We are very pleased to collaborate with the QHFZ_China this year. The QHFZ_China participated in the iGEM competition for the first time last year and achieved a gold award and a nomination for special awards. This year, the QHFZ_China team mainly uses the Water Bear preservation technology to maintain engineered bacteria. This technique can also be used in our project to expand the application scope of our landmine detection device. Through the online communications, we learned from the QHFZ_China team that the CAHS protein of the Water Bear could improve the anti-drying ability of bacteria and protect the protein activity under the freeze-drying condition to ensure that the bacteria will not be inactivated under the condition of extreme water shortage. Both teams have made significant progress in the projects through our cooperation.
We learned about each other's pilot projects through an online meeting held on July 18, 2020. Several questions about each other's projects were raised in the meeting. For example, the QHFZ_China asked how our landmine detection equipment can distinguish the number and distance of landmines around it and how to recover the device. Our team members gave detailed answers and presentations for these questions.
During the first meeting, the QHFZ_China was impressed by our project and expressed their willingness to collaborate with our team for our mutual experiments and other needs. The two teams had one to one meeting on August 5, 2020. During the meeting, the two teams exchanged ideas. We found that the bacterial preservation technology of the QHFZ_China team could be applied to our project to facilitate the long distance transportation. They suggested a strategy that would allow our engineered bacteria to be carried as lyophilized powder to desired locations. In addition, we both made suggestions to each other's projects. For example, the QHFZ_China team suggested that TNT, as a dangerous explosive, should be stored safely and more attention should be paid to the sensitivity of the device.
Mutual Aid for Arabinose Sensors
After the exchange, we established a stable relationship with the QHFZ_China team. Further experimental collaboration was carried out through the group chat with the teachers of the QHFZ_China team. We generously shared our arabinose sensor (AraC-pBAD-GFP)(BBa_K3457056) with the QHFZ_China team, which promoted the progress of their project. This year, the QHFZ_China team constructed two composite components based on this vector - AraC-pBAD-CAHS 106094 (BBa_K3457055) and AraC-pBAD-mf-Lon (BBa_K3457057). Without this plasmid, it would be difficult for the QHFZ_China team to carry out their projects smoothly. Finally, the QHFZ_China team shared their results of the constructed parts proving the modularity of the arabinose sensor.
Unboxing video from QHFZ_China.
At the same time, the iGEM headquarter encouraged the participants to evaluate their parts in different laboratories to confirm their versatility. The QHFZ_China team first measured the AraC-pBAD-GFP segment, which was sensitive to L-Arabidose and its 0.2% concentration was the optimal concentration for the induction. In addition, we tested other conditions and observed that: (1) the sensor only responded to L-Arabidose, but not D-Arabidose; (2) D-glucose and D-trehalose showed decent induction of the expression, while D-trehalose had relatively low induction. We believe that the results would improve our knowledge of the related parts.
Unboxing video from NEFU_China.
Validation of the Bacterial Storage Methods Developed by the QHFZ_China Team
During our interaction with the iGEM team from Tsinghua University High School (QHFZ_China), we learned that their projects for this year is to use with CAHS protein of the Water Bear for the preservation of bacteria. Our team has built active collaborations with the QHFZ_China team and they provided us the CAHS protein to test our bacteria. We hoped that the CAHS protein would significantly increase the survival rates of our engineered bacteria during resuscitation. Thus, we tested the effect of CAHS protein on the resuscitation of our bacteria after they were freeze-dried into powder. As shown in Fig. 1, the presence of the CAHS protein could increase the survival rate of our engineered bacteria by 3-fold during resuscitation, as compared to the condition without this protein.
Fig.1. The presence of the CAHS protein could increase the survival rates of the engineered bacteria during resuscitation.
Meanwhile, we also tested the DNT responsiveness of our engineered bacteria immediately after their resuscitation. Although the revived bacteria from the freeze-dried powder could well respond to DNT, its long-term induction reached a platform after about 6 hours (Fig. 2). Thus, the bacterial preservation based on the CAHS protein could be used in the bacterial transportation, although their long-term induction by DNT may be attenuated.
Fig.2. Comparison of the luminescence production induced by 10 mg/L of DNT in the engineered bacteria resuscitated after being freeze-dried into powder versus freeze-stored in glycerin.
The two teams also helped each other in several other ways. (1) They shared safety forms and helped each other to check them. (2) The QHFZ_China team had issues in displaying the subtitles of their promotional videos, and we provided helpful technical support. We also used the Water Bear protein lyophilization technique of the QHFZ_China team to make our engineered bacteria into dry powder, which solved the problems of long-term storage and long-distance transportation of the bacteria. Prior to the on-site use, the bacteria powder can be revived by adding a solution and then embedded in the hydrogel, which would improve the practical applications of our landmine detection device.