Difference between revisions of "Part:BBa K4088891"

Revision as of 14:46, 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 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.

Intein We used DnaE.

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