Difference between revisions of "Part:BBa K4088888"

(→‎How our system would work)
 
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==Usage and Biology==
 
==Usage and Biology==
 
'''β-Lactamase'''<br/>
 
'''β-Lactamase'''<br/>
−
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.
+
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. The molecule is “cut” between: Trp93 and His94
 
<br/>
 
<br/>
 
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).  
 
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).  
 
<br/><br/>
 
<br/><br/>
 
'''Linker'''<br/>
 
'''Linker'''<br/>
−
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.  
+
Usually, researchers use neutral amino acids like glycine and alanine as part of the linkers: it is desirable to make the linker neutral. Hydrophobic amino acids can be added, e.g. serine. Aliphatic amino acids may also be added. The length of linkers is usually 6-8 amino acids, primarily less than 10 amino acids.  We use GGGGGG because it meets these criteria.
 
<br/><br/>
 
<br/><br/>
 
'''dCas13a'''<br/>
 
'''dCas13a'''<br/>
−
Cas13a is a classic RNA-targeting nuclease which is widely used in a variety of diagnostics methods. It is one enzymes of Cas13 family which contains at least 4 subtypes, including Cas13a, used as our Parts BioBrick. We took LwaCas13a, identified from Leptotrichia wadei (Lwa).<br/>
+
Cas13a is a classic RNA-targeting nuclease that is widely used in a variety of diagnostics methods. It is one enzyme of Cas13 family which contains at least 4 subtypes, including Cas13a, used as our Parts BioBrick. We took LwaCas13a, identified from Leptotrichia wadei (Lwa).<br/>
  
−
dCas13a is a catalytically dead lwaCas13a enzyme (formerly C2c2) with R407A and R1046A mutations in HEPN-domain. 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.<br/>
+
dCas13a is a catalytically dead lwaCas13a enzyme (formerly C2c2) with R407A and R1046A mutations in HEPN-domain. dCas13a interacts with gRNA about 65 nucleotides in length. dCas13a-gRNA complex detects complementary RNA in the sample. dCas13a is a very specific variant of RNA detection, it can be customized to recognize single nucleotides on the target RNA.<br/>
  
 
LwaCas13a has been reported to mediate more robust RNA-targeting activity than other Cas13 systems like LshCas13, but it 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.<br/>
 
LwaCas13a has been reported to mediate more robust RNA-targeting activity than other Cas13 systems like LshCas13, but it 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.<br/>
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==How our system would work==
 
==How our system would work==
  
−
When the two proteins (this one and <partinfo>BBa_K4088889</partinfo>) come together due to landing on the RNA, a whole beta-lactamase is formed. It cleaves the nitrocefin and the appearance of red coloration indicates the presence of the RNA in solution.
+
When the two proteins (this one and <partinfo>BBa_K4088889</partinfo>) come together due to landing on the RNA, a whole beta-lactamase is formed. It cleaves the nitrocefin and the appearance of red coloration indicates the presence of the RNA in the solution.
 
<br/><br/>
 
<br/><br/>
  

Latest revision as of 02:00, 22 October 2021


dCas13a-Clact

С-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 1580
    Illegal BglII site found at 1790
    Illegal BglII site found at 2057
    Illegal BglII site found at 2822
    Illegal XhoI site found at 3751
  • 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. The molecule is “cut” between: Trp93 and His94
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. Hydrophobic amino acids can be added, e.g. serine. Aliphatic amino acids may also be added. The length of linkers is usually 6-8 amino acids, primarily less than 10 amino acids. We use GGGGGG because it meets these criteria.

dCas13a
Cas13a is a classic RNA-targeting nuclease that is widely used in a variety of diagnostics methods. It is one enzyme of Cas13 family which contains at least 4 subtypes, including Cas13a, used as our Parts BioBrick. We took LwaCas13a, identified from Leptotrichia wadei (Lwa).

dCas13a is a catalytically dead lwaCas13a enzyme (formerly C2c2) with R407A and R1046A mutations in HEPN-domain. dCas13a interacts with gRNA about 65 nucleotides in length. dCas13a-gRNA complex detects complementary RNA in the sample. dCas13a is a very specific variant of RNA detection, it can be customized to recognize single nucleotides on the target RNA.

LwaCas13a has been reported to mediate more robust RNA-targeting activity than other Cas13 systems like LshCas13, but it 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.


How our system would work

When the two proteins (this one and BBa_K4088889) come together due to landing on the RNA, a whole beta-lactamase is formed. It cleaves the nitrocefin and the appearance of red coloration indicates the presence of the RNA in the solution.

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