Proof of Concept

Content

Part 1：TDPs could help E. coli survive from freeze-drying and subsequent dry state in room temperature.

Part 2：TDP (CAHS 106094) showed satisfactory modularity.

Part 3：TDP (CAHS 106094) could influence the normal functions of engineered bacteria.

Part 4：TDP (CAHS 106094) degradation module.

Part 5：Conclusion and next plans.

Part 6：Proof of concept.

Part 1：TDPs could help E. coli survive from freeze-drying and subsequent dry state in room temperature.

     Our project aims at creating a new method to store various engineering bacteria. Hereafter we use E. coli to make a proof of concept (click here to see why E. coli）.

     To let the metabolism of E. coli pause without ultralow temperature, we design to make dry bacteria powder by lyophilization (click here to see why lyophilization). As a result, the bacteria must tolerate two stresses: freeze-drying process and subsequent dry state. To study whether our TDPs (Tardigrade intrinsically disordered proteins) are able to protect bacteria during such stress, we design a protocol as below (Fig. 1) (click here to see why we design it this way). 3% glucose solution was used as lyoprotectant (click here to see why 3% glucose).

     We constructed a gene circuit to let TDPs express in E. coli BL21 (DE3) strain (Fig. 2A) (click here to see why BL21）. To test whether the gene circuit worked well under the induction of 2 mM iPTG (isopropyl-β-d-thiogalactoside), we put an sfGFP protein coding sequence into the gene circuit and found that E. coli BL21 (DE3) gave green fluorescence as expected (Fig. 2B). Further, with the help of SDS-page technique, we confirmed the expression of TDPs in this system (Fig. 2C and Fig. 2D).

     Then we used several parts to test if they can protect bacteria under such stresses. The parts included several TDPs, and another two parts that was used for stress resistance before TDPs were discovered: (1) LEA, which was found in plants; (2) OtsB-OtsA, which can protect bacteria by producing trehalose (Table 1).

     Though the parts have been proved to resist desiccation by research articles and / or iGEM teams, we concluded that dry-freezing might give not quite the same stresses. As a result, several parts gave poor performance. However, several parts seemed to offer some help. Among those, we found that CAHS 106094 was best (Fig. 3A and 3B). Hereafter, we use CAHS 106094 as the principal line of this story.

     In the past, only qualitative researches about TDPs could been found. Here, we hoped to do some quantitative research to show more details about TDPs. We chose three constitutive promoters which are widely used in various strains of E. coli and hold different strength. With the help of a reporter, sfGFP, we re-confirmed that J20109 is a weak promoter, J23107 is moderate and J23100 is strong (Fig. 4A and 4B). Again, CAHS 106094 protected bacteria during freeze-drying, and the effect improved with the increase of CAHS 106094 expression (Fig. 4C). As a result, in the future, more promoters with different strengths should be tested to detect the best expression level of TDPs for every common chassis.

     Though CAHS 106094 gave a positive result, the survival rate should be futher enhanced. The more the survival rate, the less cost is needed to produce the products and the better function the bacteria will show when they are used. As for water bears (tardigrades), they express many TDPs at the same time to survive from desiccation. Therefore, we came up with an idea that we should let the bacteria express several TDPs together to increase the survival rate. As time limited, we only tried to combine two proteins, CAHS 106094 and SAHS 33020 (Fig. 5A). CAHS 106094 showed the best effect and the structure of SAHS 33020 is the most different from that of CAHS 106094 among the TDPs, so we speculated that the two proteins are most possible to show synergistic effect. However, coexpression of CAHS 106094 and SAHS 33020 could enhanced the survival rate a little bit than expression of one TDP (Fig. 5B). As a result, in the future, more combinations should be tested to find an optimum combination.

     All the results above proved that TDPs helped E. coli BL21 (DE3) strain survive in such condition. However, as time limited, we only store the bacteria power at room temperature for 2 days for most experiments, which obviously did not accord with the actual situation. We designed that the bacteria should be stored at room temperature for a long enough time, so that the bacteria could be transported and stored conveniently by ordinary people before being used. To mimic such condition, as well as to prove that TDPs can protect bacteria not only at lyophilization and short-term dry storage, but also at long-stem dry storage, we prolonged the storage time for 10 days more than that in Fig. 5 (Fig. 6A). In this situation, TDPs again showed a protective effect (Fig. 6B and 6C). In conclusion, the dryness could make the metabolism of bacteria pause and TDPs would let bacteria survive in such condition, so it is an ideal preservation method.

Part 2：TDP (CAHS 106094) showed satisfactory modularity.

     To synthetic biology, great modularity is an important standard to evaluate a genetic part. To detect if CAHS 106094 worked well in other situations, we did the following experiments. We searched the studies about TDPs in the past. All scientists and iGEM teams studied CAHS 106094 with pet28a or pet28b vectors in E. coli BL21 (DE3) strain, except an iGEM team claimed that they studied in E. coli DH5α strain but failed. To confirm that if the genetic part could be widely used beyond pet vectors and BL21 (DE3) strain. We put the coding sequence of CAHS 106094 into pYB1a vector, which was a gift from iGEM team NEFU-China 2020 and whose inducer is L-arabinose (Fig. 7A). Then we transformed it into E. coli DH5α strain and found the normal expression (Fig. 7B and 7C). As expected, the bacteria expressing CAHS 106094 held a higher survival rate than the GFP group (Fig. 7D an 7E). It is worth mentioning that because of resource and time limits, for most experiments, we did not have parallel repeats. To better calculate the effect, we have three parallel repeats in this experiment, so that there were bars (standard deviation).

Part 3：TDP (CAHS 106094) could influence the normal functions of engineered bacteria.

     Our project this year is used to store engineered bacteria before use, especially the E. coli we made last year to detect uric acid for gout patients. The results above proved that the method is effective to store bacteria. However, the addition of TDPs should not influence the normal functions of