when phages
  intruded into their host cells.
• 2) MeselsonYuan, 1968, purification of the first
  endonuclease from E.coli K12. This enzyme can
  bind to DNA specifically, but cut randomly.

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• 3) Smith et al, 1970, the first restriction
  endonuclease from H. influenzae Hind II
• 3.The presence of restriction and modification is
  a double-edged sword
• 1) Providing useful tool enzymes for gene
  engineering in vitro
• 2) Lowering the transformation efficiency in vivo

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Host-controlled restriction and modification

• 1. Functions for host cells
• 1) Modifying their own DNA
• 2) Destroying the incoming DNA

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• 2. Proofs of restriction and modification (an
  experiment)
• Phages from E.coli strain C could propagated
  by growth upon E.coli strain C with EOP(
  efficiency of plating) of 1, while the same phage
  propagated by growth upon E.coli strain K with
  EOP( efficiency of plating) of only 1/10000.

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• The phage is restricted by E.coli K. Again
  phages from E.coli K could infect E.coli K with a
  EOP of 1, this phenomenon is called modification
• 3. Result Explaining
• E.coli C EOP1
• E.coli K EOP1/10000 Restriction most of
  the phage DNA was degraded by restriction
  endonucleases in E.coli K, but some was left, the
  left DNA was modified usually by methylation,
  which showed resistance to degradation.

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• If the phage with modified DNA once again
  infects E.coli K , EOP1 , this is because the
  intruded DNA can not be digested by endonucleases.

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Types of restriction and modification (R-M) system

• 1.Types According to difference of the
  endonucleases, there are four types type I, type
  II, type III, type IIs
• 2. Characteristics of the different types of
  endonucleases
• Table 3.1

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• 3.Characteristics of type II making them useful
  for gene manipulation
• 1) They are mediated by separated enzymes
• 2) They do not require cofactors or
  S-adenosylmethionine
• 3) They recognize a defined ,usually
  symmetrical, sequence and cut within it.
• 4) when cutting DNA, they can produce
  sticking ends benefit to DNA ligation

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Nomenclature

• 1.Methods naming R-M systems( or endonucleasese)
• 1) The first letter of genus name and the first
  two letter of specific epithet to generate a
  three-letter abbreviation, usually in italics.
• 2) Where a particular strain is used, it is
  identified with a letter.
• 3) If several R-M systems are from one strain
  ,each one is identified by roman numerals.
• 4) For a homing endonuclease(???????),if it is
  encoded by an intron, it is usually given the
  prefix I(I-Ceu I).If it is an intein(???), it
  is given the prefix IP(IP-Psp I)
• 5) If R or M are needed to be clarified, R or M
  are added as a prefix. e. R.Sma I

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Recognition sequence

• 1.Recognize and cleave sequences
• 1) rotational symmetry(palindrome)
• 2) 4-8 nucleotides
• EcoR I G/AATTC or G/AATTC
• CTTAA/G
• 3) cleaved ends stick(cohensive)ends(5or3overh
  angs) or blunt(flush) ends

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• 2. Isoschizomers(???)
• restriction enzymes with the same sequence
  specificity and cut site.
• 3. Isocaudomers(???)
• Enzymes that recognize different sequences
  but produce same stick end. e.g. Bam HI(
  G/GATCC) and Bgl II (A/GATCT)
• 4. Neoschizomers(???)
• Enzymes that regconize the same sequence but
  cleaved at different points.e.g. SmaI (CCC/GGG)
  and Xma I (C/CCGGG)

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• 5. Star activity(????)
• under extreme conditions, such as elevated pH
  or lower ionic strength,restriction endonuleases
  are capable of cleaving sequences which are
  similar but not identical to their defined
  recognition sequence. e.g. EcoRI(G/AATTC),Star
  activity N/AATTN

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Number and size of restriction fragments

• 1.The number and size of restriction fragments
• To a given DNA, if its GC50,and four bases
  are distributed randomly,It can be cut to 44(256)
  bp fragments (on average)by an endonuclease
  regonizing 4 bases

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• 46 bp fragments by a 6 base-recognition
  endonuclease, 48 bp fragments by a 8
  base-recognition endonuclease. Therefore, to
  generate large DNA fragments from a genomic DNA,
  NotI(8 base-recognition endonuclease, GC/GGCCGC)
  )are usually used.
• 2. Mammalian genomic DNA CG is fivefold less
  than other DNA, therefore there are much fewer
  sites for a enodonuclease of which recognition
  site contains CG.

