In 2023, we mainly developed a strain aimed at high lycopene production through gene editing technology. We not only constructed a high lycopene-producing Corynebacterium glutamicum, but also designed a method to convert straw into glucose after treatment, and we also made a little innovation in the software. More importantly, we are willing to share our experience in wet lab and software design. It is hoped that our project will be helpful and instructive to future iGEM teams.
These parts are required for gene expression during the experiment. Our project has registered multiple parts and multiple composite parts, and also constructed a system in which CRISPR-MAD7(Cas12a) interacts with the recE/T recombinase pair.
We constructed the CRISPR-MAD7(Cas12a) gene editing system and optimized the promoter, plasmid vector, and selected the best PAM sites recognized by gRNA to improve the editing efficiency, which plays an important role in retargeted gene knockout.
By combining CRISPR-MAD7(Cas12a) with the recE/T system, researchers can achieve more controlled and targeted gene editing in C. glutamicum, thereby reducing off-target effects and improving the likelihood of obtaining the desired genetic modifications[1].
Combining the CRISPR-MAD7(Cas12a) gene editing system with the recE/T recombinase system improved the efficiency of gene editing by optimizing the system. After CRISPR-MAD7(Cas12a) nuclease was used to cleave the DNA gap, the donor DNA was repaired by homologous recombination to achieve the knockout of the target gene crtEb/crtR.
The carbon source obtained from straw treatment could improve the nutrients required for subsequent cultivation of Corynebacterium glutamicum. The carbon source obtained from straw was mainly glucose obtained by enzyme treatment after the treatment of waste straw with diluted acid.
Optimization of the promoter, plasmid vector, and selection of good PAM sites recognized by gRNA can increase the localization and binding of the target gene by CRISPR system, thereby improving the editing efficiency. With these improvements, the gene editing efficiency of CRISPR-MAD7(Cas12a) system in C. glutamicum can be further improved.
A recombinant strain with good growth ability was isolated and selected from the basic culture. After the initial culture in shaking-flasks, further scale-up culture was carried out in fermenters. The orthogonal experiment design was applied to optimize the temperature, pH, sampling time and fermentation tank speed,with a view to optimizing fermentation conditions and improving lycopene production efficiency.
Our primary aim is to simplify the process for iGEMers when it comes to committing images, reducing the need for constant switching between web browsers and their IDEs. We aspire to provide a more convenient way to access and upload content, allowing wiki builders to focus on creating and designing webpages within their IDE with fewer distractions.
Through promotion through multiple channels (Slack, Twitter, China iGEMer Community, etc.), our software has been downloaded and used many times.
For more information, please turn to the igem-uploads.
In order to help the wet group better verify the experiment, we developed a software tool for colony identification and classification called ColoVision.
It allows experimental workers without program development knowledge to upload pictures of petri dishes through the Web page and perform preliminary identification and classification.
For more information, please turn to the ColoVision.
[1] Nannan Z,Lu L,Guangjuan L, et al. Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.[J]. Journal of industrial microbiology & biotechnology,2020,47(8).
