Difference between revisions of "Part:BBa K4088891"

Revision as of 20:59, 21 October 2021

dCas13a-Nlact-int

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


Sequence and Features

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. 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 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 Npu DnaE intein. This intein is identified as a naturally occurring split mini-intein in Synechocystis sp. PCC6803 ^[4]. Its first amino acid is cysteine which is important for protein trans-splicing to take place.

How our system would work

When the two proteins (this one and 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.

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. ↑ 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