Difference between revisions of "Part:BBa K4088890"
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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>. | ||
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| − | For this part we used the DnaE intein. This intein is identified as a naturally occurring split mini-intein in Synechocystis sp. PCC6803 | + | For this part we used the 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>. |
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Revision as of 15:13, 21 October 2021
dCas13a-Clact-int
С-terminal fragment of β-lactamase fused with dCas13a and C-terminal intein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1907
Illegal BglII site found at 2117
Illegal BglII site found at 2384
Illegal BglII site found at 3149
Illegal XhoI site found at 4078 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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
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).
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.
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. This intein is identified as a naturally occurring split mini-intein in Synechocystis sp. PCC6803 [4].
How our system would work
When the two proteins (this one and BBa_K4088891) 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.
References
- ↑ Perler FB. Protein splicing mechanisms and applications. IUBMB Life. 2005 Jul;57(7):469-76. https://doi.org/10.1080/15216540500163343
- ↑ 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
- ↑ 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
- ↑ 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