Introduction
CAU-China has registered 31 basic parts and 8 composite parts during this year's wet lab work.
These components form three primary functional circuits, responsible for the deletion of PHB-related genes, the synthesis and regulation of target products as well as the suicide circuit.
Innovation
In rhizobium, there are endogenous promoters, glnK and nifH, which respond to nitrogen and oxygen levels, respectively. glnK activates downstream gene expression under low nitrogen conditions, and nifH is activated under low oxygen conditions. Both promoters become inactive in high nitrogen or high oxygen environments. By introducing these two inducible promoters, we ensured that the target bacteria can perform normal life functions during nitrogen fixation in symbiosis with plants and can effectively execute circuits for target product synthesis and self-destruction when environmental conditions change. This design allows the chassis to flexibly adapt to environmental fluctuations, ensuring the system performs optimally under the expected conditions.
Additionally, we employed the pfa biosynthetic gene cluster from Aetherobacter fasciculatus SBSr002, which was previously heterologously expressed in E. coli to produce PUFA. To ensure efficient expression of pfa biosynthetic gene cluster in Sinorhizobium fredii CCBAU45436, we optimized the gene cluster's codon usage to suit the chassis, at the same time, we added PPTase to the pfa biosynthetic gene cluster to involve in posttranslation modification..
In the design of the main circuits, we innovatively introduced elements from the CRISPRi system and the endogenous toxin-antitoxin (TA) system of the chassis organism Sinorhizobium fredii CCBAU45436. These components, combined with inducible promoters, allow the system to sense external environmental conditions and provide precise regulatory feedbacks.
In the CRISPRi system, we used the Cas12k protein, which binds reversibly to target sequences under the guidance of sgRNA. This system plays a role in both target product synthesis and the suicide circuit. When external signals reach a certain threshold, Cas12k inhibits transcription in the corresponding regions. Cas12k lacks cleavage activity, so it physically blocks transcription without cutting the target DNA sequence. As the intracellular concentration of sgRNA decreases, the bound Cas12k protein detaches from the target sequence, allowing normal transcription to resume when continuous repression is no longer needed. This ensures the system's dynamic responsiveness and long-term stability.
In the suicide circuit, vapC encodes the toxin, and vapB encodes the antitoxin. The expression of these genes is regulated by the promoters nifH and glnK. Under high oxygen conditions, expression of vapB is suppressed, and under low nitrogen conditions, the CRISPRi system is activated to suppress expression of vapC. When rhizobium escapes the symbiotic environment and enters a high-nitrogen, high-oxygen external environment, vapC will be expressed normally, while the expression of vapB is suppressed by the high-oxygen conditions. This leads to toxin levels exceeding the tolerance threshold of the antitoxin, ultimately triggering the self-destruction of rhizobium. This design prevents uncontrolled growth of rhizobium in external environments, ensuring the system's safety and precision.
In the synthesis circuit, the expression of pfa biosynthetic gene cluster is also regulated by the promoters nifH and glnK. Under high oxygen conditions (i.e., before the rhizobia form a symbiotic relationship with the plant), the expression of pfa biosynthetic gene cluster is inhibited; when the nodule structure is formed, the low oxygen environment activates the promoter nifH. However, in the early low nitrogen environment, the CRISPRi system is activated to suppress the expression of pfa biosynthetic gene cluster. After nitrogen fertilizer is applied later, the promoter glnK is deactivated, and the pfa biosynthetic gene cluster will be expressed normally.
The above design ensures coordinated interactions between different circuits, maintaining the system's overall unity. Through this innovative approach, we successfully achieved dynamic regulation of gene's deletion, target product synthesis and regulation, as well as the suicide circuit, meeting our expected experimental outcomes. This not only enhances the system's flexibility and controllability but also provides new insights for further applications in synthetic biology.
