We employed rolling circle replication (RCR) to produce DNA scaffolds and fused three enzymes—m-hTPH1, hPCBD1, and hQDPR—with zinc finger binding domains. We simulated and designed fusion proteins through protein structure modeling: Zif268-rigid linker-hTPH1, PBSII-rigid linker-hPCBD1, and Zfa-hQDPR, which recognize DNA scaffolds. Subsequently, we verified the expression of these fusion proteins in E. coli strain BL21, both individually and in combination, confirming successful protein production. Finally, we analyzed 5-HTP production using an ELISA kit.
▲ Figure 1: Protein structure simulations using AlphaFold2 and iTASSER.
We incorporated a rigid linker (rLinker) into the fusion proteins, revealing that the N-terminal Zif268 and PSBll (pink) fused with rLinker (yellow) and the C-terminal m-hTPH1 and h-PCBD1 (blue) achieved the best structural predictions. Despite using different linkers, they did not enhance ZFa protein expression, with the best structural predictions observed when ZFa (pink) was fused at the N-terminus of hQDPR (blue).
For more details, please see our Protein Modeling page.
After confirming successful cloning, we assessed the optimal expression of our fusion proteins—Zif268-rigid linker-hTPH1, PBSII-rigid linker-hPCBD1, and Zfa-hQDPR. We tested different induction times and IPTG concentrations at 37°C to induce protein expression.
▲ Figure 2: Coomassie Blue-stained SDS-PAGE analysis of the three fusion proteins at 0, 4, and 8 hours of IPTG induction. (Sup: supernatant; P: pellet)
We observed that the individual expression of PBSII-rigid linker-hPCBD1 and Zfa-hQDPR fusion proteins was successful. However, the Zif268-rigid linker-m-hTPH1 fusion protein did not express well, leading us to extend the induction time and increase IPTG concentration.
We conducted inductions for 0, 12, and 24 hours to investigate whether increased induction time would improve expression. Additionally, we assessed the impact of IPTG concentration by testing 0.2 mM, 0.4 mM, and 0.8 mM IPTG.
▲ Figure 3: Coomassie Blue-stained SDS-PAGE analysis of the fusion proteins at 0, 12, and 24 hours of IPTG induction.
▲ Figure 4: Coomassie Blue-stained SDS-PAGE analysis of the three fusion proteins with IPTG concentrations of 0.2 mM, 0.4 mM, and 0.8 mM.
Increased induction time and IPTG concentrations did not improve the expression level of the Zif268-rigid linker-m-hTPH1 fusion protein. Thus, we maintained the initial induction conditions for subsequent experiments. After confirming the successful insertion of three expression cassettes (Zif268-rigid linker-m-hTPH1, PBSII-rigid linker-hPCBD1, and Zfa-hQDPR) into the pST39 vector, we induced protein co-expression using 0.2 mM IPTG at 37°C.
▲ Figure 5: The Coomassie Blue staining of SDS-PAGE analysis of three fusion proteins' co-expression with IPTG induced.
We successfully co-expressed the fusion proteins, establishing optimal expression conditions for our constructs.
After preparing the probiotics, we will analyze 5-HTP production using an ELISA kit. Results will be provided once the kit arrives.
To regulate DNA scaffold formation, we employed the PcaUAM-pPCA3B5b Sensor to control the expression of RepA, the replication initiation protein in RCR. The RCR cassette includes RCORI 65, RCORI 105, and ZNF motifs. The PCA-regulated DNA scaffold formation aims to promote a 5-HTP biosynthesis pathway in E. coli.
▲ Figure 6: DNA digestion of the RCR cassette inserted into the pST39 vector.
After confirming the RCR cassette insertion into pST39, we proceeded with DNA sequencing and the next steps of the experiment.
We identified point mutations in the PcaUAM sequence after confirming the insertion of the PCA-regulatory cassette into pST39.
▲ Figure 7: DNA sequencing results of the PCA-regulatory cassette inserted into the pST39 vector.
Repeated experiments showed persistent point mutations, prompting us to use a different strain for plasmid transformation.
▲ Figure 8: DNA sequencing results of the PCA-regulatory cassette, inserted into pST39, transformed into E. coli strains DH5α and StbIII.
Mutations persisted even after changing the bacterial strain for transformation. We will proceed with further experiments using alternative strains and varying culture temperatures to reduce mutation rates.
We applied chitosan and sodium alginate in a layer-by-layer (LbL) assembly on EcN cells to protect probiotics from harsh gastrointestinal conditions. We enhanced the stability of these layers by adding calcium chloride, forming a gel layer via ionic cross-linking. We assessed the coating efficiency via zeta potential analysis and evaluated bacterial growth behavior after incubation at pH 2.5 and 7.0, simulating gastric and intestinal environments.
Chitosan provides positive charges, and sodium alginate provides negative charges. By detecting the zeta potential after coating each layer on E. coli, we confirmed the successful coating of the bacteria.
▲ Figure 9: Zeta potential of the chitosan-alginate layers (measured by Dynamic Light Scattering).
To simulate the gastrointestinal environment, we incubated encapsulated strains at pH 2.5 for 1, 2, and 4 hours and then at pH 7.4 for 3 hours to test bacterial survival.
(pH2.5 for 1 hour)
(pH2.5 for 2 hour)
(pH2.5 for 4 hour)
▲ Figure 10: [uncoated E. coli] pH test after incubating in pH 2.5 for 1, 2, and 4 hours.
(pH2.5 for 1 hour)
(pH2.5 for 2 hour)
(pH2.5 for 4 hour)
▲ Figure 11: [coated E. coli] pH test after incubating in pH 2.5 for 1, 2, and 4 hours.
While no uncoated E. coli survived in the acidic environment, we observed survival rates of 1.302 x 10⁷ cfu/mL, 3.74 x 10⁶ cfu/mL, and 3.7 x 10⁶ cfu/mL for coated strains at 1, 2, and 4 hours of incubation, respectively. The encapsulated strains successfully passed through the acidic environment and were released into an alkaline environment, indicating that our carriers protect probiotics effectively.
We compared coated and uncoated strains by incubating them at pH 2.5 and pH 7.0 for 1 hour, followed by observation of their growth behavior in TB broth with glass beads to simulate peristaltic movement.
▲ Figure 12 (left): Bacterial growth behavior after water bath at pH 2.5 for 1 hour.
▲ Figure 13 (right): Bacterial growth behavior after water bath at pH 7.0 for 1 hour.
The coated strain entered the log phase earlier than the uncoated strain, demonstrating higher survival rates in both acidic and neutral environments.
▲ Figure 14: Growth behavior comparison between uncoated and coated strains after water bath incubation.
The coated strain showed consistent growth behavior, while the uncoated strain experienced delayed log-phase entry in acidic conditions, highlighting the protective effect of the encapsulation process.