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BBa_K4955000

Crocetin, one of the substances we aimed to produce, is classified as a carotenoid. Many past iGEM teams have tried to biosynthesize carotenoids, but no team has succeeded in biosynthesizing crocetin.

Teams that have directly or indirectly attempted to biosynthesize crocetin in the past are listed below. Of these, WashU 2012 and Uppsala 2013 also attempted to biosynthesize picrocrocin, which we will touch on below, but were unsuccessful.

team Goal Result
WashU 2012 Production of Saffron secondary metabolites in E.coli,synechocystis Successful gene cloning but failed to demonstrate function in vivo.
Uppsala 2013 Production of Saffron secondary metabolites in lactic acid bacteria Successful Plasmid design, but enzyme folding did not work.
Uppsala 2017 Production of Saffron secondary metabolites in E. coli Although the biosynthetic pathway up to Zeaxanthin was

successfully integrated into the chromosome, the later

pathways could not be functionally demonstrated in vivo.

Latvia-Riga 2022 Production of Saffron secondary metabolites in Rhodotorula toruloides Transformants could not be obtained.

[1][2][3][4]

We have successfully biosynthesized crocetin using E. coli under special substrate-free culture conditions. We have registered the enzyme group as BioBrick (BBa_K4955000) and summarized its properties.

BBa_K4955001

Picrocrocin, one of the substances we aimed to produce, is classified as a glycoside terpenoid. We have not only succeeded in biotransforming Picrocrocin from 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC), a precursor of picrocrocin, by heterologous expression for the first time in the world, We have also succeeded in the biosynthesis of picrocrocin using E. coli under culture conditions without special substrates. We then registered the enzyme as BioBrick (BBa_K4955001) and summarized its properties.

Systems that inhibit the proliferation of E. coli

Introduction

This system was developed by Takahito Mukai of Rikkyo University’s Su’etsugu Laboratory.

Our goal was to provide the public with cookies containing E. coli bacteria that have accumulated antidepressant components. Current regulations on genetically modified organisms focus on ” reproduction”. Therefore, we hypothesized that if we could eliminate the ability of our E. coli to multiply, we would be able to lower the regulatory hurdle for implementing our cookies.

Since we were planning for social implementation, we sought a system that would minimize the incidence of escapes. It is known that Chromosome-free (i.e., non-proliferative) E. coli can be produced by using chromosome-selective restriction enzymes and a strict expression control system [5]. We began to develop a system that mimics this.

Ultimately, we demonstrated the functionality of the system on BL21(DE3) and E.coli Nissle 1917 strains. We were the first to propose the concept of using such restriction enzymes in iGEM, and we were also the first to successfully achieve Chromosome-free in E. coli. Our system has strong potential and is highly standardized, and we expect it to be used by various iGEM teams in the future.

Our System

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We implemented a Chromosome-free system using a six-base restriction endonuclease with a large number of recognition sites in the ribosomal coding region. We chose this system because of its low escape rate, and as a result we have succeeded in creating a highly standardized system that can be used in all cell types and in a variety of situations.

We also used a powerful, tight, low-cost inducer-controlled expression control system that mimics the Jungle Express system [6] to induce this enzyme.

The final inducer we used was Crystal Violet. This is also used in Gram staining and is therefore very inexpensive. By using this system, we were able to minimize the occurrence of escape mutants (bacteria that do not become chromosome-free).

However, in our experiments during iGEM, we were not able to reduce the number of escape mutants to a level suitable for social implementation. We are currently exploring the possibility of developing a more robust expression system to reduce the incidence of escape mutants.

Since we are aiming to commercialize the iGEM project this year and to obtain patents for the above-mentioned expression management mechanism and the system aimed at reducing the incidence of escape mutants in the future, we cannot give any more details at this stage. However, if we succeed in commercialization and patent acquisition, we will definitely disclose these systems to iGEM. Together with the BioBrick (BBa_K4955002), these will lead to further development of the iGEM project.

Strength

The strengths of the system we have developed are twofold

  1. The system can be used in all living cells.

  2. Even if chromosomes are degraded, the designed plasmid is not degraded.

Regarding the first point, the ribosomal coding region is conserved among all organisms, and BBa_K4955002 has many restriction enzyme sites in the ribosomal coding region, so it can be used in all cells. We have demonstrated its function in BL21(DE3) and E.coli Nissle 1917 strains.

Regarding the second point, BBa_K4955002 is a mega restriction enzyme of the 6-base recognition type. Therefore, the designed plasmid basically does not have any recognition sites (if it did, the number would be such that primers could be designed and synonymous codon substitutions could be made). This means that the chassis can be made Chromosome-free while maintaining the designed functionality. We have shown that the designed function may work in the Chromosome-free BL21(DE3) strain. The advantages of this are as follows:

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▶Advantages of not multiplying

The system we have developed may be able to run the designed functions using a non-proliferating chassis. In this case, any type of chassis and any designed function can be used. This could be a powerful tool for microbial production, biosensing, drug delivery, cell-surface display, and almost any other iGEM project.

▶Advantages as a novel chassis

The goal of synthetic biologists is to design organisms to perform their functions in a safe, reliable, and robust manner. In contrast, the goal of the organism is to continue to change through adaptation, evolution, and proliferation. This conflict leads to interference from the native gene network (variability [6], unpredictable expression [7], etc.). However, there is a great potential for Chromosome-free cells to express synthetic gene circuits in a predictable manner. This is not only a scientific advance in synthetic biology, but also one of the factors that will ensure a high resolution of the product, which is essential for social implementation.

▶Other advantages

Analysis Software

We have developed a system that we anticipate will be used by many iGEM teams in the future. Using free software called ImageJ, we have created a script that processes a series of images so that anyone can count the number of bacteria. This script allows the user to change the numerical values in each process. Each value can be adjusted for contrast, noise removal, etc., allowing for highly accurate counts. Microscopic images taken under the same conditions can be further automated, since these values do not need to be changed significantly. Microscopic images that do not need to have their numerical values changed can be placed in a single folder and automatically analyzed using a script we have created. The development of this script has made it possible to count bacteria efficiently.

The script can be obtained from the link below.

https://gitlab.igem.org/2023/software-tools/japan-united/-/blob/main/imagejMethods.ijm

References