Difference between revisions of "Part:BBa K2429007"

 
Line 4: Line 4:
  
 
Leptotrichia shahii deactivated Cas13a protein coding region. This part produces a deactivated version of the CRISPR protein Cas13a, which normally binds and cuts mRNA. With the deactivation sequence, the protein becomes catalytically inactive, and unable to cut the mRNA; however, it can still bind to the mRNA molecule.
 
Leptotrichia shahii deactivated Cas13a protein coding region. This part produces a deactivated version of the CRISPR protein Cas13a, which normally binds and cuts mRNA. With the deactivation sequence, the protein becomes catalytically inactive, and unable to cut the mRNA; however, it can still bind to the mRNA molecule.
 +
 +
This part includes the deactivated Leptotrichia shahii Cas13a protein coding region. This basic part is a pENTR vector that serves as an intermediate in the production process of a final expression vector. The final expression vector produces a deactivated version of the CRISPR protein Cas13a, which normally binds and cuts mRNA. With the deactivation sequence, the protein becomes catalytically inactive, and unable to cut the mRNA; however, it can still bind to the mRNA molecule.
 +
Without the deactivation sequence, a CRISPR protein known as Cas13a would be produced, which binds and cuts mRNA. Upon recognition of the mRNA, this protein exhibits "promiscuous" ribonuclease activity, not only cutting at base pairs along the targeted RNA molecule, but other nearby RNA molecules as well.
 +
 +
Typically, during the process of alternative splicing in eukaryotic cells, specific RNA binding proteins will bind to sequences or motifs on a pre-mRNA strand and eventually come together to form a spliceosome protein. This resulting protein will cleave the mRNA strand to exclude portions of the pre-mRNA (called introns) and the remaining sequences in the mature mRNA are known as exons.
 +
Our team used this protein in an attempt to control what exons would be included in an mRNA transcript by targeting the motifs in an intron, thus blocking splicing factors from binding and retaining an exon. Furthermore, our team tested variations of this protein (e.g. catalytically deactivated, additional domains) to see whether such variations would affect the splicing capabilities. This specific variation of the protein is catalytically inactive in bacteria, and this behavior is expected to carry over into mammalian cells. Additionally, the focus is on the protein's binding and blocking ability rather than the catalytic activity.
  
  

Revision as of 18:12, 23 October 2017


pENTR L. shahii dCas13a

Leptotrichia shahii deactivated Cas13a protein coding region. This part produces a deactivated version of the CRISPR protein Cas13a, which normally binds and cuts mRNA. With the deactivation sequence, the protein becomes catalytically inactive, and unable to cut the mRNA; however, it can still bind to the mRNA molecule.

This part includes the deactivated Leptotrichia shahii Cas13a protein coding region. This basic part is a pENTR vector that serves as an intermediate in the production process of a final expression vector. The final expression vector produces a deactivated version of the CRISPR protein Cas13a, which normally binds and cuts mRNA. With the deactivation sequence, the protein becomes catalytically inactive, and unable to cut the mRNA; however, it can still bind to the mRNA molecule. Without the deactivation sequence, a CRISPR protein known as Cas13a would be produced, which binds and cuts mRNA. Upon recognition of the mRNA, this protein exhibits "promiscuous" ribonuclease activity, not only cutting at base pairs along the targeted RNA molecule, but other nearby RNA molecules as well.

Typically, during the process of alternative splicing in eukaryotic cells, specific RNA binding proteins will bind to sequences or motifs on a pre-mRNA strand and eventually come together to form a spliceosome protein. This resulting protein will cleave the mRNA strand to exclude portions of the pre-mRNA (called introns) and the remaining sequences in the mature mRNA are known as exons. Our team used this protein in an attempt to control what exons would be included in an mRNA transcript by targeting the motifs in an intron, thus blocking splicing factors from binding and retaining an exon. Furthermore, our team tested variations of this protein (e.g. catalytically deactivated, additional domains) to see whether such variations would affect the splicing capabilities. This specific variation of the protein is catalytically inactive in bacteria, and this behavior is expected to carry over into mammalian cells. Additionally, the focus is on the protein's binding and blocking ability rather than the catalytic activity.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 294
    Illegal PstI site found at 1894
    Illegal PstI site found at 2263
    Illegal PstI site found at 2992
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 294
    Illegal PstI site found at 1894
    Illegal PstI site found at 2263
    Illegal PstI site found at 2992
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 294
    Illegal BglII site found at 638
    Illegal BglII site found at 1262
    Illegal BglII site found at 1658
    Illegal BglII site found at 1949
    Illegal BglII site found at 2039
    Illegal BglII site found at 3200
    Illegal BglII site found at 3287
    Illegal BamHI site found at 1
    Illegal BamHI site found at 4282
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 294
    Illegal PstI site found at 1894
    Illegal PstI site found at 2263
    Illegal PstI site found at 2992
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 294
    Illegal PstI site found at 1894
    Illegal PstI site found at 2263
    Illegal PstI site found at 2992
    Illegal NgoMIV site found at 4239
    Illegal NgoMIV site found at 4258
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 624
    Illegal SapI.rc site found at 730
    Illegal SapI.rc site found at 1600
    Illegal SapI.rc site found at 2434