Abstract
This year, our team has made breakthroughs in the design of endogenous timer suicide switch, filamentous fungi culture and transformation, optimization of R-body, hardware Settings, and product design. In these achievements, we have incorporated many unique inspirations and novel ideas, and we hope that our szu-contributions will help the iGEM teams in the future.
Parts
1. Upgrade of R-body components
R-body (Refractile inclusion bodies) is an amazing protein complex. The elongation of the coiled R-body at pH below 6.5 is an effective physical cleavage mechanism in E.coli, which is milder than chemical cleavage. The order of R-body gene cluster is RebA-RebB-RebD-RebC. We investigated the function of each sub-gene and proposed improvement. The redundant RebD gene was deleted and the major structural protein gene RebB was advanced to the first place of the gene cluster to regulate its expression, and the improved R-body gene cluster RebB-RebA-RebC was obtained, so as to improve the expression of R-body. We confirmed the success of the design through experiments, and the design reflects the simplicity and efficiency of the gene apparatus in synthetic biology. We hope that this modified R-body will provide a simpler and more efficient R-body synthesis device to future teams, and encourage more similar gene cluster engineering.
The improved part (BBa_K4286504) and the original existing part (BBa_K2912017) are presented below. You can click on the part number to see the details.
| Part Number | Name | Type | Part Description |
| BBa_K2912017 | R-body | Composite | Refractile inclusion bodies from Caedibacter |
| BBa_K4286504 | Improved R-body | Composite | Improved version of Refractile inclusion body |
2. Construction of an endogenous timed suicide switch in engineered bacteria
How to ensure the safety of engineered microorganisms for agricultural applications is a serious issue. In order to prevent the possible engineered microorganisms from escaping, it is a feasible method to use the conditionally triggered suicide switch. However, this is not foolproof, because the triggering conditions in the natural environment are uncertain, and there may still be the possibility of escaping. Our team proposed a solution to this potential hazard by designing a suicide switch that can be activated by endogenous factors in engineered bacteria.
We first constructed a classical oscillator element（BBa_K4286099) consisting of three repressor genes and a matching MazEF system to construct a suicide effector（BBa_K4286100). After testing and modeling predictions, we modified and improved the two devices to make them more stable. We removed the mazF expression device, which was originally located in the effector, and installed it at the oscillator element. At the same time, the protein rapid degradation tag of the oscillator element was removed and an efficient tetR binding site was added to the effector element, resulting in a new set of oscillator and effector elements(BBa_K4286101 and BBa_K4286103). If the future iGEM team faces the difficulty of the unstable environment of engineering bacteria and needs to use the endogenous suicide switch to ensure safety, you can consult our Safety and Parts pages and make personalized modifications or improvements based on the design ideas we provide. We have detailed our design and iteration process on the above two pages.
| Part Number | Name | Type | Part Description |
| BBa_K4286099 | Oscillator device | Composite | Classical oscillator composed of three genes encoding repressor protein. |
| BBa_K4286100 | Effector device | Composite | Effector device in timed suicide switch. | BBa_K4286101 | Improved oscillator device | Composite | Oscillator device for improved version of timed suicide switch. |
| BBa_K4286103 | Improved oscillator device | Composite | Effector device for improved version of timed suicide switch. |
Hardware and Software
1. 3D printing on our hardware-ENose
We constructed an electronic instrument that can obtain environmental data such as temperature, humidity, light intensity, and gas concentration through sensors, and designed a set of detection algorithms for it according to the pathological characteristics of rice sheath wilt for data analysis (see the Model page for details). At the same time, we also built a companion APP for user feedback (see the Software page for details).
In addition to the 6 gas sensors, our E-Nose hardware is also equipped with temperature and humidity sensors, wind speed sensors and WiFi communication modules. The appearance parts of the electronic nose and some other module parts can be produced by 3D printing. If future iGEM teams are interested in other fungal diseases, or want to tackle diseases that can be diagnosed by special gases, they can use our E-Nose hardware directly, and import the new data they collect to train the neural network in the detection algorithm (see the Hardware page for details).
2. Construction of Smart Farm APP
We designed a software APP called Smart Farm that matches our hardware. Its main function is to monitor farmland diseases with the electronic nose system. Users can monitor the gas concentration in the area where the equipment is located by using our APP at any time. If there is any abnormality, the APP will make an alarm in time. At the same time, we also set up a page for users to update industry information and other information on the APP to provide users with the most comprehensive services. If the latecomers are interested in our Software APP, you can go to our Software page for further details.
3. Construction of materials used to assist biocontrol bacteria to complete aquatic interface blockade
For most agricultural diseases, the pathogens are mainly latent in soil and spread through aquatic interfaces during disease outbreak. Based on this, our team has two main szu-contributions:
- 1. We proposed the concept and strategy of aquatic interface blocking, which may provide inspirations and ideas for iGEM teams who want to focus on other agricultural diseases in the future.
- 2. Combined with the knowledge of pharmacy, we developed a product form for biocontrol bacteria——TACE the Carrier, which can provide a stable environment for biocontrol spores and accurately deliver spores to the stems of crops at the aquatic interface. It may provide an efficient and easy method for iGEM teams engaged in or involved in the encapsulation and delivery of engineered microorganism in the agricultural field in the future. They can learn about our work by looking at our Product page, which may give them some inspiration.
Troubleshooting of the experiment
1. Successfully completed the transformation of Trichoderma SPP. Filamentous fungi
In order to achieve better transformation of our plasmid genes in Trichoderma atroviride or other similar filamentous fungi, we have tried many transformation methods, including protoplast transformation, transmembrane peptide transformation, agrobacterium transformation and magnesium-silicate nanoclay transformation. At the same time, we also tried to combine the protoplast transformation method with agrobacterium transformation method, and tried to use protoplasts of Trichoderma atroviride instead of spores for agrobacterium transformation.
After repeated attempts, we found that for Trichoderma atroviride, agrobacterium transformation method and protoplast transformation method are more efficient and feasible than other methods, and the combination of the two methods also has a good effect. However, it does not mean that the other methods are not of reference value, they may have better effect than agrobacterium transformation in other chassis. In order to facilitate the use of future teams, we have included the detailed protocols of various methods mentioned above in our Protocols page, and future iGEM teams can directly consult our wiki if they need to use similar methods.
2. Explored and established a complete set of LAMP-LFD detection process
This year, our team explored the appropriate PCR primers, PCR reaction system and temperature, as well as LAMP primers and reaction system (especially the magnesium ion concentration) to identify R.solani. At the same time, a whole set of LAMP-LFD detection process was also explored. We hope that our exploration of LAMP-LFD and some ideas can help iGEM teams who may apply this technology in the future. When they encounter related difficulties, We believe that our content on the Product page may give them some inspirations.