Team:Peking/Contribution

Contribution

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

sgRNA from parts.igem.org

Cutting with BsaI

processing with BsaⅠ

Assembly with target sequence oligo

fully functional sgRNA generator

Base editor circuit design

ABE

ABE

CBE

CBE

DdCBE

Core part of this circuit is the fusion protein, which is as shown below

DddA fusion

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)

DdCBE

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.