Team:NDNF China/Contribution
Previous iGEM parts
In this project, we tested the performance of Chromoproteins, mercury ion sensing circuit, and L-dopa production pathways in hydrogels, and documented the detailed data on the corresponding page of Part Registry.
Chromoproteins
We tested the performance of Chromoproteins expression strains once they were enclosed in Hidro. The results showed that the Chromoproteins are still well expressed and displayed observable corresponding colors. This further illustrates the stability of Chromoproteins.
We have tested this genetic part in the Hidro system and we found that this genetic part could be well implemented in the Hidro system for eforRed expression induced by L-arabinose. The result showed that eforRed is a stable protein working in a hydrogel environment.
Figure 1: (A) The schematic of a L-arabinose–controlled genetic switch to produce chromoprotein eforRed; (B) eforRed protein production in Hidro.
Mercury ion sensing
We tested the performance of mercury ion sensing circuit in Hidro.
First we tested the performance of this mercury ion response in LB medium independently. The results were consistent with those of BEAS_China in 2019.
In the Hidro system, the strain with mercury ion sensing circuit enclosed in Hidro is still functional. This indicated the stability of the sensing switch. The Hidro system of NDNF_China can ensure biosafety as well as being able to protect engineered strain in both acidic and alkaline environments, which is more applicable in real wastewater environments. Hidro has the potential to further assist synthetic biology design to function beyond laboratories in the real world.
We have tested this genetic circuit in the Hidro system and we found that this composite part could be well implemented in the Hidro system for heavy metal detection.
Figure 2: (A) The schematic of a Hg2+ sensing circuits in Hidro; (B) Hg2+ sensing circuits output.
L-dopa production
We tested the performance of the L-dopa synthetic production pathway in Hidro.
In the Hidro system, the L-dopa production pathway enclosed in the Hidro was still able to secrete L-dopa efficiently and secreted through the Hidro shell into the aqueous environment. This further demonstrates that the Hidro system can achieve effective secretion of the desired molecules.
Meanwhile, NDNF_China's Hidro system can also ensure biosafety and has the potential to further assist drug delivery in being safely applied in humans such as in the oral cavity or intestine.
We have tested this genetic part in the Hidro system and we found that this genetic part could be well implemented as a part of the L-dopa metabolic pathway in the Hidro system for L-dopa expression induced by caffeine.
Figure 3: (A) The schematic of caffeine–controlled genetic switch encapsulated in the Hidro system to produce L-dopa; (B) The metabolic pathway from L-tyrosine to L-Dopa. (C) L- Dopa concentration measurement in the caffeine–controlled L-Dopa production Hidro system.
Troubleshooting - Hidro
Instructions and video tutorials for Hidro system preparation
Hidro is a hydrogel system enclosing engineered bacterial strains, which can help engineered strains to work beyond the laboratory in a safe, stable and traceable way. The outer layer of Hidro is a compact shell, offering both protection and containment, preventing the strains from escaping into the wild; the inner core of Hidro provides a supportive environment for them under harsh conditions, thus enabling their stable function; A genome-integrated Tracing and Control system offers tracking and specific killing of engineered strains in case of emergencies.
Hidro can be implemented in diverse scenarios, such as heavy metal sensing, food-quality detection, drug secretion, etc. The Hidro system has the great potential to promote synthetic biology applications beyond the laboratory.
In order to help future iGEM teams or research groups to use Hidro system, especially to use Hidro to help Engineered Strains to be applied in real world environment more easily, we have documented the protocol of Hidro system in detail here, and also presented the process of making it in a tutorial video. Please visit our protocols webpage for the detailed procedure and the video:
Troubleshooting for Hidro Systems
The preparation process of Hidro is rather complex, and we document here in detail the Trouble Shooting of the Hidro system for other teams if needed.
1. The shell of hydrogel spheres is not tough enough and easily falls off from the core...
- Add Ca2+ to the MES crosslinker
2. The genetic circuits do not function as intended...
- The shell material is too toxic and could cause the death of the majority of bacteria in the core: we reduced the concentration of acrylamide in the shell, while increasing the concentration of methylene bisacrylamide, which reduces the toxicity of the reagent. The optimized gradient can also increase the speed of gel formation and reduce the contact time between acrylamide and bacteria. This optimization does not affect the containment efficiency of the shell.
- Contamination may occur during preparation process of the hydrogel core, and it is necessary to ensure that the preparation process is implemented in a sterile environment.
3. Hidro is unable to prevent the escape of strains...
- The absence of air bubbles in the shell is crucial to the bicontainment function of the shell: we have improved the degassing method by replacing the original vacuum degassing with ultrasonic degassing, which is more efficient.
- The shell is not completely wrapped around the core during preparation or the shell is too thin in some areas: Increase the thickness the shell and consider preparing several hydrogel spheres at the same time. Then select the ones appropriate for subsequent application(s).
4. Hidro preparation is time-consuming and cannot be prepared in large quantities...
- Please use molds in production instead of manual shell wrapping. You can produce 50-60 Hidro in a single batch, which greatly reduced the production time.
