Our primary objective is to engineer Nissle 1917 to produce 5-HTP, a precursor to serotonin that humans can utilize. We designed an improved Zif268-m-hTPH1 protein sequence within its plasmid to achieve this. Additionally, we aim for Nissle 1917 to regenerate the Zif268-m-hTPH1 protein autonomously, so we incorporated the sequences for ZFa-hQDPR and PBSII-hPCBD1 proteins. To enhance the efficiency of this process, we also designed a system to bring these proteins into close spatial proximity using Zinc finger proteins and a DNA scaffold.
Design:
To ensure continuous production of 5-HTP in Nissle 1917, we introduced the gene sequence for Zif268-m-hTPH1, which is essential for 5-HTP synthesis. Additionally, we inserted the gene sequences for PBSII-hPCBD1 and ZFa-hQDPR proteins, enabling Nissle 1917 to synthesize the coenzyme BH4 necessary for 5-HTP production.
Result:
▲ After confirming the insertion of the expression cassette into the pST39 vector, we induced protein expression by treating with 0.2 mM IPTG at 37°C. To determine whether the expression level increases over time, we conducted inductions for 0, 4, and 8 hours, using pST39 as the control group
Design:
We designed a DNA sequence containing a zinc finger protein binding motif to optimize the spatial arrangement of Zif268-m-hTPH1, PBSII-hPCBD1, and ZFa-hQDPR proteins. This sequence is then replicated into circular ssDNA using the rolling circle replication (RCR) mechanism.
Build:
Initially, we utilized modeling to predict the feasibility of the fusion proteins and designed the corresponding sequences. We constructed a plasmid to express the three fusion proteins and designed the DNA scaffold sequence on a separate plasmid.
Learn:
Through this design, Nissle 1917 may produce an excess of 5-HTP, potentially leading to symptoms like irritable bowel syndrome. Therefore, we decided to incorporate a switch within the DNA scaffold system to regulate 5-HTP production more effectively.
▲ We used the T7 promoter to drive the sequences of Zif268-m-hTPH1, PBSII-hPCBD1 and ZFa-hQDPR proteins.
Design:
To align with our stress reduction theme, we identified a sensor responsive to protocatechuic acid (PCA) and its associated promoter. We integrated two promoters within one plasmid: one for continuous expression of the sensor, PcaUAM protein, and the other for the PCA promoter. A terminator was also included to separate the two promoters.
Build:
We positioned the PcaUAM gene downstream of the LacIQ promoter to ensure its constant expression. Following the PcaUAM gene, the T7Hyb1 terminator was inserted to demarcate the two promoters. When PCA enters Nissle 1917, it binds to the PcaUAM sensor, activating the PCA promoter and triggering the downstream expression of RepA genes. Ultimately, the RepA protein recognizes the RCORI 105 sequence and initiates the RCR reaction, amplifying the DNA scaffold to enhance 5-HTP production.
▲ The three fusion proteins bound to their respective motifs on the DNA scaffold, bringing them closer together.