Basic Parts
| Part name | Registry Code | Part Type | Source | Brief introductions |
|---|---|---|---|---|
| PnuoA | BBa_K5300013 | Regulatory | Sinorhizobium fredii CCBAU45436 | Constitutive promoter for maintaining downstream gene transcription. |
| mCherry | BBa_K5300014 | Reporter Element | Bradyrhizobium diazoefficiens USDA 110 (Bradyrhizobium japonicum USDA 110) | Reporter fluorescent protein, serves as a target of sgRNA to verify circuit construction and CRISPRi functionality. |
| RBS | BBa_K5300012 | RBS/SD | Sinorhizobium fredii CCBAU45436 | Ribosome binding site, ensures proper expression of the vapC gene cluster in rhizobia. |
| vapC | BBa_K5300019 | Protein coding sequences(TA) | Sinorhizobium fredii CCBAU45436 | Toxin gene in the toxin-antitoxin system; triggers cell death when toxin levels exceed the tolerance threshold. |
| Terminator | BBa_K5300020 | Terminator | Sinorhizobium fredii CCBAU45436 | Endogenous terminator of vapC, ensures proper termination of gene expression in the chassis. |
| PnifH | BBa_K5300016 | Regulatory | Sinorhizobium fredii CCBAU45436 | Promoter responsive to low oxygen signals, activates downstream gene transcription under low oxygen conditions and becomes inactive in high oxygen environment. |
| vapB | BBa_K5300018 | Protein coding sequences(TA) | Sinorhizobium fredii CCBAU45436 | Antitoxin gene in the toxin-antitoxin system; triggers cell death when toxin levels exceed the tolerance threshold. |
| PglnK | BBa_K5300017 | Regulatory | Sinorhizobium fredii CCBAU45436 | Promoter responsive to low nitrogen signals, activates downstream gene transcription under low nitrogen conditions and becomes inactive in high nitrogen environment. |
| sgRNA-mCherry | BBa_K5300024 | DNA (CRISPRi) | Bradyrhizobium diazoefficiens USDA 110 (Bradyrhizobium japonicum USDA 110) | sgRNA targeting the mCherry sequence, pairs with tracrRNA to guide Cas12k protein to the target sequence. |
| tracrRNA | BBa_K5300026 | DNA (CRISPRi) | Scytonema hofmannii | tracrRNA, integrated with sgRNA to form a complex that works with Cas12k proteins to form the core mechanism of the CRISPRi system. tracrRNA enhances the ability to recognise the target DNA by helping the sgRNA to form a specific structure, and plays a key role in the accuracy and efficiency of the CRISPRi system. |
| Cas12k | BBa_K5300021 | Protein coding sequences(CRISPRi) | Scytonema hofmanni | A member of the Cas protein family, lacks cleavage activity and inhibits transcription through physical binding without cutting the target sequence. When intracellular sgRNA levels decrease, the bound Cas12k protein detaches from the target sequence, allowing normal transcription to resume. |
| B0015 double terminator | BBa_K5300022 | Terminator | Scytonema hofmanni | Endogenous terminator of Cas12k, ensures proper termination of gene expression in the chassis organism. |
| UP-feo-suicide | BBa_K5300027 | DNA | Sinorhizobium fredii CCBAU45436 | Upstream homology arm element for integrating the suicide circuit sequence into the S. fredii CCBAU45436 genome. |
| DOWN-feo-suicide | BBa_K5300028 | DNA | Sinorhizobium fredii CCBAU45436 | Downstream homology arm element for integrating the suicide circuit sequence into the S. fredii CCBAU45436 genome. |
| sgRNA-GFP | BBa_K5300025 | DNA (CRISPRi) | Bradyrhizobium diazoefficiens USDA 110 (Bradyrhizobium japonicum USDA 110) | sgRNA targeting the gfp sequence, pairs with tracrRNA to guide Cas12k protein to the target sequence. |
| gfp | BBa_K5300015 | Reporter Element | Bradyrhizobium diazoefficiens USDA 110 (Bradyrhizobium japonicum USDA 110) | Reporter fluorescent protein, serves as a sgRNA target to verify circuit construction and CRISPRi functionality. |
