Team:CPU CHINA/partsOverview

This year, among the 30 parts designed, 19 of them are basic parts and the rest are composite parts. All of the parts are designed on our own, and most of them have been tested to ensure their reliability.

Click here to learn more about our project to understand the purpose of each parts! Click the part number to see detailed information of the parts we design!

Basic Parts

Among the 19 basic parts designed this year by ourselves, BBa_K3853008 is our favorite one, which is the coding sequence of manganese peroxidase derived from Phanerochaete chrysosporium. The translated product of this part enables our multi-enzyme complex to degrade polyethylene efficiently. Besides, many other basic parts have also been designed to construct our project.

Favorite	Part Name	Type	Description	Designer	Length
	BBa_K3853000	coding	MnP	Peng Luo	1074
	BBa_K3853001	coding	AAO	Peng Luo	1719
	BBa_K3853002	coding	HFB1	Peng Luo	246
	BBa_K3853003	coding	dCas9	GongYu Liu	4104
	BBa_K3853004	coding	SpyTag	Peng Luo	39
	BBa_K3853005	coding	SpyCatcher	Peng Luo	351
	BBa_K3853006	DNA	dsDNA	GongYu Liu	189
	BBa_K3853007	DNA	gRNA scaffold	GongYu Liu	80
♥	BBa_K3853008	coding	his-tag-SpyTag-MnP	Peng Luo	1248
	BBa_K3853009	coding	his-tag-SpyTag-AAO	Peng Luo	1893
	BBa_K3853010	coding	his-tag-SpyTag-HFB1	Peng Luo	420
	BBa_K3853011	coding	dCas9-SpyCatcher	GongYu Liu	4530
	BBa_K3853012	coding	sfGFP	Peng Luo	720
	BBa_K3853013	coding	MnP(E74P)	RuYi Ma	1074
	BBa_K3853014	coding	MnP(E74M)	RuYi Ma	1074
	BBa_K3853015	coding	MnP(D182T)	RuYi Ma	1074
	BBa_K3853016	coding	MnP(S232P)	RuYi Ma	1074
	BBa_K3853017	coding	MnP(E74L)	RuYi Ma	1074
	BBa_K3853018	coding	MnP(S78P)	RuYi Ma	1074
	BBa_K3853019	coding	his-tag	Peng Luo	1074

Composite Parts

A series of composite parts have been designed by us this year, and most of them are used for the assembly of the PE-degrading complex in our project throughout the season.

To achieve this assembly, we designed fusion enzymes and proteins containing SpyTag and fusion deactivated CRISPR-associated protein 9 (dCas9) containing SpyCatcher, enabling the incubation-induced connection of multi-protein complexes via covalent bond formation mediated by the two Spy domains. Subsequently, these multi-protein complexes could anchor to designated positions on the designed double-stranded DNA (dsDNA) template through the guidance of dCas9-combined sgRNAs.

Our favorite composite part is BBa_K3853051 , which contains the sequence of manganese peroxidase (MnP) for polyethylene degradation, and the sequence of SpyTag for multi-domain complex formation, as well as sequences of AOX1 promoter and AOX1 terminator, enabling its efficient inducible expression in Pichia pastoris.

Favorite	Part Name	Type	Description	Designer	Length
	BBa_K3853050	composite	P_AOX1-α-factor-sfGFP-AOX1 Terminator	Peng Luo	2136
♥	BBa_K3853051	composite	P_AOX1-α-factor-his-tag-SpyTag-MnP-AOX1 Terminator	Peng Luo	2664
	BBa_K3853053	composite	P_AOX1-α-factor-his-tag-SpyTag-AAO-AOX1 Terminator	Peng Luo	3309
	BBa_K3853054	composite	P_AOX1-α-factor-his-tag-SpyTag-HFB1-AOX1 Terminator	Peng Luo	1836
	BBa_K3853055	composite	R0085-C0012-B2002-his-tag-dCas9-SpyCatcher-B0012	Peng Luo	5758
	BBa_K3853056	composite	P_AOX1-α-factor-his-tag-MnP(E74P)-AOX1 Terminator	Peng Luo	2508
	BBa_K3853057	composite	P_AOX1-α-factor-his-tag-MnP(E74M)-AOX1 Terminator	Peng Luo	2508
	BBa_K3853058	composite	P_AOX1-α-factor-his-tag-MnP(D182T)-AOX1 Terminator	Peng Luo	2508
	BBa_K3853059	composite	P_AOX1-α-factor-his-tag-MnP(S232P)-AOX1 Terminator	Peng Luo	2508
	BBa_K3853060	composite	P_AOX1-α-factor-his-tag-MnP(E74L)-AOX1 Terminator	Peng Luo	2508
	BBa_K3853061	composite	P_AOX1-α-factor-his-tag-MnP(S78P)-AOX1 Terminator	Peng Luo	2508

Part Collection

This year, we developed a part collection related to the CRSIPR/dCas9 anchoring system. By introducing SpyTag into the N-terminus of different proteins to develop fusion proteins, their connection to dCas9-SpyCatcher and the following sgRNA-guided anchoring on dsDNA can be realized. The type, quantity and proportion of anchoring components will remarkably affect their individual function, as well as the overall performance of the complex. It depends on how the system designer would like to arrange the dCas9-binding sequence on dsDNA, and how the assembly process is conducted through specific order for acquiring expected component array of the complex.

For our part collection, we characterized three different fusion proteins, i.e. SpyTag-MnP, SpyTag-AAO, SpyTag-HFB1, and uploaded their complete and detailed characterization data to the iGEM part registry. We hope these organized information can be instructive for the design and charaterization of upcoming teams who wish to adopt these protein-nucleic-acid complex.

Peptide segment	Fusion	Part Name	Type	Description	Designer	Length
SpyTag	N-terminal	BBa_K3853051	composite	P_AOX1-α-factor-his-tag-SpyTag-MnP-AOX1 Terminator	Peng Luo	2664
		BBa_K3853053	composite	P_AOX1-α-factor-his-tag-SpyTag-AAO-AOX1 Terminator	Peng Luo	3309
		BBa_K3853054	composite	P_AOX1-α-factor-his-tag-SpyTag-HFB1-AOX1 Terminator	Peng Luo	1836