• •       Description
  •       Design
  •       Proof Of Concept
  •       Results
  •       Implementation
  •       Experiments
  •       Notebook
• •     Modeling
  •     Protein
• • Intergrated
  • Education
• •                         Parts
  •                         Contribution
  •                         Engineering
  •                         Collection
• •                  Safety
• •            Judging
• •           Team
  •           Attributions
  •           Collaboration
  •           Partnership
  •           Acknowledgement

   TOP

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 SDS-PAGE 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 be 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 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-C). 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-term 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 engineered bacteria. To our application scenario, expression of TDPs should not influence the gene circuit that responses to uric acid. To test it, we modified the gene circuit we used last year. Briefly, at the right of uric-acid detection gene circuit, we added a sequence that could expressed CAHS 106094 (Fig. 8A and 8B). As control, the part, which we used last year, could sense uric acid. However, once CAHS 106904 was added, the basic expression of reporter gene (dsRed) was too high and the gene circuit could not sense uric acid (Fig. 8C and 8D).

     Obviously, HucR was influenced. We speculate that there are two possible causes: (1) During vector construction, the promoter of HucR, was influenced. So the expression of HucR, which can repress the expression of dsRed, reduced. (2) CAHS 106094 binded HucR and hindered the binding of HucR and DNA (PhucR). To the first possible cause, as the insertion site of CAHS 106094 expression sequence was more than 40 bp from the promoter of HucR (CP6 promoter), and CP6 is a modularized part which has been widely used, we speculate that the cause is not right. To the second possible cause, from our human practice with Guangyuan Song, who is a doctoral student of structural biology, he told us the structures of TDPs are similar to molecular chaperones, which can interact with other proteins. As a result, the second cause might be right, which means the interaction of TDPs and other proteins could protect the proteins from freez-drying, but also influence the functions of these proteins. Summing up the above, some strategies should be taken.

Part 4：TDP (CAHS 106094) degradation module.

     From the introduction above, TDPs are important for storing bacteria, but redundant for engineered bacteria to perform their functions. Moreover, just as the result in part 3, TDPs might affect the normal functions of certain engineered bacteria. To avoid such potential influence, we came up with an idea that we should let TDPs degrade as quickly as possible when they are not needed.

     For the first version, we added an AAV degradation tag at the C-terminal of CAHS 106094. AAV tag can be recognized by endogenous protease of E. coli. As a result, if CAHS 106094 is needed, iPTG is added and the expression and degradation of CAHS 106094 will achieve equilibrium and the protein level will be stable at a certain concentration; If CAHS 106094 is not needed, iPTG is removed and CAHS 106094 is not expressed, residual CAHS 106094 will be degraded very fast as it has an AAV tag and a very short half-life period. In contrast, if AAV tag do not exist, the half-life period of exogenous proteins would be very long and can only be diluted by cell growth and division (Fig. 9A). With a Western Blotting experiment, we confirmed the effect of AAV tag in such condition (Fig. 9B).

     However, as endogenous protease of E. coli exists at any time and cannot be controlled, the protein of CAHS 106094 would not be enough if it is needed. To control CAHS 106094 level more accurately, we used a pdt tag instead of the AAV tag. Pdt tags can be recognized by protease mf-Lon, which comes from a kind of mycoplasma, but can not recognized by endogenous protease of E. coli. iGEM team William_and_Mary 2017 measured several pdt tags and as a proof of concept, we used pdtA tag here. However, the other tags are also candidates because the requirement of different consumers would be different, which we will explain in Model page.

     As an exogenous protein, we could control the expression of mf-Lon to specifically degrade CAHS 106094 with a pdtA tag. As we mainly use iPTG to induce CAHS 106094 expression, we chose another inducer, L-Arabinose, to induce mf-Lon. iGEM team NEFU-China 2020 and Jilin 2020 shared us two different parts that sense L-Arabinose. We tested the parts and found 0.2% L-Arabinose was sufficient to activate the expression of reporter gene (GFP or sfGFP), while D-Arabinose was invalid (Fig. 10A-D). We imagined that before freeze-drying, iPTG is used to induce CAHS 106094; when adding water to recover the bacteria, L-Arabinose is used to induce mf-Lon to degrade CAHS 106094. However, we used 100 μL 3% D-Glucose to do the freeze-drying and 1000 μL water or LB medium to recover, so the L-Arabinose based gene regulation system should work in 0.3% D-Glucose. We induced the bacteria with 0.2% L-Arabinose in different D-Glucose concentration. As expected, glucose reduced the activity of L-Arabinose sensor, but in 0.3% D-Glucose, 0.2% L-Arabinose could still induce the expression of the reporter gene (GFP) (Fig. 10E), which meant our design was reasonable. It is worth mentioning that trehalose, which is a well-known protective agent, badly reduced L-Arabinose sensor (Fig. 10E and 10F) (click here to see the possible cause), which again meant trehalose was not a suitable protective agent in our system.