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• 3.Certain restriction endonucleases showed
  preferential cleavage of some sites in the same
  DNA molecules
• ? DNA 4 Sac II sites, central 3 sites are
  cleaved 50 times faster than the remaining one.

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Variations on cutting and joining DNA molecules

• 1.DNA fragments cut with the same endonuclease
  can re-join together.
• 2. DNA fragments cut with two endonucleases
  producing compatible cohensive ends(
  isoschizomers,isocaudomers) or blunt ends can
  also join together.e.g. Age I(A/CCGGT) and Ava
  I(C/CCGGG) or EcoR V(GAT/ATC) and Alu I(AG/CT)

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• 3. DNA fragments with different stick ends
  digested by two different endonucleases can be
  joined together after the stick ends are filled
  in by DNA polymerase I or blunted by mung bean
  nuclease, but they cannot re-cut by the two
  enzymes.

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4.MethyltransferaseM.SssI

• 1) Source Spiroplasma(????)
• 2) Application A methylate CpG
  B Label DNA isolated from
  organisms other than vertebrates(????) and
  echinoderms(????)

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The Dam and Dcm mehtylases of E.coli

• 1. Three site-specific DNA methylases
• 1) Dam methylase encoded by dam
  gene,transfer a methyl group to the N6 position
  of A in GATC
• 2)Dcm methylase encoded by dcm gene,modify
  the internal C at the position C5 position in
  CCAGG or CCTGG
• 3)M.EcoKI ,rare sites.

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• 2.The importance of the specific methylases
• First, DNA isolated from E.coli(dam or dcm)
  may not be cut by some endonucleases. E.g. MboI,
  but Sau3A(GATC).
• Secondly, the modification of plasmid can affect
  the frequency of transformation (reduced).
• Thirdly, direct repetitive sequences deletion
  appears to Dam methylation.

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The importance of eliminating restriction systems
in E.coli strains used as hosts for recombinant
molecules
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• 1. A suitable E.coli cloning host strain is a
  restriction-deficient strain, otherwise, the
  transformed DNA might be degraded.
• 2. Genes structure of the immigration control
  region of E.coli strain K12(wild type) Fig3.4

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• 1) hsdRMS encoding EcoKI restriction cutting
  non-methylated foreign DNA.
• 2) mcrA, mcrBC and mrr encoding three
  endonucleases cutting DNA modified by M.SssI,
  Mrr will attack DNA with methylated A at some
  specific sites.
• 3) E.coli HB101 this region is completely
  deleted

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The importance of enzyme quality

• 1.QC(quality control) no contaminants
• e.g. exonucleases can digest cohensive ends
  phosphatases can remove the phosphatase residues
  at 5 end, which makes following ligation
  impossible.

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• 2.A simple method QC
• a-complementation
• Plasmid containing lacZ gene was over-digested
  with a enzyme recognizing a site in LacZ, then
  re-ligated and transformed into LacZ- strain. If
  all of colonies are blue, the endonuclease is
  qualified.

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Joining DNA molecules

• 1. To create artificially recombinant molecules
• 2. Three methods for DNA ligation
• 1) by T4 DNA ligase
• 2) by E.coli DNA ligase
• 3) utilizing terminal deoxynucleotidyltransferase
  to synthesize homopolymeric 3 single stranded
  tail

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DNA ligase

• 1.Function seal single-stranded nicks between
  adjacent nucleotides in a duplex DNA chain.
• 2.Difference between T4 DNA ligase and E.coli DNA
  ligase
• Cofactors T4 DNA ligase requires ATP
• E.coli DNA ligase requires NAD
  

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3. Mechanism
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Alkaline phosphatase

• Alkaline phosphatase treatment can prevent
  plasmids self-ligation without insertion of
  foreign DNA. Fig 3.8

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Adaptors
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Homopolymer tailing

