Difference between revisions of "Part:BBa K1323002"

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.

@@ Line 11: / Line 11: @@
 <strong>Contribution</strong>
 <br>
-Section below is the contribution Team Tsinghua made to this part. We characterized this part by transforming the part into <i>E. coli</i> and validated its sequence. In addition, in order to perform imaging related experiments, we fused a green fluorescence protein to this construct. As is indicated below, the nuclear localization of dCas9 protein can be well visualized.
+Group: Team Tsinghua 2016.
+Author: Tianyang Mao.
-<div style="margin-left:0px;height:5%;width:5%;">
+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.
-https://static.igem.org/mediawiki/parts/3/3f/T--Tsinghua--part--data1.jpg
-https://static.igem.org/mediawiki/parts/9/9f/Picture1111.png
-</div>
 <span class='h3bb'>Sequence and Features</span>