Team:QHFZ-China/Proof Of Concept

Proof of Concept

Proof of Concept




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

     Our project aims to create a new method of storing various engineered bacteria. Hereafter we use E. coli to make a proof of this concept.

     To let the metabolism of E. coli pause without using ultralow temperatures, we planned to make dry bacteria powder through lyophilization. As a result, the bacteria must tolerate two types of stress: the freeze-drying process and the subsequent dry state. To study whether or not our TDPs (Tardigrade intrinsically disordered proteins) can protect bacteria during such stress, we designed a protocol as shown below (Fig. 1). A 3% glucose solution was used as a protectant during the lyophilization process.


Fig. 1 The protocol of our experiment to test the relative survival rate.

     We constructed a gene circuit to let TDPs express in E. coli BL21 (DE3) strain (Fig. 2A). 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).


Fig. 2 TDPs were expressed with modified pet28a+ vector in E. coli BL21 (DE3) strain. (A) The Schematic cartoon of the vector we used. (B) GFP showed the target gene in the vector can be induced by 2 mM iPTG. (C) A quantitative data of Fig. 2B. (D) SDS-PAGE showed the expression of TDPs.

     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).

Table. 1 Proteins that were studied in this project

Proteins Description References
sfGFP a green fluorescence protein (Control Group) -
SAHS 33020 a TDP 1
CAHS 89226 a TDP 1
CAHS 94205 a TDP 1
CAHS 106094 a TDP 1
CAHS 107838 a TDP 1
LEA a plant resistance relative protein 2
OtsA and OtsB two enzymes from bacteria that can convert glucose into trehalose 3

     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.


Fig. 3 CAHS 106094 had the best protective effect. (A) A screening of 5 TDPs, LEA and OtsBA. (B) The statistical graph of Fig. 3A.

     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 that are widely used in various strains of E. coli and hold different strengths. 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 standard chassis.


Fig. 4 The protective effect was enhanced with the expression level of CAHS 106094. (A) GFP showed the relative strength of J23109, J23107 and J23100 promoters. (B) A quantitative data of Fig. 4A. (C) The relative strength of the promoters. The strength of J23109 was set as 1. (D) The lyophilization-recovery test of E/ coli BL21 (DE3) strain expressing CAHS 106094 with different promoters. (E) The statistical graph of Fig. 4E. (F) Another statistical graph of Fig. 4E. The X-axis is the relative expression level of CAHS 16094.

     Though CAHS 106094 gave a positive result, the survival rate should be further enhanced. The more the survival rate, the less cost is needed to produce the products, and the better function the bacteria will show when used. As for water bears (tardigrades), they express many TDPs at the same time to survive from desiccation. Therefore, we came up with the idea that we should let the bacteria express several TDPs together to increase the survival rate. As a 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 more possible to show a synergistic effect. However, the co-expression of CAHS 106094 and SAHS 33020 could enhance 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.


Fig. 5 To combine different TDPs (SAHS 33020 AND CAHS 16094) gave exhibited a better preventive effect. (A) The Schematic cartoon of the gene part we used. (B) The lyophilization-recovery test of E/ coli BL21 (DE3) strain expressing single of double TDPs. (C) The statistical graph of Fig. 5C.

     All the results above proved that TDPs helped E. coli BL21 (DE3) strain survive in such condition. However, since the time is limited , we only store the bacteria power at room temperature for 2 days for most experiments, which did not match 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. TTo mimic such condition and prove that TDPs can protect bacteria at lyophilization and short-term dry storage and long-term dry storage, we prolonged the storage time for ten 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.


Fig. 6 TDPs showed protective effect during long-term dry storage at room temperature. (A) The flow chart showed the change of the protocol. (B) The lyophilization-recovery test of E/ coli BL21 (DE3) strain expressing TDPs. (C) The statistical graph of Fig. 6B.



Part 2：TDP (CAHS 106094) showed satisfactory modularity

     To synthetic biology, great modularity is a necessary standard to evaluate a genetic part. To detect if CAHS 106094 worked well in other situations, we did the following experiments and 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 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).


Fig. 7 TDP (CAHS 106094) showed satisfactory modularity. (A) The Schematic cartoon of the vector we used. (B) GFP showed the target gene in the vector can be induced by 0.2% L-arabinose. (C) A quantitative data of Fig. 7C. (D) The lyophilization-recovery test of E/ coli DH5α strain expressing CAHS 106094. CAHS 106094 showed protective effect. (E) The statistical graph of Fig. 7D. Data are shown via mean ± SD. N = 3.



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 sufficient to store bacteria. However, the addition of TDPs should not influence the normal functions of engineered bacteria. In our application scenario, the 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 uric-acid detection gene circuit's right, we added a sequence that could express CAHS 106094 (Fig. 8A and 8B). As a 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).


