Difference between revisions of "Part:BBa K1062004"
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<partinfo>BBa_K1062004 parameters</partinfo>
 
<partinfo>BBa_K1062004 parameters</partinfo>
 
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<partinfo>BBa_E0040 AddReview number</partinfo>
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<I>UsernEnter the review inofrmation here.ame</I>
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We used this part as a reporter protein in a cell free system. We found that there was observable fluorescence from this part and experienced no issues with expressing it in a cell free system.
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<partinfo>BBa_E0040 AddReview 3</partinfo>
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<I>2018 OUC-China</I>
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===[https://parts.igem.org/Part:BBa_K2615003 Csy4 (Csy6f)], a member of CRISPR family.===
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  Csy4 is a 21.4 kDa protein that binds and cleaves at the 3' side of a stable RNA hairpin structure via sequence- and structure-specific contacts. Csy4 binds its substrate RNA with extremely high affinity and functions as a single-turnover enzyme. Tight binding is mediated exclusively by interactions upstream of the scissile phosphate that allow Csy4 to remain bound to its product. Substrate specificity is achieved by RNA major groove contacts that are highly sensitive to helical geometry, as well as a strict preference for guanosine adjacent to the scissile phosphate in the active site. A highly basic a-helix docks into the major groove of the hairpin and  contains multiple arginine residues that form a network of hydrogen.
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[[Image:T--OUC-China--complex.jpg|center|thumb|250px|'''Fig.1  The Csy4/Hairpin complex.''']]
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===Background of 2018 OUC-China' project===
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  This year, we design a toolkit focused on translational regulation, which is composed of a RNA endoribonuclease (Csy4) and a RNA module (hairpin). In our project, the cleavage function of Cys4 releases a cis-repressive RNA module ([https://parts.igem.org/Part:BBa_K2615020 crRNA], paired with RBS) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. A Ribosome Binding Site (RBS) is an RNA sequence to which ribosomes can bind and initiate translation.
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  We want to achieve precise expression of proteins by using different Csy4 mutants. The aim is using one system to realize diverse expression. We focus on the sites which play an important role in binding and cleavage. Gln104 is located in the linker segment connecting the body of Csy4 to the arginine-rich area, which makes sequence-specific hydrogen bonding contacts in the major groove of the RNA stem to nucleotides G20 and A19. His29 is in its deprotonated form and functions as a general base during cleavage by activating the 2′-hydroxyl nucleophile through proton abstraction. The side chain of Tyr176 points into the active site and stacks on top of the His29 imidazole group, which plays a role in orienting His 29. Phe155 is to recognize the ssRNA-dsRNA junctions in RNA hairpin. Based on the molecular simulation and the theory of fluctuations, four mutants are chosen rationally: [https://parts.igem.org/Part:BBa_K2615004 Q104A], [https://parts.igem.org/Part:BBa_K2615007 H29A], [https://parts.igem.org/Part:BBa_K2615005 Y176F], [https://parts.igem.org/Part:BBa_K2615006 F155A].
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[[Image:T--OUC-China--Csy4complex.jpg|center|thumb|400px|'''Fig.2  Four key sites of wild type Csy4.''']]
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Revision as of 11:58, 3 October 2018

Csy4

Csy4 is an enzyme that is essential to the creation of gRNAs. Csy4 is a member of CRISPR family.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 353
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 69


Usage and Biology

Conjugation Project

Conjugation project.jpg

Synthetic Circuit

Synthetic Circuit.jpg









No review score entered. UsernEnter the review inofrmation here.ame

We used this part as a reporter protein in a cell free system. We found that there was observable fluorescence from this part and experienced no issues with expressing it in a cell free system.

•••

2018 OUC-China

Csy4 (Csy6f), a member of CRISPR family.

Csy4 is a 21.4 kDa protein that binds and cleaves at the 3' side of a stable RNA hairpin structure via sequence- and structure-specific contacts. Csy4 binds its substrate RNA with extremely high affinity and functions as a single-turnover enzyme. Tight binding is mediated exclusively by interactions upstream of the scissile phosphate that allow Csy4 to remain bound to its product. Substrate specificity is achieved by RNA major groove contacts that are highly sensitive to helical geometry, as well as a strict preference for guanosine adjacent to the scissile phosphate in the active site. A highly basic a-helix docks into the major groove of the hairpin and contains multiple arginine residues that form a network of hydrogen.

Fig.1 The Csy4/Hairpin complex.

Background of 2018 OUC-China' project

This year, we design a toolkit focused on translational regulation, which is composed of a RNA endoribonuclease (Csy4) and a RNA module (hairpin). In our project, the cleavage function of Cys4 releases a cis-repressive RNA module (crRNA, paired with RBS) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. A Ribosome Binding Site (RBS) is an RNA sequence to which ribosomes can bind and initiate translation.

We want to achieve precise expression of proteins by using different Csy4 mutants. The aim is using one system to realize diverse expression. We focus on the sites which play an important role in binding and cleavage. Gln104 is located in the linker segment connecting the body of Csy4 to the arginine-rich area, which makes sequence-specific hydrogen bonding contacts in the major groove of the RNA stem to nucleotides G20 and A19. His29 is in its deprotonated form and functions as a general base during cleavage by activating the 2′-hydroxyl nucleophile through proton abstraction. The side chain of Tyr176 points into the active site and stacks on top of the His29 imidazole group, which plays a role in orienting His 29. Phe155 is to recognize the ssRNA-dsRNA junctions in RNA hairpin. Based on the molecular simulation and the theory of fluctuations, four mutants are chosen rationally: Q104A, H29A, Y176F, F155A.

Fig.2 Four key sites of wild type Csy4.