iZJU-China

Inspired by social practice

Unlike most other iGEM teams, the inspiration for our team's projects doesn't come from brainstorming sessions alone; instead, it arises from real-world experiences. Some of our team members embarked on a social practice research trip to the 0799 Art District in Pingxiang, Jiangxi in January 2023. While touring oil painting studios, we noticed a strong, unpleasant odor caused by certain oil paints and the use of turpentine, a common thinner. As medical students, we were concerned about potential health risks.

As the saying goes, "Genuine knowledge comes from practice." We understand that confirming project ideas can often be a long and challenging process. Therefore, we encourage future teams to explore and conduct research visits to different places when seeking project inspiration. Along the way, you may find unexpected inspiration and pleasant surprises.

Figure 1. The practice group came to 0799 Art District.

Figure 2. Practice members visited the 0799 Art Space exhibition.

Wet lab contributions

Useful new parts

During our project, we have added a number of new parts to the iGEM registry to facilitate access and to be used by future teams.

The following components will facilitate the oxidation and biodegradation of terpenes, especially α-pinene:

P450BM-3QM

It encodes a monooxygenase mutant that can accept α-pinene as a substrate for selective oxidation, and oxidize it to α-pinene oxide, trans-verbenol and myrtenol.

Prα-POL

It encodes an α-pinene oxide lyase that can catalyze the decyclizing reaction of α-pinene oxide to form formcis-2-methyl-5-iso-propylhexa-2,5-dienal (also called Isonovalal).

The following components can constitute an NADPH regeneration system to enhance the activity of NADPH-dependent enzymes:

GLF

It encodes a glucose facilitator that can transport unphosphorylated glucose into the cell without consuming metabolic energy in the form of proton potential or phosphoenolpyruvate.

GlcDH-II

It encodes an NADP-dependent glucose dehydrogenase that oxidizes unphosphorylated glucose in the cytoplasm to gluconolactone and simultaneously converts NADP+ to NADPH.

Simplified aqueous-organic two-phase system

The aqueous-organic two-phase system has been shown as an efficient system for whole-cell catalytic culture. It can help us to achieve better whole-cell catalysis of low water-soluble substrates and reduce the toxicity of toxic organic compounds to engineered bacteria (1).

However, by reading the relevant literature, we found that most of the literature had very vague introductions on how to build the experimental device of the aqueous-organic two-phase system (2). In addition, professional instruments such as bioreactors are usually needed to complete the construction of the system (3). These make it difficult for small laboratories and undergraduate students to conduct relevant experiments.

After several trials and improvements (details can be found in the lab notebook), we formed a simplified device that can meet the construction of an aqueous-organic two-phase system suitable for reaction of small volume of substrates. The instruments and reagents used, such as culture tubes, decolorization shaker, methanol, etc. are all easy to get. We also wrote a detailed protocol, which can be found in the protocol section of experiment. It can provide inspiration for future iGEM teams who need to build aqueous-organic two-phase systems.

Figure 3. The structure of simplified aqueous-organic two-phase system.

The components of the device are indicated in the figure.

Troubleshooting: Purification of transmembrane proteins

We expressed a heterologous transmembrane protein called GLF in E. coli. However, after protein purification, SDS-PAGE gel electrophoresis did not reveal any protein bands (Figure 2).

Figure 4. SDS-PAGE results of the GLF proteins before the purification step was modified. The result suggested that the expression of GLF protein was not successfully detected. The cell lysate (CL) was discarded by mistake, so this time the result of CL was not shown on the gel.

FT (Flow through), W1-3 (wash 1-3), Protein Marker, E1-6 (elution 1-6).

After excluding the possibility of transformation failure, we wondered whether the purification steps for transmembrane proteins were different from those for intracellular proteins. After consulting our PI and reading the relevant literatures, we found that the purification of membrane proteins required the addition of detergent such as Triton X-100 during the cell lysis step (4).

Therefore, we tried incubating the bacterial solution in 1% (v/v) Triton X-100 for 1 h instead of ultrasonic cell crushing. The rest of the experimental procedures were unchanged. In addition, we also lowered the temperature of the protein denaturation treatment to 37 ° C to avoid degradation of membrane proteins. Surprisingly, the target band of GLF appeared in SDS-PAGE gel electrophoresis after protein purification this time, confirming our hypothesis (Figure 3).

Figure 5. SDS-PAGE results of the GLF protein after the purification step was modified. The result suggested that the expression of GLF protein was successfully detected, which was indicated by the corresponding bands around 50kDa. However, the bands of protein GLF were still relatively blurry in all lanes.

CL (Cell lysate), FT (Flow through), W1-3 (wash 1-3), Protein Marker, E1-6 (elution 1-6). 

Although our final band is still not very clear, this troubleshooting is still meaningful. This can provide useful experience for future iGEM teams who want to perform membrane protein purification. For details, see the protocol in the Experiments part.

Dry lab

During our project, we have developed mathematical models to simulate α-pinene degradation pathway and the co-culture of two strains of Escherichia coli that can degrade α-pinene by two-step reaction. We used the Lotka-Volterra competition system to describe the population dynamics of the two strains in the same reaction container, where they may compete for limited resources and space. We also considered the effects of glucose uptake, NADPH regeneration, and α-pinene oxide toxicity on the growth and survival of the two strains.

We estimated the values of the model parameters by performing separate and co-culture experiments with the two strains and measuring their absorbance values over time. We then fitted our model to the experimental data using python and obtained good agreement between the model predictions and the observed results.

Our model can provide useful insights into the optimal conditions for achieving efficient and stable α-pinene biodegradation by co-culturing engineered bacteria. It can also help us to design better strategies for improving the performance and robustness of our system. Our model may be extended to other substrates or bacteria that share similar characteristics with our system. We hope that our model can inspire future iGEM teams who want to explore the potential of co-culture systems for biocatalysis and bioremediation.

Safety

We summarized what we have done to achieve lab safety and how we designed to guarantee the product safety. For lab safety, we first confirmed that each member of the wet lab group had the qualification to enter the laboratory for experiments, that is, we have learned the relevant knowledge of laboratory safety and passed the assessment. Next, we had a review and intensive training. During the experiment, we also reminded each other and practiced constantly to ensure safety while improving our experimental skills (Figure 4).

Figure 6. Summary of lab safety

For project safety, we considered both the project design and product structure. Our project design learned from previous research results, which could be a solid support. Our design of biological air purifier guaranteed product security from three aspects: monitoring techniques, control of the release and waste management (Figure 5). These two workflow charts can be used as a reference for future iGEM team.

Figure 7. Summary of product structure regarding biosafety