Team:Peking/Contribution
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Parts list
| Registry | Type | Description |
|---|---|---|
| BBa_K3645000 | coding | G1333 DddA(tox)-C |
| BBa_K3645001 | coding | G1333 DddA(tox)-N |
| BBa_K3645002 | coding | G1397 DddA(tox)-C |
| BBa_K3645003 | coding | G1397 DddA(tox)-N |
| BBa_K3645004 | coding | MS2 Coat Protein |
| BBa_K3645005 | coding | UGI |
| BBa_K3645006 | coding | Mutated TadA |
| BBa_K3645007 | coding | dCas9 for ABE |
| BBa_K3645008 | coding | TadA-dCas9 |
| BBa_K3645009 | coding | CDA-UGI |
| BBa_K3645010 | coding | dCas9 for CBE |
| BBa_K3645011 | coding | dCas9-CDA-UGI |
| BBa_K3645014 | coding | 10AA linker |
| BBa_K3645015 | coding | 15AA linker |
| BBa_K3645018 | coding | G1333C-linker-MS2CP-linker-UGI |
| BBa_K3645019 | coding | G1333N-linker-MS2CP-linker-UGI |
| BBa_K3645020 | coding | G1397C-linker-MS2CP-linker-UGI |
| BBa_K3645021 | coding | G1397N-linker-MS2CP-linker-UGI |
| BBa_K3645012 | composite | ABE circuit |
| BBa_K3645013 | composite | CBE circuit |
| BBa_K3645016 | RNA | sgRNA generator |
| BBa_K3645017 | RNA | sgRNA(with 2xMS2) generator |
Parts design
sgRNA assembly
This below is the sgRNA generator(assembly method below is the same for sgRNA(with 2xMS2) generator)
Getting the part
Cutting with BsaI
Assembly with target sequence oligo
Base editor circuit design
ABE
CBE
DdCBE
Core part of this circuit is the fusion protein, which is as shown below
here, we show how it would be assembled into the dCas9 circuit, but actual functional DdCBE needs a pair of DddA fusion protein (C- & N- terminal)
Citation
[1] Madej T, Lanczycki CJ, Zhang D, Thiessen PA, Geer RC, Marchler-Bauer A, Bryant SH. " MMDB and VAST+: tracking structural similarities between macromolecular complexes. Nucleic Acids Res. 2014 Jan; 42(Database issue):D297-303
[2] Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature. 2017 Nov 23;551(7681):464-471. doi: 10.1038/nature24644. Epub 2017 Oct 25. Erratum in: Nature. 2018 May 2;: PMID: 29160308; PMCID: PMC5726555.
[3] Banno S, Nishida K, Arazoe T, Mitsunobu H, Kondo A. Deaminase-mediated multiplex genome editing in Escherichia coli. Nat Microbiol. 2018 Apr;3(4):423-429. doi: 10.1038/s41564-017-0102-6. Epub 2018 Feb 5. PMID: 29403014.