     Then we used mf-Lon coding sequence instead of the reporter (GFP) downstream the L-Arabinose sensor (Fig. 11A). By western blotting, we proved that the level of CAHS 106094-pdtA could be easily controlled by the two inducers, iPTG and L-Arabinose (Fig. 11B). In addition, As pdt tags and mf-Lon protease make up a complete protein degradation system, they make it convenient to apply the TDPs in other chassis other than E. coli.

Part 5：Conclusion and next plans.

     To conclude the above four parts, we proved that with TDPs, engineered bacteria can be stored without any equipments. The bacteria can be easily transported and stored until being used. You only need to add some medium when using them. What's more, TDPs can be quickly degraded to avoid any influence. As a result, the bacteria are siccostabile before being used while have no difference from the bacteria without TDPs when being used. The convenient method would make engineered bacteria closer to our daily life.

     However, there is a lot more work to be done. (1) First, to be honest, our results are not quite stable, so we should improve our experiment skills, use more professional equipments and take more repeats to confirm the conclusion. For instance, as we observed, calculating cell numbers by OD600 is not quite accurate, which means more appropriate methods and equipments should be used. (2) Second, Some DNA parts of certain engineered bacteria should be adjusted to coordinate the TDP system. For example, to the uric acid biosensor, the reporter gene, deRed, should also hold a degradation tag so that the accumulation of dsRed during bacteria preparation could be removed as quickly as possible. (3) Third, we should improve the protocol to enhance the survival rate further. For example, the cooling rate and drying time of the lyophilization, the formula of the freeze-dried liquid. There have been many studies to improve freeze-dried liquid, but we should look for the ones that fit our system and the constituent are not needed to be removed when the bacteria are used. (4) At last, more gene combinations should be tried and the gene expression level should be optimized. In this project, we mainly paid attention to the genes to resist desiccation. However, during freeze-drying, the freeze and vacuum stresses should not be ignored. Water bear is a kind of miraculous organism, which can resist many stresses. Actually, we also tried a gene from water bear, Dsup, in human Hela cell line, and found it can resist ultraviolet radiation (Fig. 12A-C). Though it is not related to the storyline of our project, it proved that water bear is a treasure and we can find many useful genes in it. With the development of genomics, it is possible to find the genes related to freeze and vacuum. We hope that one day we can find the genes and apply them to engineered bacteria.

Part 6：Proof of concept.

     To our human practice with Shaojie Li, nowadays the survival rate of freeze-dried microorganism is around 10%. So there is a huge space to improve it. It is obvious that if some new methods can be used to double the survival rate, the cost to produce freeze-dried microorganism turns half. There are many dried microbiological products, such as lactobacillin tablets, yeast tablets and nitrifying bacteria capsule. With our project, the cost of the products above can be reduced manyfold.

     In our imagined application scenario, day powder condition could help people store the bacteria conveniently, and TDPs could help alive engineered bacteria keep a reasonable quantity and all-right physical state. As a result, once some medium is added, the bacteria would show their functions soon. Therefore, we designed a uric-acid detection kit for gout patients (Fig. 13). As is shown, in 2019, our team has designed a hardware and found saliva was a suitable sample; and this year, we made the uric-acid sensitive bacteria into dry power. When patients use it, they only need to add a bottle of solution A (1 mL, containing LB medium for bacterial culture, L-Arabinose for inducing mf-Lon to degrade TDPs and the reporter) and the saliva sample into the dry bacteria, then bring the test tube into the hardware and wait for 4 hours, the hardware will show the uric-acid concentration. No refrigerator, centrifuge or other professional equipments are needed.

     It is obvious that TDPs has a wider applied range. To explain this, we collaborated with NEFU-China 2020 iGEM team. NEFU-China designed a hardware for minesweeping. Its core is a hydrogel containing a kind of engineered bacteria that senses TNT. To their design, the bacteria are loaded into hydrogel in the laboratory, and then transported to the minefields for use. As designed, hydrogel has a storage life for about 7 days, and needs cold storage. Because minefields are usually remote, if minesweepers want to use it, they need to order some hydrogels, wait for about 3 days, and immediately use them in about 4 days (Fig. 14A). To make it more convenient, we try to use TDPs and freeze-drying to collaborate with NEFU-China (Fig. 14B). NEFU-China told us that the hydrogel is not difficult to make, so it can be made at the minefields by minesweepers. So we designed a kit for this aim (Fig. 14C). The bacteria was freeze-dried and have a long storage life, so that the minesweepers can order a large amount and use it at any time. They only need to make a hydrogel with the kit and put it into the hardware. At the same time, the transport is easier as the cold chain is no longer needed (Fig. 14D). Team NEFU-China thought that it was very helpful (Score: 4 out of 5).

     Namely, we designed a convenient kit for uric-acid detection at home, and also showed that TDPs and freeze-drying can be used in other products to make them easier closer to practical use.