Difference between revisions of "Part:BBa K4088889"
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<partinfo>Ba_K4088889 short</partinfo>
 
<partinfo>Ba_K4088889 short</partinfo>
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<br/><br/>
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N-terminal fragment of β-lactamase fused with dCas13a.
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<br/><br/>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4088889 SequenceAndFeatures</partinfo>
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<br/><br/><br/><br/>
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==Usage and Biology==
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'''β-Lactamase'''<br/>
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β-Lactamase (AmpR) - the gene encoding beta-lactamase is a protein known for providing antibiotic resistance of bacteria to ampicillin, because it is able to destroy the beta-lactam ring. This ability allows beta-lactamase to be used as a reporter protein (reaction with nitrocefin, which changes color when the beta-lactam ring is destroyed). Simple, reliable, readily available, it is not difficult to express  in cells. These advantages have led us to use this particular reporter protein.
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<br/>
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N_lac and C_lac are genes encoding N and C fragments of beta-lactamase, respectively. We selected their sequence in a special way, taking into account the mechanism of protein splicing <ref> Perler FB. Protein splicing mechanisms and applications. IUBMB Life. 2005 Jul;57(7):469-76. https://doi.org/10.1080/15216540500163343</ref> . For this, we purposefully divided the ampR sequence in front of cysteine (you can also use threonine, the main thing is that they carry nucleophiles in the radical).
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<br/><br/>
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'''Linker'''<br/>
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Usually researchers use neutral amino acids like glycine and alanine as part of the linkers: it is desirable to make the linker neutral and important to not use proline and other charged amino acids. Hydrophobic amino acids can be added, e.g. serine. Aliphatic amino acids may also be added. Length of linkers is usually 6-8 amino acids, primarily less than 10 amino acids.  We use GGGGGG because it meets these criteria. (look at alphafold for it: but it does not start there and gives an error. we even asked our acquaintances to start it with our sequence - without success). So we apparently choose the approach of experimental verification of the linker.
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<br/><br/>
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'''dCas13a'''<br/>
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LwaCas13a has been reported to mediate more robust RNA-targeting activity than the LshCas13 system but requires a stabilizer fusion, for example, msfGFP for efficient interference activity <ref> Mahas, A., Aman, R. & Mahfouz, M. CRISPR-Cas13d mediates robust RNA virus interference in plants. Genome Biol 20, 263 (2019). https://doi.org/10.1186/s13059-019-1881-2</ref>. As a stabilizer fusion we use fragments of beta-lactamase thus improving targeting activity.
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<br/>
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dCas13a is a catalytically dead lwaCas13a enzyme (formerly C2c2) with R407A and R1046A mutations in HEPN-domain. The Cas13 family of enzymes contains at least 4 subtypes, including Cas13a, which is used in this Part. dCas13a interacts with gRNA about 65 nucleotides in length. dCas13a-gRNA complex detects complementary RNA in the sample. dCas13a is very specific variant of RNA detection, it can been customized to recognize single nucleotides on the target RNA.
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<br/><br/>
  
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N-terminal fragment of β-lactamase fused with dCas13a.<br/><br/>
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==References==
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β-Lactamase (AmpR) - <br/>
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N-terminal fragment of β-lactamase - <br/><br/>
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dCAS13a is a catalytically dead Cas13a enzyme (formerly C2c2). The Cas13 family of enzymes includes at least 4 families, for example Cas13a, which is used in this biobrick. dCas13a interacts with gRNA about 65 nucleotides in length. dCas13a protein complexes with gRNA detect complementary RNA in the sample. dCas13a is very specific variant of RNA detection, it can been customized to recognize single nucleotides on the target RNA.<br/><br/>
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4088889 SequenceAndFeatures</partinfo>
 
  
  

Revision as of 14:20, 21 October 2021


dCas13a-Nlact

N-terminal fragment of β-lactamase fused with dCas13a.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1346
    Illegal BglII site found at 1556
    Illegal BglII site found at 1823
    Illegal BglII site found at 2588
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]





Usage and Biology

β-Lactamase
β-Lactamase (AmpR) - the gene encoding beta-lactamase is a protein known for providing antibiotic resistance of bacteria to ampicillin, because it is able to destroy the beta-lactam ring. This ability allows beta-lactamase to be used as a reporter protein (reaction with nitrocefin, which changes color when the beta-lactam ring is destroyed). Simple, reliable, readily available, it is not difficult to express in cells. These advantages have led us to use this particular reporter protein.
N_lac and C_lac are genes encoding N and C fragments of beta-lactamase, respectively. We selected their sequence in a special way, taking into account the mechanism of protein splicing [1] . For this, we purposefully divided the ampR sequence in front of cysteine (you can also use threonine, the main thing is that they carry nucleophiles in the radical).

Linker
Usually researchers use neutral amino acids like glycine and alanine as part of the linkers: it is desirable to make the linker neutral and important to not use proline and other charged amino acids. Hydrophobic amino acids can be added, e.g. serine. Aliphatic amino acids may also be added. Length of linkers is usually 6-8 amino acids, primarily less than 10 amino acids. We use GGGGGG because it meets these criteria. (look at alphafold for it: but it does not start there and gives an error. we even asked our acquaintances to start it with our sequence - without success). So we apparently choose the approach of experimental verification of the linker.

dCas13a
LwaCas13a has been reported to mediate more robust RNA-targeting activity than the LshCas13 system but requires a stabilizer fusion, for example, msfGFP for efficient interference activity [2]. As a stabilizer fusion we use fragments of beta-lactamase thus improving targeting activity.
dCas13a is a catalytically dead lwaCas13a enzyme (formerly C2c2) with R407A and R1046A mutations in HEPN-domain. The Cas13 family of enzymes contains at least 4 subtypes, including Cas13a, which is used in this Part. dCas13a interacts with gRNA about 65 nucleotides in length. dCas13a-gRNA complex detects complementary RNA in the sample. dCas13a is very specific variant of RNA detection, it can been customized to recognize single nucleotides on the target RNA.

References

  1. ↑ Perler FB. Protein splicing mechanisms and applications. IUBMB Life. 2005 Jul;57(7):469-76. https://doi.org/10.1080/15216540500163343
  2. ↑ Mahas, A., Aman, R. & Mahfouz, M. CRISPR-Cas13d mediates robust RNA virus interference in plants. Genome Biol 20, 263 (2019). https://doi.org/10.1186/s13059-019-1881-2