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| == Restriction Enzymes candidates == | | == List of links == |
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| We found 1 megaendonuclease (18 recognition sites) and 5 endonucleases (8 recognition sites):
| | *[[IGEM:Paris_Bettencourt_2012/Notebook/RE_group/Restriction_Enzymes_candidates | Restriction Enzymes Candidates]] |
| | *[[IGEM:Paris_Bettencourt_2012/Notebook/RE_group/Promoter_Candidates | Promoter Candidates]] |
| | *[[IGEM:Paris_Bettencourt_2012/Notebook/RE_group/Parts | Parts]] |
| | *[[IGEM:Paris_Bettencourt_2012/Notebook/RE_group/Construction_design | Construction design]] |
| | *[[IGEM:Paris_Bettencourt_2012/Notebook/RE_group/Lab_notes | Lab Notes]] |
| | *[[IGEM:Paris_Bettencourt_2012/Notebook/RE_group/Goals | Our goals]] |
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| * I-SceI (no copies in the genome sequences of E.Coli)
| | == External Links == |
| * Abs I (8 copies in the genome sequences of E.Coli)
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| * Fse I (4 copies in the genome sequences of E.Coli)
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| * Rig I (4 copies in the genome sequences of E.Coli)
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| === I-SceI === | | # Codon optimization: [https://bioinfo.invitrogen.com/geneartgenes/projectmgmt GeneArt] |
| | # Colicins Project: [http://2008.igem.org/Team:Heidelberg/Project/Killing_II Heidelberg 2008] |
| | # Self Destructing Plasmid: [http://www.doomoney.com/debrid/proxy2/fichier.php?u=Oi8vMjAwOS5pZ2VtLm9yZy9UZWFtOlRVRGVsZnQvU0RQX092ZXJ2aWV3&b=5 TUDelft 2009 ] |
| | # Assembly: [http://partsregistry.org/Assembly:RBS-CDS_issues RBS-CDS issues] |
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| *Copy numbers in E.Coli: 0
| | ==Google Document Links == |
| *Came Frome: ''Saccharomyces cerevisiae YJM789''
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| ''Saccharomyces cerevisiae YJM789'':
| | #Biobricks retrieved by our group: [https://docs.google.com/a/igem-paris.org/spreadsheet/ccc?key=0Aqqw6ards8CndHpBa2RPWE1mM29IcFptZUxncnU2MWc Here] |
| | | #Primers ordered by our group: [https://docs.google.com/a/igem-paris.org/spreadsheet/ccc?key=0Aqqw6ards8CndHljSmNIcGJlN0hnRGZscTR6V2gxVWc Here] |
| *Recognition site: TAGGGATAACAGGGTAAT
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| *Sequence: [http://www.ncbi.nlm.nih.gov/gene/5405835 NCBI]
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| ''Saccharomyces cerevisiae S288c'':
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| *Name of the gene: I-SceIV
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| *Abstract:
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| ** Starting with crude yeast mitochondria, the intron homing endonuclease, I-SecIV, was purified to near homogeneity. This highly purified enzyme differs from some other well-characterized yeast mitochondrial intron-encoded endonucleases in terms of its structure and DNA cleavage specificity. The enzyme is a heterodimer with a native molecular mass of 92 kDa. A small catalytic subunit (32 kDa) is probably encoded largely or entirely by intron 5 alpha of the cytochrome oxidase subunit I gene. A larger polypeptide subunit (60 kDa) may be a nuclear factor necessary for intron mobility. '''I-SceIV exhibits a low DNA sequence specificity as it cleaves a variety of DNA substrates.''' Analysis of kinetic parameters shows that the purified enzyme has a very high affinity for DNA and exhibits low turnover which may have implications for subsequent steps in the intron homing process. [http://www.ncbi.nlm.nih.gov/pubmed/9675098]
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| ''E. Coli'':
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| * Systematic Mutagenesis of E.coli K-12 MG1655 ORFs: [http://www.genome.wisc.edu/functional/tnmutagenesis.htm], [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC451658/pdf/1592-03.pdf pdf]
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| * Supporting information appendix ([http://www.pnas.org/content/suppl/2011/07/28/1104681108.DCSupplemental/sapp.pdf full]):
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| **The chromosomal I-SceI endonuclease expression system used previously to introduce DSBs into F’128 in the Lac system for stress-induced mutagenesis was used to induce DSBs into the E. coli chromosome. The 18bp I-SceI cutsite sequence is normally absent from the E. coli genome, and was engineered into a sight near 3.9Mb in the genome. The site was included in primers used to amplify a kanamycin-resistance cassette and recombined into the genome. Three Chi sites were engineered adjacent to one side of the cutsite in the active orientation to interact productively with RecBCD to promote DSB repair, GG3’ side of the 5’GCTGGTGG Chi sequence towards the DSB site. The chromosomal I-SceI gene is under the control of the <math>P_{BAD}</math> promoter and so is induced strongly by arabinose and weakly in the absence of glucose which represses that promoter. In all experiments reported, measuring mutagenesis and/or efficiency of DSB formation by the I-SceI system, terminal cultures used in the experiments were shown to retain the functional regulatable I-SceI gene and cleavage site by quantitative measurement of arabinose-sensitivity of the cells by comparing cfu titers on arabinose and glucose plates. Typical frequencies of arabinose-resistant mutants, which have acquired a mutation in the I-SceI cutsite or gene, were usually between <math>10^{-4}</math> and <math>10^{-5}</math>, and never more than <math>10^{-3}</math>, as previously, demonstrating that the vast majority of cells examined in our experiments is DSB-competent. We found previously that the arabinose-resistant mutants consist mostly of cutsite mutants, presumably the result of low-level Ku-independent nonhomologous end-joining in ''E. coli''.
