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

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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:




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*Usage:
*Usage:
Integration works in E.coli, mammalian cell and in vitro.
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==
==questions/notes==

Revision as of 03:06, 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: 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.

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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