Results
Antidepressant Component Production
Final Results
- We have successfully demonstrated the functionality of Parts (BBa_K4955000). Crocetin production was confirmed under conditions that did not involve any specific substrates, using BL21(DE3) and DH5α strains.
- We have also successfully validated the functionality of Parts (BBa_K4955001). For the first time worldwide, we achieved in vivo production of Picrocrocin under conditions that did not involve any special substrates, using the BL21(DE3) strain.
- Furthermore, we achieved carotenoid production in the E. coli Nissle 1917 strain.
Production of Crocetin
The Parts (BBa_K4955000) cloned into the pRK404 vector were introduced into BL21(DE3) and cultured in TB medium (50 mL culture) for 72 hours.
The culture supernatants were extracted and subjected to analysis using HPLC-MS. As a result, we were able to confirm the production of Crocetin in the culture supernatants of both the DH5α and BL21(DE3) strains.
Production of Picrocrocin
We initially attempted to confirm substance production separately by introducing Parts (BBa_K4955000) carrying the enzymes up to HTCC and Parts (BBa_K4955001) carrying the enzymes up to Picrocrocin into BL21(DE3) strains. However, this approach was unsuccessful. Subsequently, we assembled Parts (BBa_K4955001) downstream of Parts (BBa_K4955000) that had been assembled into pRK404, and then introduced this construct into BL21(DE3) strains.
We cultured the strains in TB medium (50 mL culture) for 72 hours. The culture supernatants were then suspended in an equal volume of methanol, and after centrifuging at 15000 rpm for 5 minutes, we analyzed the supernatant using HPLC-MS, successfully confirming the production of Picrocrocin. Parts (BBa_K4955001) encodes a sugar transfer enzyme from Crocus sativus (Saffron). The heterologous in vivo conversion of Picrocrocin from HTCC using the enzymes mentioned above, as well as the biosynthesis of Picrocrocin under conditions that did not involve any special substrates, represents a world-first achievement.
Production of target substance in E.coli Nissle 1917 strain
We introduced the Parts (BBa_K4955000) cloned into the Duet-1 vector, pRK404, into E. coli Nissle 1917 strain and cultured it in TB medium (50 mL culture) for 72 hours.
The culture supernatant appeared distinctly yellow, suggesting the production of carotenoids, but we were unable to detect Crocetin.
Preparation of non-proliferating E. coli
Final Results
- In the BL21(DE3) strain, induction of (BBa_K4955002) resulted in suppressed growth of the bacterial cells compared to those that did not have this induction.
- In the BL21(DE3) strain, we observed chromosomal degradation in the bacterial cells induced with (BBa_K4955002) using fluorescence microscopy.
- In the E. coli Nissle 1917 strain, induction of (BBa_K4955002) resulted in suppressed growth of the bacterial cells compared to those that did not have this induction. Based on our experience with BL21(DE3), it is inferred that chromosome-free conversion has been successful.”
Background
Our goal was to accumulate antidepressant compounds inside bacterial cells and directly incorporate them into cookies for general consumption. Current regulations on genetically modified organisms primarily focus on ‘proliferation.’ Therefore, we hypothesized that if we could eliminate the proliferation ability of the engineered E. coli we designed, it could potentially reduce regulatory barriers when implementing our cookie production.
Considering the prospect of societal implementation, we aimed to develop a system that minimizes the occurrence of escape events to the greatest extent possible. By using selective restriction enzymes on the chromosome and a strict expression control system, it is known that it is possible to create _E. coli _that are chromosome-free, meaning they do not proliferate. We began developing a system inspired by this concept.
Ultimately, we successfully demonstrated the functionality in the BL21(DE3) strain and E. coli Nissle 1917 strain. The restriction enzyme we used during this process was summarized as a BioBrick (BBa_K4955002). In the context of iGEM, we were the first to propose the concept of using such restriction enzymes, and we were also the first to achieve chromosome-free conversion in E. coli. Our system holds significant potential and is highly standardized, making it suitable for use by various iGEM teams in the future.
Production of non-proliferative BL21(DE3) and E.coli Nissle 1917 strains
We introduced bacterial cells with Parts (BBa_K4955002) into LB agar plates containing Crystal Violet (CV) at 1μM and LB agar plates without CV (negative control) for both BL21(DE3) and E. coli Nissle 1917 strains. After overnight incubation, we determined the presence or absence of growth based on the differences in colony formation.
We observed that colony formation was significantly inhibited in LB agar plates containing CV, confirming that CV effectively hindered colony formation. Some escape mutants did manage to form colonies; however, we attribute this to the incomplete nature of the expression control mechanism of (BBa_K4955002).
Confirmation that BL21(DE3) is Chromosome-free
Bacterial cells carrying Parts (BBa_K4955002) were introduced into TB cultures containing CV at 1μM and TB cultures without CV (negative control) in BL21(DE3) strain. These cultures were subjected to shaking incubation for a specific duration, after which fluorescence microscopy was employed for observation. Chromosome-free status was confirmed using DAPI staining.
We induced CV at OD600=0.2 and monitored the progress over time through fluorescence microscopy, thereby confirming the success of chromosome-free conversion.”
Integration~Objective substance production in Chromosome-free E. coli~.
Final Results
- We confirmed Crocetin production in cultures where we had previously confirmed the chromosome-free status of bacterial cells through fluorescence microscopy in the BL21(DE3) strain.
Our goal was to achieve Crocetin production in_ E. coli_ that had become chromosome-free in the BL21(DE3) strain. We conducted the basic cultivation conditions identical to those in which Crocetin production had been previously confirmed, but we introduced CV induction (addition of 1 μM CV) at the same time as IPTG induction.
We confirmed the chromosome-free state of cultures induced with CV at OD600=8. In samples taken 84 hours after CV induction, the proportion of chromosome-free E. coli decreased significantly. This is believed to be due to the proliferation of escape mutants.
Samples taken after 72 hours of CV induction were extracted and analyzed using HPLC-MS, confirming the presence of Crocetin production. However, it is possible that this production was due to escape mutants or occurred before the achievement of chromosome-free status.