Regulatory

Part:BBa_K2926003

Designed by: Ina Schmitt   Group: iGEM19_Bielefeld-CeBiTec   (2019-09-21)
Revision as of 22:01, 21 September 2019 by Ischmitt (Talk | contribs)


GALL yeast promoter


Cas 13a Lsh This Leptotcichia shahii

For the full characterization of this part, see BBa (Lbu) 


Cas13a Lwa

Leptotrichia wadei



For the full characterization of this part, see BBa (Lbu)


Cas 13a Lbu

Usage and Biology

Cas13a from Leptorichia bucallis (Lbu) is a single-effector RNA guided ribonuclease. Upon recognition of its RNA target the activated Cas13a performs RNA cleavage of nearby non-target RNAs. This is called "colleteral" RNA cleavage and can lead to cell death in prokaryotes.(Liu et al., 2017) CRISPR (clustered regulary interspaced short palindormic repeat) is an adaptive immune systems that provides protection against vaious threats to the cell. Cas13a is part of a CRISPR-Cas system which protects the cell against mobile genetic elemens like viruses, transposable elements and conjugative palsmids. (Koonin and Zhang, 2017) So far different verions of the protein where found in Leptorichia bucallis, Leptotrichia wadei and Leptotcichia shahii. (Gootenberg et al., 2017) The CRISPR cluster, containing the guide RNAs for Cas13a is transciped as pre-CRISPR RNA (pre-crRNA) and processed into crRNA. Unlike other Cas effectors, Cas13a is capable of processing the pre-crRNA by itself. It is cleaving the pre-crRNA 4 bais pairs upstram of the stem-loop, binding the cr-RNA sequence and then building the cas13a:crRNA surveillance complex. (East-seletsky et al., 2017)

File:T--Bielefeld-CeBiTec--Background Cas13a Background.png
As soon as the Cas13a:crRNA surveillance complex is formed, it can be activated by the detection of its target RNA. Aktivation leads to colleteral cleavage of all nearby RNA strands, resulting in cell death.

Grafik zu pre-crRNA processing

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 AgeI site found at 47
  • 1000
    COMPATIBLE WITH RFC[1000]



Computational Analysis of Cas13a Lbu

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Plasmid Design

Sequencing Results

Transciption Analyses via (q)RT-PCR


Protein Purification

Intein dependtend purification

SDS PAGE

MALDI Analyses


blabla in vitro Assay


gRNA Modeling???



Cultivations

Shake Flask Cultivation

Micocultivations

Outlook

References

[edit]
Categories
//chassis/eukaryote/yeast
//promoter
Parameters
biology