User:Pakpoom Subsoontorn/Notebook/Genetically Encoded Memory/2008/10/10: Difference between revisions

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Name: FLP, Host: Saccharomyces cerevisiae
 
*Classification: tyrosine recombinase
*structure
**Length
**Amino-Acid-Sequence
**Domains
***six tyrosine kinase conserved residues: R191, K223, H305, R308, W330, Y343 (nucleophilic tyrosine and catalytic pentad)
*Target sequence: two identical simple sequence < 50 bp
*Mechanism:
*Engineering
**Mutation
***Mutation could change specificity to HK022's site
**Chimeric
**Directed-Evolution
*Exision:
**Unnatural-Host




Revision as of 03:03, 9 November 2008

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Summary Note on site-specific recombinase system

  • Insert content here...

Classification

Keywords for classification, which can be overlapped

  • By Structure
    • Tyrosine recombinase
    • Serine recombinase
    • Large Serine recombinase
  • By Function
    • Integrase
    • Excisionase
    • Invertase
    • Resolvase
    • Transposase
  • other stuffs
    • Integron

Methods

  • Discovering new site-specific recombinase
  • Characterizing protein domains
    • DNA binding domain
    • Catalytic domain
  • Finding the target-site
    • Minimal specific sequence for targeting
  • Looking into recombination mechanism
    • DNA topologies
    • Protein-DNA interaction
    • Chronology of events
    • Chemistry at active site
  • Evaluating Efficiency
    • Recombination yield
    • Specificity

Site Specific recombinases

(see more experiment detail)







Name: P22, Host Salmonella tryphimurium

  • Action: tyrosine integrase
  • Structure: length:: 387 aa
  • Target sequence:

attB:: has two inverted repeated sequence flanking an overlap region. inverted repeated sequence (core-type). The overlap region shares sequence with attP. Crossing over occurs at the overlap region. overlap-region::TTCGTAA attP has arm-type sites flanking core-type site.

  • Mechanism:

IHF is required.


Name: HP1, Host: Haemophilus influenzae

  • Action: tyrosine integrase
  • Structure:

length:: 337 aa crystal structure of C-terminal catalytic domain is known

  • Target sequence:

attB:: has two inverted repeated sequence flanking an overlap region. inverted repeated sequence (core-type). The overlap region shares sequence with attP. Crossing over occurs at the overlap region. overlap-region::TTTTAAA attP has arm-type sites flanking core-type site.

  • Mechanism:



Name: L5, Host: Mycobacterium smegmatis





Name: R4, Host: Streptomyces

  • Action: tyrosine integrase
  • Structure:

length:: 469 aa crystal structure is unknown.

  • Target sequence: attB:: has two inverted repeated sequence (core-type) flanking an overlap region .

attP:: has core-type sites and overlap region, without an arm-type site. Minimal attB and attP site is <56 bps.

  • Excision:

Excisionase has not yet been found.

  • Usage:

Integration works in mammalian cell.



Name: TP901, Host: Lactococcus lactis

  • Action: tyrosine integrase
  • Structure:

length:: 485 aa. crystal structure is unknown.

  • Target sequence: attB:: has two inverted repeated sequence (core-type) flanking an overlap region.

attP:: has core-type sites and overlap region, without an arm-type site. Minimal attB and attP site is <56 bps

  • Mechanism:
  • Excision: Excisionase has already been indentified.
  • Usage:

Integration works in E.coli, mammalian cell and in vitro.







  • Classification: tyrosine recombinase
  • structure
    • Length
    • Amino-Acid-Sequence
    • Domains
      • six tyrosine kinase conserved residues: R212, K235, H308, R311, Y342, H308 (nucleophilic tyrosine and catalytic pentad)
  • Target sequence: two identical simple sequence < 50 bp
  • Mechanism:
  • Engineering
    • Mutation
      • Mutation could change specificity to HK022's site
    • Chimeric
    • Directed-Evolution
  • Exision:
    • Unnatural-Host








Name: XerC, Host: Escherichia coli








Name: Tn3, Host: Klebsiella pneumoniae Action: resolvase


Name: gin (Phage Mu), Host: Escherichia coli Action: invertase

questions/notes

  • play with bacteriophage and microbial genome database, look for recombinase genes
  • look at Tn4451 mechanism for boosting up integration, Lyras and Rood 2000

Database

Reference

  1. Groth AC & Calos MP (2004), Phage Integrases: Biology and Applications. J.Mol.Biol. 335:667-678
  2. Sclimenti CR, Thyagarajan B & Calos MP, Directed evolution of a recombinase for

improved genomic integration at a native human sequence. Nucl Acids Res, 29 5044-5051

  1. Grainge I & Sherratt DJ (2007), Site-Specific recombination. Molecular Genetics of Recombination, Springer


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