• 1.Enzyme
• terminal deoxynucleotidyltransferase
• 2.Sustrates
• 1)DNA with 3exposed OH group, arising from
  cutting by ?exonuclease or restriction
  endonucleases
• 2)one of dNTPs

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Joining PCR products

• 1.Taq DNA polymerase adds a single 3 A overhang
  to each end of PCR product.
• 2.T/A cloning using vector with a 3 T overhangs

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• 3. Incorporation of extra sequence at the 5 end
  of a primer into amplified DNA

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Joining DNA without DNA ligase

• 1.vaccinia topoisomerase cutting activity and
  rejoining activity
• 2. DNA fragment with CCCTT overhangs can be
  inserted into a vector with AAGGG .
• 3.Especially useful for T/A cloning.

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Chapter 4

• Basic biology of plasmid and phage vector

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Plasmid biology and simple plasmid vector

• 1.Plasmids they are replicons which are stably
  inherited in an extrachromosomal state.
• 2.Four forms of plasmids
• CCC DNAcovalently closed circles DNA(two strands
  are intact)
• OC DNA Open circles DNA(only one strand is
  intact)
• SC DNA Supercoiled DNA
• L DNA linear DNA( both strands are cleaved)

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• 3. Interconversion of different forms
• DNA gyrase, endonucleases, DNA ligase, EB

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• 4.Phenotypes
• Phenotypes plasmids confer on their host cell
• Antibiotic resistance, protein degradation,
  heavy-metal resistance, enterotoxin production

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• 5.Catogories
• According to mobilization
• conjugative plasmids (with tra genes) and
  non-conjugative plasmids.
• According to copy numbers
• relaxed plasmids multiple copies
• stringent plasmids a limited number of copies

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Host range of plasmids

• 1.Host range is determined by its ori region
• 1)plasmids whose ori is derived from plasmid
  ColE1 have a restricted host range.
• 2)promiscuous plasmids (????)have a broad host
  range.

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Plasmid copy numbers

• Two mechanisms controlling copy numbers through
  regulating the initiation of plasmid replication
• 1. Regulation by antisense RNA
• ori region transcripts a RNA called RNA II,
• It acts as a primer during replication only after
  cleaved by RNase II

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• Replication is also mediated by RNA I encoded by
  the same ori region.
• RNA I and RNA II are complementary, they can form
  a duplex RNA, which interferes with the
  processing of RNA II by RNase H resulting in the
  replication termination.
• More plasmids transcripts more RNA I.

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• A protein called Rop encoded by plasmids
  participates in the regulation of RNA I

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• 2. Regulation by binding of essential proteins to
  iterons (repeat sequences)
• A protein called RepA encoded by repA near the
  ori region binds to an iteron sequence in the
  ori region, and initiates the replication.

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• Two mechanisms concerning RepA regulation
• If copy number is high, RepA binds to its own
  promoter and blocks its expression.
• RepA can link two plasmids together by binding to
  their iteron sequences, thereby preventing them
  from initiating replication.

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Partitioning and segregative stability of plasmids

• 1. Segregative instability
• The loss of plasmids due to defective
  partitioning.
• 2. Factors affecting segregative instability
• 1) par region in plasmids(par mutation,less)
• 2) Nutrition and stress conditions (less)

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• 3) DNA superhelicity (mutation, less)
• 4) Formation of plamids multimers (less)

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Incompatibility of plasmids

• 1. Incompatibility of plasmids
• the inability of two different plasmids to
  coexist in the same cell in the absence of
  selection.
• 2. Plasmids with the same ori or par region
  are incompatible.

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The purification of plasmid DNA

• 1. CsCl-EB density gradient centrifugation.
• The chromosome DNA can bind more EB resulting
  in a lower density while plasmid DNA (covalent
  circle) only binds less EB resulting in a higher
  density.

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• 2.Alkaline denaturation method.
• 1) Cells are lysed with NaOH and SDS
• 2) The lysates are neutralized with acidic NaAC,
  Chromosome DNA and proteins are precipitated .
• 3) Centrifuge, the plasmid DNA remains in the
  supernatant.
• 4) Plasmids DNA can be precipitated with
  isopropanol or ethanol.

