Difference between revisions of "Part:BBa K1323002"
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<partinfo>BBa_K1323002 short</partinfo> | <partinfo>BBa_K1323002 short</partinfo> | ||
| − | 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 ( | + | 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. |
This part was DNA-synthesized by Bio Basic. | This part was DNA-synthesized by Bio Basic. | ||
| − | |||
===Usage and Biology=== | ===Usage and Biology=== | ||
| + | <br> | ||
| + | <strong>Contribution</strong> | ||
| + | <br> | ||
| + | Group: Team Tsinghua 2016. | ||
| + | <br> | ||
| + | Author: Tianyang Mao. | ||
| + | <br> | ||
| + | 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. | ||
| − | |||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K1323002 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1323002 SequenceAndFeatures</partinfo> | ||
| − | |||
==References== | ==References== | ||
| − | + | Qi, L.S. et al., (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. ''Cell'', 1173-1183. | |
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1476
Illegal BamHI site found at 4894 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.