Team:NEFU China/Parts

As the most basic module, the iGEM parts have significantly influenced the direction and improvement of our project. Plenty of parts have been used by the iGEMers every year that contribute to the success of many cases. This year, we have used several previous parts in our virus detection project. For the purpose of virus detection, we developed a colorimetric virus detection system based on G-quadruplex. We used recombinase polymerase amplification (RPA) to specifically amplify the gene III of M13 phage. Then, a short single-stranded DNA (ssDNA) is replaced by engineered DNA polymerases through targeted deletion technique. The replaced fragments are subsequently used as primers in the following rolling circle amplification (RCA). Finally, multiple G-quadruplex motifs produced by the RCA binds hemin to act as an active peroxidase on the substrate, 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS). Through this sequential and amplifying system, the virus DNA can be detected by the color change of the substrate. All of these parts have helped us to make our platform convenient and sensitive. At the same time, we also want to bring a number of novel parts to the iGEM to facilitate the research of the future teams.

Basic parts

Name	Number	Type	Description	Length
Phi29	BBa_K3894014	Coding	Phi29 is used to displace strand that are targeted, and to initiate chromogenic processes by polymerizing and displacing DNA in subsequent Rolling Circle Amplification(RCA).	1716bp
Klenow-SH3	BBa_K3894022	Coding	Klenow is mainly used for single strand replacement in our project.	2973bp
nCas9	BBa_K3894015	Coding	nCas9 is a mutation of Cas9 and is used to slice single-stranded DNA.	4107bp
ZF	BBa_K3894016	Coding	Through designing a ZF to bind a specific sequence and attaching it to a single strand-cleavage enzyme, we could generate a guided nickase to create a nick at a desirable site.	360bp
pBAD	BBa_K2913022	Promoter	The pBAD expression system allows tightly controlled, titratable expression of your protein through the regulation of specific carbon sources such as glucose, glycerol, and arabinose. pBAD is ideal for expressing toxic proteins and optimizing protein solubility in E. coli.	330bp
TrrnB	BBa_K2913020	Terminator	A terminator works well.	158bp
BbvCI-R1	BBa_K3894017	Coding	BbvCI-R1 is one of two heterogeneous subunits of the R.BbvCI enzyme.	855bp
ssDNA-G-quadruplex	BBa_K3894023	DNA	Single-stranded DNA-G-quadruplex is used as the template of the RCA, which amplifies G-quadruplex sequence.	74bp

Composite parts

Name	Number	Type	Description	Length
Nb.BbvCI-R2-Phi29	BBa_K3894024	Coding	The iGEM 2021NEFU_China designed Phi29 to connect Nb.BbvCI-R2, and achieved the connection between Phi29 and ZF through the assembly of the BbvCI-R1 and R2 subunits.	2607bp
BbvCI-R1-ZF	BBa_K3894025	Coding	The iGEM 2021NEFU_China designed the ZF-linked BbvC-R1 to replace nCas9-slig that could achieve anchoring and chain cleavage.	1221bp

Improved parts

Name	Number	Type	Description	Length
Klenow.mut-SH3	BBa_K3894012	Coding	The iGEM 2021NEFU_China created the D424A mutant of Klenow and constructed Klenow.mut-SH3 that theoretically lost its 3'→5' exonuclease activity.	2973bp
Nb.BbvCI-R2	BBa_K3894018	Coding	The iGEM 2021NEFU_China created the E177G mutant of R.BbvCI-R2. We used the R1⁺R2^- mutant, named as Nb.BbvCI, which can only cleaves the bottom chain.	885bp