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 and 2C). Further, with the help of Western blotting technique, we confirmed the expression of TDPs in this system (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 J23109 is a weak promoter, J23107 is moderate and J23100 is strong (Fig. 4A-C). Again, CAHS 106094 protected bacteria during freeze-drying, and the effect improved with the increase of CAHS 106094 expression (Fig. 4D-F). 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 t