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• 3. Factors affecting plasmid yield.
• 1) Copy number in the cells( growth medium,
  stage of growth, genotype of host cell)
• 2) Operation whether cells are lysed
  completely.
• 3) endA of host cells
• endA encodes endonuclease I whose optimum
  substrate is double-stranded DNA.

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• 4) Isolation of large plasmid
• Cell are treated with lysozyme , subsequently
  with SDS , lysates are run an agarose gel,
  plasmids can be extracted from the gel.

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Desirable properties of plasmid cloning vehicles

• 1.Three properties for an ideal cloning vector
• 1) low molecular weight
• 2) of selectable phenotypic traits
• 3) multiple cloning sites (MCS)

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• 2. Advantages of a low molecular weight of
  plasmid
• 1) easily handle e.g. more resistant to damage
• 2) multiple copies
• 3) MCS has more choices for restriction
  endonuclease sites

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• 3. MCS sometimes located within a gene.
• If an insertion is cloned into this site, the
  gene will be inactivated.

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pBR322, a purpose-built cloning vehicle

• 1.Origins of plasmid pBR322

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• 2.Using pBR322 to identify a promoter
• Hind III site lies within the promoter of
  TcR(Pribnow box) , insertion of a gene usually
  leads to the inactivation of TcR gene. But if the
  fragment inserted carries a promoter-like
  sequence , TcR is retained.

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• 3. Knowing the origins of a plasmid helps you to
  decide whether two plasmids can be co-exited in
  one host cell.
• whether oris of the two plasmids are the same.

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Improved vectors derived from pBR322

• pUC vectors(pUC7,pUC8, pUC18, pUC19,
  pUC118,pUC119)
• The MCS is inserted into LacZ sequence.
• LacZ encodes a-peptide of ß-galactosidase,
  a-peptide can be complementary with the remaining
  part of ß-galactosidase encoded by chromosome
  DNA, and recombinants can be detected by
  blue/white screening.

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Bacteriophage ?

• Essential features
• 1.linear duplex molecule , 48.8kb
• 2.the entire sequence has been determined by
  F.Sanger(1982)
• 3.of cohesive termini(cos sites), they can be
  circularized when injected into a host cell.

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• 4. Genes on the left of linear map code for head
  and tail protein.
• 5. Genes on the right of a linear map are
  concerned with recombination and lysogenization.
• 6.Many genes in the central region are not
  essential, and can be replaced with other genes
  when ?DNA is used as a cloning vector.

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Promoters and control circuits
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? transcription occurs in three stage early
middle late

• Fig4.12

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Vector DNA

• 1.Wild type DNA is not suitable as a vector.
• 2.Replacement vector a fragment (stuffer) in the
  vector at the ends of both sites having
  restriction sites can be replaced by foreign DNA.
• 3.Insertional vector Having a site at which a
  foreign DNA can be inserted.

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• 4. The shortest ?DNA which can produce a plaque
  of normal size is 25 deleted DNA.
• 5. Phage ?can accommodate only about 5 more than
  its normal size.

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Improved phage- ?vector

• 1.The aim to improve wild-type phage- ?vector
• 1) to increase the capacity for foreign DNA
  fragments and restriction sites
• 2) to allow probes to be conveniently prepared
• 3) to develop vectors for expressing cDNA

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• 2. To increase the capacity , using replacement
  vector
• Problem how to easily separate the wild-type
  vector from a recombinant one?
• Solution Using E.coli strains lysogenic for
  phage P2 ,which wild-type phage- ? cannot infect.
• Phenotype for wild-type phage- ? Spi
  (sensitive to P2 inhibition), Spi is determined
  by red and gam genes in the central stuffer.

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• If the central stuffer is replaced with foreign
  DNA ,the recombinant phage- ?will show
• a phenotype of Spi- and can infect E.coli
  strains lysogenic for phage P2 .
• Consequences gam is responsible for the switch
  from bidirectional replication to rolling-circle
  replication. Gam- phage could not generate
  concatemeric linear DNA,

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• As a result, ? DNA could not be packed into
  phage heads, and gam- phage could not form
  plaques ,therefore it is unsuitable for a vector
  .
• But gam- phage do form plaques if the host
  strain is rec, and this genes function can be
  enhanced by chi.