Fig. 8 TDP (CAHS 106094) could influence the normal functions of uric acid sensor. (A) The Schematic cartoon of the part we construct. The binding site was very close, so Pcp6 may be affected. (B) The DNA sequencing ensured the construction of this vector. (C) dsRed showed that the constitutive expression of dsRed is over high. (D) The statistical graph of Fig. 8D.

     Thus, 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 is reduced. (2) CAHS 106094 bound 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 freeze-drying, and 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 essential for storing bacteria but redundant for engineered bacteria to perform their functions. Moreover, just as a result, in part 3, TDPs might affect certain engineered bacteria's normal functions. 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 the 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 ata specific 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 concise a brief half life. In contrast, if AAV tag does 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 SDS-page experiment, we confirmed that AAV tag extremely promoted the degradation (Fig. 9B).


Fig. 9 AAV module. (A) The Schematic cartoon the function of AAV tag. (B) SDS-PAGE showed the low expression of CAHS 106094-AAV, which may be because of a fast degradation.

     However, the 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. As what is shown in Fig. 9B, After adding AAV tag, we could hardly see the expression of CAHS 106094. To maintain 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 be recognized by the 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 different consumers' requirements would be additional, which we will explain in Model page.

     As an exogenous protein, we could control the expression of mf-Lon to degrade CAHS 106094 with a pdtA tag specifically. 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.

     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_China 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 the 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 the 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, a well-known protective agent, badly reduced the L-Arabinose sensor (Fig. 10E and 10F), which again meant trehalose was not a suitable protective agent in our system.


Fig. 10 Measurement of Arabinose sensor. (A) GFP showed the gene circuit from NEFU-China can be induced by L-arabinose. (B) A quantitative data of the part from NEFU-China. L-arabinose induced the part, and 0.2% was enough, but-D-arabinose did not work. (C) GFP showed the gene circuit from Jilin_China can be induced by L-arabinose. (E) A quantitative data of the part from Jilin_China. (E) A quantitative data showed glucose and trehalose, repressed the promoter. At 0.2%, the affect of trehalas’ was worse. (F) A hypothetical reason to explain Fig. 10E. Trehalose can be degraded into two glucose, so that affect the part.

     Then we used mf-Lon coding sequence instead of the reporter (GFP) downstream the L-Arabinose sensor (Fig. 11A). Unfortunately, the synthesis of mf-Lon was full of complications. Finally, we got mf-Lon sequence and finished the construction of the vectors: (1) adding a pdtA tag sequence at the 3' end of CAHS 106094 sequence (but before the termination codon); (2) ligating the ParaBAD promoter and mf-Lon. (Fig. 11B) However, we did not have enough time to get more data with the vectors. As a result, we constructed a math model to explain how it works.


Fig. 11 Design of degradation module. (A) The Schematic cartoon of the designed module. (B) The DNA sequencing data showed the successful construction of CAHS 106094-pdt and mf-Lon parts.



Part 5：Conclusion and next plans

     To conclude the above four parts, we proved that with TDPs, engineered bacteria could 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 will be quickly degraded to avoid any influence. As a result, the bacteria are siccostabile before being used while having no difference from the bacteria without TDPs. The convenient method would make engineered bacteria closer to our daily life.

     However, there is a lot more work to be done. (1) First, our results are Relatively unstable. For instance, as we observed, calculating cell numbers by OD600 is not entirely 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, the reporter gene, deRed, should also hold a degradation tag to the uric acid biosensor 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 withstand many pressure. We also tried a gene from the water bear, Dsup, in human Hela cell line and found it can resist ultraviolet radiation (Fig. 12A-C). Though it is not related to our project's storyline, it proved that a 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.


Fig. 12 Dsup protected human HeLa cells during ultraviolet radiation. (A) Cells before ultraviolet radiation. GFP showed the successfully transfection. (B) Cells 48 h after ultraviolet radiation. The red circles were the region that could exposure UV.



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, dry 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.


Fig. 13 A designed kit for uric acid detection. The bacteria do not need a freezer to be stored.

     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. Team NEFU-China thought that it was very helpful (Score: 4 out of 5).


Fig. 14 A designed protocol for NEFU-China 2020. (A) Their plan is to make hydrogel in lab and is should be used in 7 days. (B) We gave an alternative solution is to make dry powder in lab and to make hydrogel just before use, so the shelf life is prolonged. (C) Th kit we designed.

     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.


References

[1] G.J., Koshland, D., and Goldstein, B. (2017). Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. Mol Cell 65, 975-984 e975.
[2] iGEM Team Valencia 2010, https://2010.igem.org/Team:Valencia
[3] iGEM Team Imperial_College_London 2009, https://2009.igem.org/Team:Imperial_College_London

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