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| ***'''Strain''': SMR7270 | MG1655 ∆araBAD567 ∆attλ::PBADI-SceI
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| '''Referenses:'''
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| #''«On Spontaneous DNA Damage in Single Living Cells»'', Jeanine M. Pennington, Ph.D. thesis, Baylor College of Medicine, Houston (2006):
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| #''«A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation»'' [http://www.sciencedirect.com/science/article/pii/S1097276505015224], Susan M. Rosenberg, Ph.D.
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| #*'''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).
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| #**'''Strain''': PBADI-SceI SMR6316/SMR6318
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| #*** '''SMR6316''': SMR6272 Δattλ::PBADI-SceI [F′ΔtraI::dhfr] | This study
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| #*** '''SMR6318''': SMR6272 [F′ΔtraI::dhfr] | This study
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| #''«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) [http://ac.els-cdn.com/009286748690262X/1-s2.0-009286748690262X-main.pdf?_tid=3c069ae6c415e9206eac886baf7aff4f&acdnat=1340715583_e5677f62cb58c3b8ede1e378885e7fae], [http://ac.els-cdn.com/009286748690262X/1-s2.0-009286748690262X-main.pdf?_tid=95b1f6677122222459de88e5e65860fd&acdnat=1340717118_eb8ab049b238ee41f634a763072cab40 pdf].
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| === Abs I ===
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| Copy numbers in E.Coli: 8
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| Came Frome: ''Arthrobacter species7M06'' [http://science.sibenzyme.com/article8_article_23_1.phtml]
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| === Fse I ===
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| Copy numbers in E.Coli: 4
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| Recognition site:
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| [[Image:Fse-I-cutsite_1_v1_00001.gif]]
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| Source:
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| A E. coli strain that carries the FseI gene from Frankia species Eul1b (NRRL 18528) [http://nar.oxfordjournals.org/content/18/8/2061.abstract Original paper].
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| 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.
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| 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.
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| Gene sequence: [http://www.ncbi.nlm.nih.gov/nuccore/JF323049.1 FseI]
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| === Rig I ===
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| Copy numbers in ''E.Coli'': 4
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| ***It is isoschizomer [http://en.wikipedia.org/wiki/Isoschizomer]of FseI
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| Organism: ''Rhizobium yangligense''
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| Recognition site: GGCCGG^CC
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| * 3' Sticky End: GGCC
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| * 5' Sticky End:
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| Gene sequence: N/A
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| == Inducible promoters candidates ==
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| Our main objective is to find inducible promoters that are not leaky.
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| === P bad ===
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| Overview of PBAD Promoter Family: [http://partsregistry.org/PBAD_Promoter_Family]
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| *Pbad Part: BBa_I13453 [http://partsregistry.org/wiki/index.php/Part:BBa_I13453 Read More]
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| *Pbad+AraC Part:BBa_I0500 [http://partsregistry.org/Part:BBa_I0500 Read More]
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| *Characterization by the [http://2009.igem.org/Team:Cambridge/Project/Amplification/Characterisation Cambridge 2009 team] of different Pbad/araC promoters
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| *Plasmid: Part [http://partsregistry.org/Part:pSB3K3 pSB3K3]
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| === P lac ===
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| *We will get the plasmid construct from [http://ac.els-cdn.com/S0167701204000958/1-s2.0-S0167701204000958-main.pdf?_tid=56e38983168f46867e2b27e632169e7f&acdnat=1340707948_a14ba1d4913920cc608eb38d5fd732c6 this paper]: "Tightly regulated vectors for the cloning and expression of toxic genes". Larry C. Anthony*, Hideki Suzuki, Marcin Filutowicz
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| *LacIq Part:BBa_K091111 [http://partsregistry.org/Part:BBa_K091111 Read More]
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| *LacI Part BBa_C0012 [http://partsregistry.org/wiki/index.php/Part:BBa_C0012 Read More]
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| *Lac Operon Part BBa_R0010 [http://partsregistry.org/wiki/index.php/Part:BBa_R0010 Read More]
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| *Lac Operon Part BBa_K611024 (mutant of the BBa_R0010 part. Lower leakage, but also lower expression) [http://partsregistry.org/Part:BBa_K611024 Read More]
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| === P tet ===
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| *Characterization by the [http://2011.igem.org/Team:EPF-Lausanne/Our_Project/Reporter_Systems/ptet EPF-Lausanne 2011 team]
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| *Ptet Part:BBa_R0040 [http://partsregistry.org/Part:BBa_R0040 Read More]
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| *TetR Part: BBa_C0040 [http://partsregistry.org/wiki/index.php/Part:BBa_C0040 Read More]
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| === Interesting Articles ===
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| *"Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements."
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| Rolf Lutz and Hermann Bujard* [http://nar.oxfordjournals.org/content/25/6/1203.full Read It]
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