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3.Examples EMBL3 and EMBL4

• Fig 4.13

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Packaging phage- ?in vitro

• 1.Basic procedure
• Recombinant ?DNA in concatemeric form head
  precursor ( encoded by gene E) Endonuclease(encod
  ed by geneA) products of gene D
  head products of gene W and gene FII
  tail
• phage particles

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• Fig 4.14

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• 2. Sources of head precursor, products of gene D
  and gene E, tails
• One lysogenic strain in which gene D is an amber
  mutation, could accumulate head precursor (gene
  E), tails and assembly proteins.
• Another lysogenic strain in which gene E is an
  amber mutation, could accumulate tail ,assembly
  proteins and gene D.

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DNA cloning with single-stranded DNA vector

• M13,fl and fd are filamentous coliphages
  containing a circular single-stranded DNA
  molecules.

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The biology of the filamentous coliphages

• 1. M13, single-stranded DNA, 6407bases
• fd, 6408 bases, 97 homologous to M13 DNA.
• 2. The filamentous phages only infect strains
  of enteric bacteria harboring F pilus.
• 3. Infected cells continue to grow and divide ,
  but at a slower rate, in the end, about 1000
  particles can be generated by one cell.

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• 4. When the single-stranded DNA enter the cell,
  it is converted to a double stranded replicative
  form(RF) and begin to replicate.
• 5. When the copy number of RF is about 100,it
  begin to replicate in the form of rolling circle,
  due to the accumulation of gene 5 protein.
• 6. The single-stranded DNA is released and packed
  with capsid protein.

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Why use single-stranded vector

• 1.Application of single-stranded vector
• 1) Preparation of sequencing template
• 2) Preparation of probes
• 3) Used for site-directed mutation
  (Kunkel,1989)

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• 2. Advantages of filamentous phages
• 1) The DNA has RF, and can be manipulated like
  a plasmid.
• 2) Both RF and single-stranded DNA can
  transfect competent E.coli cells to yield either
  plaques or infected colonies.

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• 3) The phage particle size is governed by the
  size of viral DNA, there are no packing
  constraints for the length of foreign DNA .
• 4) Its easy to determine the orientation of an
  insert, the two single strands isolated can
  hybrid if the foreign DNA are inserted in
  opposite directions .

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Development of filamentous phage vectors

• 1. Unlike ?DNA , the wild type filamentous
  coliphage is not suitable as a vector because
  there are fewer restriction sites and no
  non-essential stuffer which can be replaced by
  foreign genes.
• 2. Widely used vector M13mp18

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• Fig 4.17

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Chapter 5

• Cosmids, phasmids and other advanced vectors

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Introduction

• 1. There are many vectors of different
  characters.
• 2. There is urgent need for cloning large DNA
  fragments.

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Vectors used for cloning large fragments of DNA

• Cosmid vector
• 1.cosmid plasmid with ?DNA cohensive sites.
  
  
  
• 2.Accommodate 32-47 kb of DNA fragment
• Can be packed in vitro, useful for construct
  genome library.
• 3. Avoid two or more fragments are ligated into
  one vector.

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• Fig 5.1

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• Fig 5.2

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• Fig 5.3

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BACs and PACs as alternatives to cosmids

• 1. PAC Phage P1 artificial chromosome, 100kb

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• Fig 5.4

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• 2. BAC bacterial artificial chromosome, 300kb

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Choice of vector

• 1. Disadvantages of YACs
• 1) 50 YACs show structural instability
  of inserts.
• 2) Two or more fragments can incorporate
  into one clone.

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• 2. Advantages of BAC and PAC over YAC
• 1) lower levels of chimerism
• 2) ease of library generation
• 3) ease of isolation and manipulation of
  inserts

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Specialist-purpose vectors

• Vectors that can be used to make a
  single-stranded DNA for sequencing

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• pUC-based phagemid
• 1. A vector derived from pUC containing M13
  ori and pUC(ColE1) ori
• 2. This kinds of phagemid replicate as
  double-stranded molecules.
• 3.When helper phage such as M13K07 exits,
  Single stranded DNA for sequencing can be
  produced.

