Difference between revisions of "Part:BBa K4088891"

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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.
 
<br/>
 
<br/>
−
N_lac is a gene encoding N fragment of beta-lactamase. Its sequence does not affect the process 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> .  
+
N_lac is a gene encoding N-fragment of beta-lactamase. Its sequence does not affect the process 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> .  
 
<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 without any 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.
+
Usually, researchers use neutral amino acids like glycine and alanine as part of the linkers: it is desirable to make the linker neutral and without any charged amino acids. 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 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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Intein is a segment of a protein that can self-catalytically excised out and ligate the remaining parts of the protein (N- and C-exteins) with a peptide bond <ref>Anraku, Y., Mizutani, R. and Satow, Y. (2005), Protein Splicing: Its Discovery and Structural Insight into Novel Chemical Mechanisms. IUBMB Life, 57: 563-574. https://doi.org/10.1080/15216540500215499</ref>.
 
Intein is a segment of a protein that can self-catalytically excised out and ligate the remaining parts of the protein (N- and C-exteins) with a peptide bond <ref>Anraku, Y., Mizutani, R. and Satow, Y. (2005), Protein Splicing: Its Discovery and Structural Insight into Novel Chemical Mechanisms. IUBMB Life, 57: 563-574. https://doi.org/10.1080/15216540500215499</ref>.
 
<br/>
 
<br/>
−
For this part we used the Npu DnaE intein. This intein is identified as a naturally occurring split mini-intein in Synechocystis sp. PCC6803 <ref>Wu, H., Hu, Z., & Liu, X. Q. (1998). Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proceedings of the National Academy of Sciences of the United States of America, 95(16), 9226–9231. https://doi.org/10.1073/pnas.95.16.9226</ref>. Its first amino acid is cysteine which is important for protein trans-splicing to take place.
+
For this part, we used the DnaE intein. DnaE - alpha subunit of the DNA polymerase III intein. This intein is identified as a naturally occurring split intein in Nostoc punctiforme <ref>Oeemig JS, Aranko AS, Djupsjöbacka J, Heinämäki K, Iwaï H. Solution structure of DnaE intein from Nostoc punctiforme: structural basis for the design of a new split intein suitable for site-specific chemical modification. FEBS Lett. 2009 May 6;583(9):1451-6. https://doi.org/10.1016/j.febslet.2009.03.058</ref>. Split inteins can also be created artificially by separating the inteins' primary sequence into two pieces on the genetic level<ref>Zettler, Joachim, Schütz, Vivien and Mootz, Henning D.(2009), The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction, FEBS Letters, 583, doi: 10.1016/j.febslet.2009.02.003</ref>.<br/>
 +
The rates for protein trans-splicing of the Npu DnaE intein that are 5.5-fold higher (at 37 °C) than the rate reported for the split Sce VMA intein and up to about 33 to 170-fold higher than found for the Ssp DnaE intein. The splice product yields obtained with the Npu intein were nearly unaffected in the temperature range: 6°, 12°, 25°, 37° with the highest rate observed at 37 °C. Split inteins can also be created artificially by separating the inteins’ primary sequence into two pieces on the genetic level. <br/>
 +
The most important properties of the split intein tools are [5]:  <br/>
 +
1. the yield of the protein trans-splicing reaction <br/>
 +
2. its rate constant <br/>
 +
3. the solubility of the intein fragments <br/>
 +
4. their tolerance towards different fused “substrate” exteins. <br/>
 +
 
 +
For this part we used the N fragment of the Npu DnaE intein (<partinfo>BBa_K4088892</partinfo>).
 