| RBS1(NifD) | BBa_K5300003 | RBS/SD | Sinorhizobium fredii CCBAU45436 | Ribosome binding site (Shine-Dalgarno sequence), ensures proper expression of the pfa biosynthetic gene cluster in rhizobia. |
| pfa1 | BBa_K5300004 | Protein coding sequences | Aetherobacter fasciculatus SBSr002 | Member of the pfa biosynthetic gene cluster, encodes the pfa1 enzyme, which works with other PUFA synthase in the cluster to induce DHA production. |
| RBS2(NifK) | BBa_K5300006 | RBS/SD | Sinorhizobium fredii CCBAU45436 | Ribosome binding site (Shine-Dalgarno sequence), ensures proper expression of the pfa biosynthetic gene cluster in rhizobia. |
| pfa2 | BBa_K5300007 | Protein coding sequences | Aetherobacter fasciculatus SBSr002 | Member of the pfa biosynthetic gene cluster, encodes the pfa2 enzyme, which works with other PUFA synthase in the cluster to induce DHA production. |
| RBS3 (NifE) | BBa_K5300008 | RBS/SD | Sinorhizobium fredii CCBAU45436 | Ribosome binding site (Shine-Dalgarno sequence), ensures proper expression of the pfa biosynthetic gene cluster in rhizobia. |
| pfa3 | BBa_K5300009 | Protein coding sequences | Aetherobacter fasciculatus SBSr002 | Member of the pfa biosynthetic gene cluster, encodes the pfa3 enzyme, which works with other PUFA synthase in the cluster to induce DHA production. |
| RBS4 (NifN) | BBa_K5300010 | RBS/SD | Sinorhizobium fredii CCBAU45436 | Ribosome binding site (Shine-Dalgarno sequence), ensures proper expression of the pfa biosynthetic gene cluster in rhizobia. |
| ppt | BBa_K5300011 | Protein coding sequences | Aetherobacter fasciculatus SBSr002 | Gene encoding the PPTase enzyme, located downstream of the pfa biosynthetic gene cluster. PPTase involves in posttranslation modification. |
| Up-Arm phaC2 | BBa_K5300029 | DNA | Sinorhizobium fredii CCBAU45436 | Up-Arm phaC2, is a DNA sequence located upstream of the phaC2 gene, used as one of the homologous sequences for conducting rhizobia genetic experiment. |
| Down-Arm phaC2 | BBa_K5300030 | DNA | Sinorhizobium fredii CCBAU45436 | Down-Arm phaC2, is a DNA sequence located downstream of the phaC2 gene, used as one of the homologous sequences for conducting rhizobia genetic experiment. |
| bgc33 | BBa_K5300031 | Protein coding sequences | Clostridium sp. CAG:567 (Human metagenome) | bgc33 encodes nonribosomal peptide synthetase (NRPS), which catalyzes the synthesis of N-octanoyl-Met-Phe-H. |
| sfp | BBa_K5300032 | Protein coding sequences | Clostridium sp. KLE:1755 (Human metagenome) | The sfp gene encodes a PPTase that is responsible for performing post-translational modifications of the protein. |
| nifB upstream | BBa_K5300033 | SD | Sinorhizobium fredii CCBAU45436 | Ribosome binding site (Shine-Dalgarno sequence), ensures proper expression of the sfp gene in rhizobia. |
Composite Parts
| Composite Part Name | Registry Code | Part Type |
|---|---|---|
| PHB deletion: Up-Arm phaC2 + Down-Arm phaC2 | BBa_K5300040 | Composite |
| PnuoA-mCherry-RBS-vapC-Terminator-PnifH-vapB-PglnK-sgRNA-mCherry-tracrRNA-PnuoA-Cas12k-double Terminator | BBa_K5300041 | Composite |
| PglnK-sgRNA-GFP-tracrRNA-PnifH-gfp-pfa biosynthetic gene cluster | BBa_K5300042 | Composite |
| PglnK-sgRNA-GFP-tracrRNA-PnifH-gfp | BBa_K5300043 | Composite |
| PnuoA-Cas12k-double Terminator-PglnK-sgRNA-GFP-tracrRNA-PnifH-gfp | BBa_K5300044 | Composite |
| PnuoA-pfa biosynthetic gene cluster | BBa_K5300037 | Composite |
| PglnK-gfp | BBa_K5300045 | Composite |
| PnifH-dipeptide aldehyde | BBa_K5300039 | Composite |
Cloning vectors
| Part Name | Registry Code | Origin | Length (bp) |
|---|---|---|---|
| pJQ200SK | BBa_K5300034 | Artificial synthesized from Quandt and Hynes, 1993 | 5374 |
| pBBR1MCS-2 | BBa_K5300023 | Artificial synthesized from Kovach et al., 1995 | 5148 |