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Expression vectors

• 1. Vectors carrying cis-acting elements such as
  promoter, terminator, RBS.
• 2.Basic structure of RNA polymerase
• core enzyme 2 a, 1ß,1ß
• holoenzyme core anzyme and s
• 3.Function of RNA polymerase
• Transcription of genes

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Vectors for making RNA probes

• 1.Advantage of RNA probes over single-stranded
  DNA probes
• The rates of hybridization and the stability
  are far greater for RNA-DNA hybrids compared with
  DNA-DNA hybrids.
• 2. How to make RNA probes?

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• 1) Relevant gene was cloned into a plasmid and
  under the control of phage promoter.
• 2) The recombinant plasmid was cut with an
  endonuclease.
• 3) Supply with phage RNA polymerase and NTP mix
  (one was labelled )
• 4) RNA probes will be transcripted out.

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• Fig 5.8

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• 3.Why use a phage promoter to produce RNA probes?
• 1) Such promoters are very strong.
• 2) The phage promoter is not recognized by E.coli
  RNA polymerase , RNA probes cannot be produced
  in E.coli.
• 3) Phage RNA polymerase such as T7 RNA polymerase
  is simple, containing only one peptide.

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• 4. How to prepare a special RNA probe?
• 1) The relevant gene was inserted between
  two phage promoters.
• Fig 5.9(a)

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• Fig 5.9(a)

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• 2) The relevant gene was inserted between two
  similar promoters with different restriction
  sites.
• Fig 5.9 (b)

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• Fig 5.9(b)

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Vectors for overexpressing recombinant proteins

• 1.Factors affecting the expression of a cloned
  gene
• 1) Promoter strength
• 2) Transcriptional termination
• 3) Plasmid copy number
• 4) Plasmid stability
• 5) Host cell physiology

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• 6) Translational initiation sequences
• 7) Codon choice
• 8) mRNA structure

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Table 5.2
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Table 5.3
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• 2.Regulated promoter
• 1) Some recombinant proteins such membrane
  proteins are toxic to host cells even in small
  amounts.
• 2) Overexpression of proteins are toxic even
  if they are nontoxic in small amounts.
• 3) Frequently used controllable promoters
• ?PL, ?PR , PT7, Ptac, Ptrc table 5.3

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• 3. pET vectors
• Host cells E.coli BL21(DE3) plyS

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Fig5.10
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Vectors to facilitate protein purification

• 1. Affinity chromatography to make the
  recombinant protein be expressed with another
  protein (called tag)
• 2. The recombinant protein fused with MBP
  (maltose binding protein). MBP can be bound
  specifically to resin covalently linked with
  maltose.

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• 3. The recombinant protein fused with His-tag.
  His-tag can be bind specifically to Ni2 Resin
• 4. The recombinant protein fused with
  GST(glutathione S- transferase). The recombinant
  proteins can be bound to glutathione resin.
• 5. The recombinant protein fused with biotin.
• Biotin can be bind specifically to
  streptavidin resin.

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• Fig 5.13

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Fig5.14
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Inteins, exteins and protein splicing

• 1. Intein a protein splicing element encoded by
  S.cerevisiae VMA1 gene.
• 2. It can cut itself at the end of its
  N-terminal at low temperatures in the presence of
  thiols.
• 3. If intein are fused with a target protein at
  its N-terminal and Chitin binding protein at its
  C-terminal, the fused protein can be bound to
  chitin beads and the target protein can be cut
  down from the fused protein.

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• Fig 5.15

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Vectors to promote solubilization of expressed
proteins

• 1. Some recombinant proteins appear in soluble
  forms, while others appear in inclusion bodies.
• 2. Soluble proteins are usually fully refolded
  and show biological activities, while proteins in
  the form of inclusion bodies are not refolded and
  show no activity.

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• Fig 5.16

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• 3. Measures to increase the yield if soluble
  proteins
• 1) Lowering the growth temperature
• 2) Changing media compositions and pH to
  reduce the growth rate.
• 3) Reducing the concentration of inducer
  e.g.IPTG

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• 4) Co-expression with chaperonines e.g. GroEL
• 5) Secretion expression using vectors with
  signal peptide coding region. The recombinant
  protein is expressed with signal peptide at the
  end of N-terminal and secreted into periplasm or
  medium.After secretion, the signal peptide is
  released.