This year, our team has made new characterization of the existing parts and create new parts for improvement, which contributed a lot of meaningful data and conclusions. Besides, we introduced new chassis and operation protocols into the iGEM. Lastly, by presenting our project, we present new concepts and compatible platforms for others to use. We have put a lot of effort into providing new design ideas and broadening the application of iGEM projects, so we hope that our contribution will help iGEM teams in the future.

To achieve the nutrient starvation response, we selected three carbon starvation promoters as the sensors: PyciG (BBa_K4115018), PcstA (BBa_K4115003 or BBa_K118011), and PcsiE (BBa_K4115016). To check if the selected promoters can be activated by nutrient limitation (starvation), we constructed report genes as Figure 1A. The fluorescence intensities normalized by OD₆₀₀ indicate the relative promoter activities.

sfGFP has a very long half-life in the cytoplasm, which makes it not suitable for indicating some immediate change in gene expression. At the suggestion of our partnership BUCT_China, we decide to add an LVA degradation tag on the C-terminal of sfGFP (To see more collaborations with BUCT_China, you can go to our partnership page.). LVA tag can reduce the half-life. So in principle, using sfGFP with LVA tag as the reporter can more realistically reflect changes in promoter activity (Figure 1B).

Figure 1. sfGFP (A) or sfGFP with LVA (B) are used as a reporter to indicate starvation promoter activity.

The following data in Figure 2 demonstrates that all three selected promoters have a higher promoter activity under the high glucose concentration. Also, sfGFP with LVA is a better reporter for PcstA and PcsiE (Figure 2B). After adding the LVA tag, the relative FI/OD₆₀₀ decreased since the shorter half-life. Promoter activity fold changes at different glucose concentrations were also increased with the addition of the LVA tag. Unexpectedly, LVA tag increases the relative FI/OD₆₀₀ of PyciG abnormally (Figure 7A). This may be due to LVA tag competitively inhibiting the degradation of sigmaS, for RpoS degrades in the same pathway with LVA-tagged proteins. Another interesting finding is on J23101. It is usually known as a constitutive promoter, but its activity changes with the global metabolism level. At high glucose concentrations, the activity of J23101 was significantly higher than that at low glucose concentration.

Figure 2. Relative FI/OD₆₀₀ of the three selected starvation promoters. (A) PyciG. (B) PcstA and PcsiE. J23101 (a constitutive promoter) is used as the positive control. Empty in Figure 2 is used as the negative control and the automatic FI/OD₆₀₀ of Empty can seem as a background signal. The background signal has been removed from FI/OD₆₀₀ of all groups shown in this figure. Then these data are normalized with J23101 (under 4.0 g/L glucose) and shown as relative FI/OD₆₀₀. (LVA) indicates that in this group, the reporter is sfGFP with LVA. All the constructs were cloned into pUC high-copy number backbone.

We mainly evaluate the quality of promoters from two aspects: First, promoter activity is an important evaluation index. Empirically, we believe that promoter activity not lower than the J23101 (a constitutive promoter with moderately strong activity) is necessary for production and genetic circuits. Second, fold-change is another important perspective for promoters, especially for those being used in complicated genetic circuits. Fold-change can be defined as the ratio of the activity in the activated state to the non-activated state. For our starvation promoters, the practical definition is the ratio of promoter activity under low glucose concentration to that under high glucose concentration. We want the fold change to be as large as possible.

According to the above data in Figure 2, we find that PcstA has the largest fold-change (6 folds) and proper activity among the three starvation promoters. To further increase its fold change and activity, we make some mutants on its CRP-binding sequence and Fis-binding sequence. To know more, please go to our Improve page.

To achieve the intercellular communication, we choose a widely used quorum sensing system, lux for this purpose. The whole lux system can be separated into two parts: the signal sender and the signal receiver. 3O C6 HSL is the signal molecule of quorum sensing. The signal sender expresses an AHL synthetase, LuxI (BBa_C0161). The signal receiver needs to express a composite part device BBa_K4115039, which is mainly based on LuxR (BBa_C0062).

To examine whether the signal receiver can function properly, we induced the reporter (sfGFP) expression in the receiver with 3OC6 HSL at concentrations ranging from 10^-4 to 10^-14 M (Figure 3). After 3 hours induction, the cultures are used for measuring FI and OD₆₀₀. Our data fitted the logistical curve successfully (Figure 4). Concentration-dependent changes in promoter activity were observed between 10^-10 and 10^-7 M. 10^-7 M 3OC6 HSL is sufficient to induce the maximum activity of lux pR (BBa_R0062).

