In this project, we constructed 13 new parts - while applying an existing part - to construct a knockout system for the sweet potato IbGBSSI gene, and achieved the expected results. We had successfully designed and annealed the single guide RNA (sgRNA (IbGBSSI), BBa_K4887002) oligos targeting gen IbGBSSI (BBa_K4887001) and inserted it into the plasmid psgR-Cas9-At to construct the backbone of IbGBSSI knockout system (BBa_K4887021). And the backbone system was then combined with the binary vector pCAMBIA1301s to obtain the expression box of IbGBSSI knockout system (BBa_K4887021). After transforming this knockout construct into embryogenic callus of sweet potato using the method of Agrobacterium-mediated transformation, IbGBSSI-deficient mutants were received.
The following are the list of parts we constructed and used. Please go to our DESIGN , ENGINEERING and PARTS pages for more details.
| No. | Part Number | Type | Description |
| 1 | BBa_K4887001 | Basic | IbGBSSI, the encoding gene of granule-bound starch synthase I (GBSSI) in sweet potato (Ipomoea batatas). |
| 2 | BBa_K4887002 | Basic | sgRNA (IbGBSSI), the single-guide RNA designed targeting for the gene IbGBSSI. |
| 3 | BBa_K4887003 | Basic | 3*FLAG, the FLAG gene harboured in the backbone of plasmid psgR-Cas9-At. |
| 4 | BBa_K4887004 | Basic | NLS, the nuclear localization signal harboured in the backbone of plasmid psgR-Cas9-At. |
| 5 | BBa_K4887005 | Basic | Cas9, the gene of CRISPR-associated nuclease 9, a dual RNA-guided DNA endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). |
| 6 | BBa_K4887006 | Basic | HygR, the hygromycin resistance gene. |
| 7 | BBa_K4887007 | Basic | pAtU6, he U6 promoter from Arabidopsis thaliana. |
| 8 | BBa_K4887008 | Basic | pAtUBQ, the Arabidopsis ubiquitin-1 promoter. |
| 9 | BBa_K4887009 | Basic | p35S, CAMV 35S promoter, a very common constitutive promoter used in plant biotechnology. |
| 10 | BBa_K4887010 | Basic | tUBQ, an ubiquitin terminator from Arabidopsis thaliana. |
| 11 | BBa_K4887011 | Basic | GUS, a commonly used reporter gene in plant transgenic engineering |
| 12 | BBa_K4887021 | Composite | pAtU6-sgRNA-pAtuBQ-3*FLAG-NLS-CAS9-NLS-tUBQ, the backbone of IbGBSSI knockout system |
| 13 | BBa_K4887022 | Composite | pAtU6-sgRNA-pAtuBQ-3*FLAG-NLS-CAS9-NLS-tUBQ-p35S-HygR-tNOS, the expression box of IbGBSSI knockout system |
| 14 | BBa_K1537031 | Basic | tNOS, the terminator nopaline synthase (TNOS) gene from Agrobacterium tumefaciens. |