Difference between revisions of "Part:BBa K1323002"
 
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<partinfo>BBa_K1323002 short</partinfo>
 
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dCas9 or ‘dead’ cas9 is a mutated version of Cas9 that has lost its endonuclease activity - it no longer cuts double stranded DNA, instead simply binding to it. This means it can function as a repressor after complexing with sgRNA and the sgRNA-target site. dCas9 contains mutations in the RuvC1 and HNH nuclease domains; these changes are what bestow this modified Cas9 with it’s ability to bind DNA instead of cleaving it (Lei S. Qi, 2013). It has a Xylose Inducible Promoter [https://parts.igem.org/Part:BBa_K1323014 BBa_K1323014] and a sodA RBS [https://parts.igem.org/Part:BBa_K1323022 BBa_K1323022]. The illegal sites have been removed, and the sequence was codon optimized for expression in S. epidermidis using JCat software.  
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dCas9 or ‘dead’ cas9 is a mutated version of Cas9 that has lost its endonuclease activity - it no longer cuts double stranded DNA, instead dCas9 simply binds to it. This means dCas9 can function as a repressor after complexing with sgRNA ([https://parts.igem.org/Part:BBa_K1323000 BBa_K1323000], [https://parts.igem.org/Part:BBa_K1323001 BBa_K1323001]) and the sgRNA-target site. dCas9 contains mutations in the RuvC1 and HNH nuclease domains; these changes are what bestow this modified Cas9 with its ability to bind DNA instead of cleaving it (Qi et al., 2013). BBa_K1323002 has a Xylose Inducible Promoter [https://parts.igem.org/Part:BBa_K1323014 BBa_K1323014] and a ''sodA'' RBS [https://parts.igem.org/Part:BBa_K1323022 BBa_K1323022]. The illegal sites have been removed, and the sequence was codon optimized for expression in ''S.epidermidis'' using JCat software.  
  
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This part was DNA-Synthesized.
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This part was DNA-synthesized by Bio Basic.
  
  
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===Usage and Biology===
 
===Usage and Biology===
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<br>
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<strong>Contribution</strong>
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<br>
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Group: Team Tsinghua 2016.
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<br>
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Author: Tianyang Mao.
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<br>
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Summary: Section below is the contribution Team Tsinghua made to previous parts . We characterized three parts, one of which belongs to this page (https://parts.igem.org/Part:BBa_K1323002, https://parts.igem.org/Part:BBa_K1493504 and https://parts.igem.org/Part:BBa_K1470002) by transforming the part into <i>E. coli</i> and validated its sequence using enzymatic digestion as well as sequencing. We functionally improved these parts by fusing three parts together (dCas9 from BBa_K1323002, GFP from BBa_K1493504, and Gal4BD from BBa_K1470002). In a sentence, the nuclear localization of dCas9 protein can be well visualized. More controlled experiments suggest we successfully repurposed CRISPR/Cas9 for transcriptional control of an exogenous suicidal system contingent upon the fidelity of canonical mRNA sequences. For detailed documentation, please refer to the part page https://parts.igem.org/Part:BBa_K1923011.
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1323002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1323002 SequenceAndFeatures</partinfo>
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==References==
 
==References==
  
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Lei S. Qi, M. H. (2013). Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 1173-1183.
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Qi, L.S. et al., (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. ''Cell'', 1173-1183.
  
 
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Latest revision as of 16:35, 24 October 2016

dCas9: Expression cassette under a xylose inducible promoter

dCas9 or ‘dead’ cas9 is a mutated version of Cas9 that has lost its endonuclease activity - it no longer cuts double stranded DNA, instead dCas9 simply binds to it. This means dCas9 can function as a repressor after complexing with sgRNA (BBa_K1323000, BBa_K1323001) and the sgRNA-target site. dCas9 contains mutations in the RuvC1 and HNH nuclease domains; these changes are what bestow this modified Cas9 with its ability to bind DNA instead of cleaving it (Qi et al., 2013). BBa_K1323002 has a Xylose Inducible Promoter BBa_K1323014 and a sodA RBS BBa_K1323022. The illegal sites have been removed, and the sequence was codon optimized for expression in S.epidermidis using JCat software.

This part was DNA-synthesized by Bio Basic.


Usage and Biology


Contribution
Group: Team Tsinghua 2016.
Author: Tianyang Mao.
Summary: Section below is the contribution Team Tsinghua made to previous parts . We characterized three parts, one of which belongs to this page (https://parts.igem.org/Part:BBa_K1323002, https://parts.igem.org/Part:BBa_K1493504 and https://parts.igem.org/Part:BBa_K1470002) by transforming the part into E. coli and validated its sequence using enzymatic digestion as well as sequencing. We functionally improved these parts by fusing three parts together (dCas9 from BBa_K1323002, GFP from BBa_K1493504, and Gal4BD from BBa_K1470002). In a sentence, the nuclear localization of dCas9 protein can be well visualized. More controlled experiments suggest we successfully repurposed CRISPR/Cas9 for transcriptional control of an exogenous suicidal system contingent upon the fidelity of canonical mRNA sequences. For detailed documentation, please refer to the part page https://parts.igem.org/Part:BBa_K1923011.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1476
    Illegal BamHI site found at 4894
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

Qi, L.S. et al., (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 1173-1183.