<br/><br/>
 
<br/><br/>
  
 
==How our system would work==
 
==How our system would work==
−
When the two proteins (this one and [https://parts.igem.org/Part:BBa_K4088890 BBa_K4088890]) come together due to landing on RNA, trans splicing occurs, this process needs to be stimulated with DTT or 2-mercaptoethanol. The result of trans splicing is a peptide bond between the beta lactamase fragments to form the active enzyme. It cleaves nitrocefin and the appearance of red coloration indicates the presence of the RNA in solution.
+
Our viral RNA protein detection system comprises the chimeric proteins, dcas13a-C_lac-C_int (<partinfo>BBa_K4088890</partinfo>) and dcas13a-N_lac-N_int (this one). Detection is possible through protein trans-splicing, during which parts of the intein are assembled into a whole intein, which is cut out, forming a peptide bond between the beta-lactamase fragments, thereby creating a functional reporter protein. We stimulate trans-splicing with DTT or 2-mercaptoethanol. The problem of proper functioning of proteins in fused protein is solved by introducing a flexible Polyglycine linker sequence between the dcas13a gene and the gene of the beta-lactamase fragment.  
 
<br/><br/>
 
<br/><br/>
 +
 
==References==
 
==References==
  

Latest revision as of 02:08, 22 October 2021


dCas13a-Nlact-int

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


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
    Illegal BglII site found at 4082
  • 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 is a gene encoding N-fragment of beta-lactamase. Its sequence does not affect the process of protein splicing[1] .

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 without any charged amino acids. 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 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.


Intein
Intein is a segment of a protein that can self-catalytically excised out and ligate the remaining parts of the protein (N- and C-exteins) with a peptide bond [3].
For this part, we used the DnaE intein. DnaE - alpha subunit of the DNA polymerase III intein. This intein is identified as a naturally occurring split intein in Nostoc punctiforme [4]. Split inteins can also be created artificially by separating the inteins' primary sequence into two pieces on the genetic level[5].
The rates for protein trans-splicing of the Npu DnaE intein that are 5.5-fold higher (at 37 °C) than the rate reported for the split Sce VMA intein and up to about 33 to 170-fold higher than found for the Ssp DnaE intein. The splice product yields obtained with the Npu intein were nearly unaffected in the temperature range: 6°, 12°, 25°, 37° with the highest rate observed at 37 °C. Split inteins can also be created artificially by separating the inteins’ primary sequence into two pieces on the genetic level.
The most important properties of the split intein tools are [5]:
1. the yield of the protein trans-splicing reaction
2. its rate constant
3. the solubility of the intein fragments
4. their tolerance towards different fused “substrate” exteins.

For this part we used the N fragment of the Npu DnaE intein (BBa_K4088892).

How our system would work

Our viral RNA protein detection system comprises the chimeric proteins, dcas13a-C_lac-C_int (BBa_K4088890) and dcas13a-N_lac-N_int (this one). Detection is possible through protein trans-splicing, during which parts of the intein are assembled into a whole intein, which is cut out, forming a peptide bond between the beta-lactamase fragments, thereby creating a functional reporter protein. We stimulate trans-splicing with DTT or 2-mercaptoethanol. The problem of proper functioning of proteins in fused protein is solved by introducing a flexible Polyglycine linker sequence between the dcas13a gene and the gene of the beta-lactamase fragment.

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
  3. ↑ Anraku, Y., Mizutani, R. and Satow, Y. (2005), Protein Splicing: Its Discovery and Structural Insight into Novel Chemical Mechanisms. IUBMB Life, 57: 563-574. https://doi.org/10.1080/15216540500215499
  4. ↑ Oeemig JS, Aranko AS, Djupsjöbacka J, Heinämäki K, Iwaï H. Solution structure of DnaE intein from Nostoc punctiforme: structural basis for the design of a new split intein suitable for site-specific chemical modification. FEBS Lett. 2009 May 6;583(9):1451-6. https://doi.org/10.1016/j.febslet.2009.03.058
  5. ↑ Zettler, Joachim, Schütz, Vivien and Mootz, Henning D.(2009), The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction, FEBS Letters, 583, doi: 10.1016/j.febslet.2009.02.003