In this year's project, our team designed a total of 20 parts, 14 of which were basic parts and the rest were composite parts. All of the parts are designed around our project, and every one can browse the detailed information on the CPU_CHINA 2022 iGEM Team Parts page or by clicking on the part numbers below.
Among all the basic parts, our favorite one is BBa_K4308005, which we discovered and decided to refine its application in in vitro diagnostics using the CRISPR-CasΦ system. To improve its cleavage efficiency and sequence recognition specificity, our team optimized the design of key active sites of CasΦ protein and its crRNA, obtained some of their mutants, and screened the CasΦ with the fastest cleavage BBa_K4308017 and the crRNA with the lowest mismatch rate (H4) BBa_K4308023.
| Part number | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K4308000 | Primer | Forward Primer for RPA of LINC00857 | Jun Zhang | 35 |
| BBa_K4308001 | Primer | Reverse Primer for RPA of LINC00857 | Jun Zhang | 30 |
| BBa_K4308002 | Primer | Primer for RCA | Jun Zhang | 24 |
| BBa_K4308003 | DNA | Padlock probe for RCA | Jun Zhang | 78 |
| BBa_K4308004 | Coding | MBD-SA | Jun Zhang | 798 |
| BBa_K4308005 | Coding | CasΦ/ Cas12j | Jun Zhang | 2301 |
| BBa_K4308006 | Coding | Cas12f / Cas14a | Jun Zhang | 2784 |
| BBa_K4308007 | RNA | Cas12a1_crRNA | Jun Zhang | 41 |
| BBa_K4308008 | RNA | CasΦ_crRNA | Jun Zhang | 45 |
| BBa_K4308009 | RNA | Cas12f_crRNA | Jun Zhang | 181 |
| BBa_K4308015 | Coding | CasΦ(n) | Jun Zhang | 2292 |
| BBa_K4308016 | Coding | CasΦ(v) | Jun Zhang | 2241 |
| BBa_K4308017 | Coding | CasΦ(Neg-K) | Jun Zhang | 2292 |
| BBa_K4308023 | RNA | CasΦ_crRNA(H4) | Jun Zhang | 53 |
Our team has designed a series of Composite Parts, most of which are assembled for the CRISPR-CasΦ system to provide better cleavage efficiency and sequence recognition specificity. In addition, we designed a fusion protein for the assembly of our nanostructures to further improve the overall detection system.
| Part number | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K4308031 | Composite | Cas12a1-crRNA-LINC00857 | Jun Zhang | 3782 |
| BBa_K4308032 | Composite | CasΦ-crRNA-LINC00857 | Jun Zhang | 2346 |
| BBa_K4308033 | Composite | Cas12f-crRNA-LINC00857 | Jun Zhang | 2965 |
| BBa_K4308034 | Composite | CasΦ(n)-crRNA-LINC00857 | Jun Zhang | 2337 |
| BBa_K4308035 | Composite | CasΦ(v)-crRNA-LINC00857 | Jun Zhang | 2286 |
| BBa_K4308036 | Composite | CasΦ(Neg-K)-crRNA-LINC00857 | Jun Zhang | 2337 |
Our team focused on the refinement of CRISPR-CasΦ system in in vitro diagnostics. To improve its cleavage efficiency and sequence recognition specificity, our team optimized the design of key active sites of CasΦ and its crRNA to obtain greater clinical value for nucleic acid diagnosis. Finally, we successfully obtained CasΦ protein BBa_K4308005 with its three mutants BBa_K4308015, BBa_K4308016, and BBa_K4308017. Among them, BBa_K4308017 (Mut-4 mutant) has the fastest cleavage rate. Meanwhile, we designed several crRNAs with hairpin structures BBa_K4308008, and screened the crRNA with the lowest mismatch rate (H4) BBa_K4308023 by adjusting the length of the hairpin structure to improve the detection efficiency of the CRISPR-Cas system for single-base mismatch targets. finally, we optimized the CasΦ(Neg-K)/crRNA(H4) also known as Mut-4/H4 system BBa_K4308032 for subsequent disease detection.
| Part number | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K4308005 | Coding | CasΦ/ Cas12j | Jun Zhang | 2301 |
| BBa_K4308008 | RNA | CasΦ_crRNA | Jun Zhang | 45 |
| BBa_K4308015 | Coding | CasΦ(n) | Jun Zhang | 2292 |
| BBa_K4308016 | Coding | CasΦ(v) | Jun Zhang | 2241 |
| BBa_K4308017 | Coding | CasΦ(Neg-K) | Jun Zhang | 2292 |
| BBa_K4308023 | RNA | CasΦ_crRNA(H4) | Jun Zhang | 53 |
| BBa_K4308032 | Composite | CasΦ-crRNA-LINC00857 | Jun Zhang | 2346 |
| BBa_K4308034 | Composite | CasΦ(n)-crRNA-LINC00857 | Jun Zhang | 2337 |
| BBa_K4308035 | Composite | CasΦ(v)-crRNA-LINC00857 | Jun Zhang | 2286 |
| BBa_K4308036 | Composite | CasΦ(Neg-K)-crRNA-LINC00857 | Jun Zhang | 2337 |