Figure 3. sfGFP is used as a reporter to indicate lux pR promoter activity.

Figure 4. Induced sfGFP expression of the receiver with 3O C6 HSL at concentrations ranging from 10^-4 to 10^-14 M.

After confirming that our receiver is valid, we used the supernatant of the signal sender culture to activate the signal receiver (Figure 5). 10^-7 M 3O C6 HSL is supplied for positive control. The supernatant successfully induced the receiver to produce efficient sfGFP expression compared to the negative control (Blank). Referring to Figure 4, the concentration of 3O C6 HSL in the supernatant is about 10^-9 M.

Figure 5. Induced sfGFP expression of the receiver with the supernatant of the signal sender culture. Blank: Culture of receiver strain without treating with 3O C6 HSL

The experiments above demonstrate our quorum-sensing constructs can achieve intercellular communication between bacteria. Then, we applied this system to the immobilized E. coli to perform further characterization to see if the intercellular communication between immobilized bacteria is workable.

In the experiment, three kinds of immobilized E. coli: one carrying the LuxI gene can synthesize the signal molecule 3OC6HSL; one carrying the LuxR-Plux-sfgfp sequence can respond to the 3OC6HSL signal by expressing sfGFP; the last one carrying no functional part (empty). Immobilized E. coli (LuxI) and E. coli (LuxR-Plux-sfgfp) were placed into the same dishes as the experimental group and E. coli (empty) and E. coli (LuxR-Plux-sfgfp) were placed together to be the negative control. After two days of co-culture and induction, the two experimental groups showed obvious fluorescence enhancement compared with the negative control groups, which indicated the success of intercellular communication in immobilized E. coli (Figure 6).

Figure 6. Intercellular communication in immobilized E. coli through LuxR system. The top dishes are the negative control group while the bottom dishes are the experimental groups. There are two parallel groups.

This year, we developed two new chassis strains to build our ternary microbial symbiosis system, S. elongatus HL7942, and A. caulinodans ORS571. We hope to provide more chassis choices and convenience for future iGEM teams through our introduction of chassis and sharing of operating methods.

Figure 7. The two new chassis strains we developed.

S. elongatus has been used as the autotrophic part of microbial symbiosis systems to provide carbon sources many times. The HL7942 is a gift from Dr. Xuefeng Lu, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences. This is an artificially engineered strain, which could be naturally transformed into an allogeneic DNA sequence. In this case, the genetic operation of S. elongatus HL7942 will be much easier than any other S. elongatus strain.

A. caulinodans ORS571 is a type of rhizobium, which can perform nitrogen fixation by nitrogenase. However, it is quite different from many other rhizobia, because It can perform nitrogen fixation in the free state while other rhizobia only do this when are symbiotic with plants. Besides, A. caulinodans has been relatively well studied and genetically modified in previous literature, which provides us with sufficient experience and guidance to carry out the operation. Therefore, A. caulinodans ORS571 is undoubtedly the best choice for future iGEM teams who would like to propose projects related to microbial nitrogen fixation.

In our experiment, we completed the culture and genetic operation of the two microorganisms above and verified the results, as well as summarized the experimental methods. To see more details about our results and methods, please go to the Results and Experiments page.

We propose an innovative Separate-Immobilized Fermentation pattern. "Separate" means different engineered microorganisms ferment in separate fermenters, and "Immobilized" means microorganisms are immobilized into the medium as the stationary phase, and the culture fluid can flow between microorganisms as the mobile phase. Besides, we introduced hardware design including temperature control, flow rate control, gas control, and solution condition monitoring to the system. To see more details on the hardware design, please go to the Hardware page. Hopefully, this interesting and "crazy" proposal could inspire future teams to come up with more innovation in the manufacturing track.

What's more, due to the compatibility and universality of our project, it could serve as a production platform for future iGEM teams. They could involve our symbiotic system in their project to achieve the purpose of engineering microorganisms to work continuously without an external energy supply. In the process, we expect that future teams will give feedback and suggestions for improvement on MBCS-Mars, and even launch new versions of our project by engineering success. To see how we implement MBCS-Mars, please go to the Implementation page.