Part:BBa_K3853055
R0085-C0012-B2002-his-tag-dCas9-SpyCatcher-B0012
| Part Information | |
|---|---|
| RFC standard | RFC 25;RFC 1000 |
| Partner part | BBa_K3853008:SpyTag-MnP |
| BBa_K3853009:SpyTag-AAO | |
| BBa_K3853010:SpyTag-HFB1 | |
| Composite part | BBa_K3853055 |
| Submitted by | CPU_CHINA |
This part consists of defective endonuclease dCas9(BBa_K3853003), modified immunoglobulin-like domain SpyCatcher and Ser/Gly linker(S/G linker). S/G linker is applied to link dCas9 with SpyCatcher. dCas9 can specifically target and bind dsDNA under the mediation of single-guide RNA (sgRNA), meanwhile, SpyCatcher is usually used for protein ligation. dCas9-SpyCatcher has been reported as connection system to assemble protein-nucleic-acid complex. We use BBa_K3853055 to construct the expression system to express and purify the protein.
Biology
The dCas9 gene use in this experiment was derived from two nuclease cleavage active domains of Streptococcus pyogenes Cas9 gene, RuvC and HNH, and simultaneously inactivate by artificial single point mutation (D10A & H840A). dCas9 can recognize with single-guide RNA (sgRNA), and combine with it to form a complex but does not play a cutting role. Through complementary base pairing between sgRNA and artificially design dsDNA, multiple dCas9 can be indirectly connect to dsDNA to form a complex.
The SpyCatcher/SpyTag system is a bio-coupling technology connect by isopeptide bonds, which is derive from the modification of immunoglobulin like collagen adhesin domain (CnaB2)[1]. In the CnaB2 domain, Glu77 catalyzes the nucleophilic attack on the carbonyl carbon of Asp117 by unprotonic amines in nearby Lys31, resulting in the spontaneous formation of intramolecular isopeptide bonds. Based on the principle of isopeptide bond formation, CnaB2 is split into two parts that could be recombined and covalently reacted: a peptide containing active Asp representing the c-terminal β chain and a protein partner from the rest of the protein. After modification, the peptide SpyTag is formed with 13 amino acids (AHIVMVDAYKPTK) and the protein Spycatcher with 138 amino acids (15 kDa). The two can spontaneously form isopeptide bond and bond stably[2](Fig. 1). This part is the fusion of dCas9 and SpyCatcher.
Fig. 1 Schematic diagram of the working mechanism of protein fusion using the SpyTag/SpyCatcher system.
Usage
In 2021 CPU_CHINA project, we fuse dCas9 with SpyCatcher, making it into a biological module that can fuse with various "standardized enzymes" link to SpyTag. By virtue of its binding ability with DNA, some enzymes and proteins that can play a synergistic role can be fixed in a relatively close space distance, in order to obtain a more efficient degradation of PE plastic molecular machine. we use the T7 promoter (BBa_R0085) with lac operator (BBa_C0012) to regulate protein expression. We use BBa_K3853055(Fig. 2) to construct the expression system to express and purify the protein.
Fig. 2 Gene circuit of dCas9-SpyCatcher.
Characterization
1. Identification
We used PCR to obtain three homologous recombination fragments (dCas9; vector of pET-28a; G3S*4-SpyCatcher) (Fig. 3) for the construction of expression plasmid. The successfully recombined plasmid was transformed into E.coli DH5α, and the monoclonal colonies with positive transformation results were selected for subsequent sequencing verification ( File 1 ). The experimental results showed that the plasmid was successfully constructed.
Fig. 3 Agarose gel electrophoresis of PCR products. Vector refers to plasmid pET-28a. G3S*4 refers to quadruple Ser/Gly link.
2. Purification and Proof of the expression
we uesd T7 promoter to express dCas9-SpyCatcher in E.coli DH5α in our composite part BBa_K3853055. we used Ni-NTA affinity column to purify dCas9-SpyCatcher. Target bands could be observed at the position of about 176.2 kDa (Fig. 4), which means the protein of dCas9-SpyCatcher could be successfully expressed, and the related gene worked well.
Fig. 4 SDS-PAGE of purified products of dCas9-SpyCatcher. 50 mM imidazole and 500 mM imidazole represent corresponding eluates with different imidazole concentrations, and the binding buffer is generated by equilibrating the Ni column after elution with 500 mM imidazole.
Since we had introduced 6×His tag at the N-terminus of dCas9-SpyCatcher, we used his-antibody as the primary antibody to perform Western Blot analysis to detect the expression status of the target protein. The protein band of dCas9-SpyCatcher could be observed clearly (Fig. 5), meaning that the related gene functioned well.
Fig. 5 Western Blot of dCas9-SpyCatcher. The concentration of dCas9-SpyCatcher: lane 1: 0.25 mg/ml; lane 2: 0.5 mg/ml; lane 3: 1 mg/ml.
3. Functional Verification
We incubated SpyTag-MnP and dCas9-SpyCatcher together to verify the combination of SpyCatcher and SpyTag. Ensure that dCas9-SpyCatcher functions normally. The samples before and after the incubation were run for verification. SDS-PAGE (Fig. 4) showed that SpyTag-MnP and dCas9-SpyCatcher were normally combined.
Fig. 6 SDS-PAGE of the combination of SpyTag-MnP and dCas9-SpyCatcher. Lane 1: SpyTag-MnP; Lane 2: dCas9-SpyCatcher; Lane 3: SpyTag-MnP mixed with dCas9-SpyCatcher.
References
[1] Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109, E690-697, doi:10.1073/pnas.1115485109 (2012).
[2] Reddington, S. C. & Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Current opinion in chemical biology 29, 94-99, doi:10.1016/j.cbpa.2015.10.002 (2015).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1170
Illegal BglII site found at 4790 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 5174
Illegal AgeI site found at 4518 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 5601
| None |