IGEM:Paris Bettencourt 2012/Notebooks/RE group/Restriction Enzymes candidates

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We found 4 candidates: 1 meganuclease and 3 endonucleases


  • Copy numbers in E. coli genome sequence: 0
  • Recognition site: TAGGGATAACAGGGTAAT (18)
  • Source: Saccharomyces cerevisiae YJM789

  • Sequence:
    1. Saccharomyces cerevisiae YJM789: NCBI
      Name of the gene in Saccharomyces cerevisiae S288c: I-SceIV.
      Note: I-SceIV exhibits a low DNA sequence specificity as it cleaves a variety of DNA substrates.
    2. SMR6316: SMR6272 Δattλ::PBADI-SceI [F′ΔtraI::dhfr] | This study
      Now we are waiting for SMR6316 with genome sequence
    3. Self Destructive Plasmid based on I-SceI: iGEM TUDelft-2009
    4. p(LacI) controlled I-SceI homing endonuclease generator: BBa_K175041
    5. p(TetR) controlled I-SceI restriction site + GFP-LVA under: BBa_K175044 (WORKS!)
      Read Careful: http://2009.igem.org/Team:TUDelft/SDP_Results
      Cloning Strategy: Read more
    6. From iGEM projects (or not!?): NCBI

  • References:
    1. «Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV», Wernette C. M. Biochem Biophys Res Commun. 1998 Jul 9; 248(1):127-33. [1]
    2. «Systematic Mutagenesis of E.coli K-12 MG1655 ORFs», [2], pdf
      Supporting information appendix: (full)
    3. «On Spontaneous DNA Damage in Single Living Cells», Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):
    4. «A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation» [3], Susan M. Rosenberg, Ph.D.
      A Chromosomally Encoded Inducible I-SceI Endonuclease for Specific DSBs in E. coli To make DSBs inducibly at specific sites in the genome, we cloned the I-SceI double-strand-endonuclease universal open reading frame (Colleaux et al., 1986) behind the E. coli arabinose-inducible PBAD promoter and placed it in the E. coli chromosome (Gumbiner-Russo et al., 2001). I-SceI makes a specific DSB with 4 bp, 3′ overhangs at an 18 bp cutsite that is not normally present in the E. coli genome (Monteilhet et al., 1990). Strains that carry both the PBADI-SceI cassette and a cutsite either in the chromosome (data not shown) or the F′ episome, but not strains carrying each singly, display 40- to 100-fold reductions in colony-forming units (cfu) on arabinose medium (2% ± 0.9% survival, mean ± SEM of data from strains SMR6304, SMR6308, SMR6310; available in the Supplemental Data with this article online) and arabinose-induced linearization of DNA (Meddows et al., 2004), indicating that most receive a DSB. Our inducible chromosomal construct has also been used for in vivo gene cloning (Zhang et al., 2002) and studies of DSBR (Meddows et al., 2004), and I-SceI expression has been a mainstay of in vivo mammalian DSBR studies (Johnson and Jasin, 2001).
    5. «Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease», Colleaux et al., (1986) [4], pdf.

Abs I

  • Copy numbers in E.Coli: 8
  • Source: Arthrobacter species7M06 [5]

Sequence: N/A

Fse I

  • Copy numbers in E.Coli: 4
  • Recognition site: Fse-I-cutsite 1 v1 00001.gif
  • Source: E. coli strain that carries the FseI gene from Frankia species Eul1b (NRRL 18528) Original paper.
    • No recorded usage in E. coli.
    • FseI is one of the least stable restriction enzymes when stored at -20°C. We recommend storage at -70°C if it is to be stored for longer than 30 days.
    • FseI is strongly inhibited by certain salts, including NaCl or ammonium acetate. If you perform alcohol precipitation before digestions with FseI, use sodium acetate (or potassium acetate) as the salt in the precipitation.
  • Sequence:
    1. FseI
      There are no E, X, S, P and FseI sites in the gene sequence of fseIR.

Rig I

  • Copy numbers in E.Coli: 4
    It is isoschizomer [6]of FseI
  • Organism: Rhizobium yangligense
  • Recognition site: GGCCGG^CC
  • Sequence: N/A