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 at creating a new method to store various engineering bacteria. Hereafter we use E. coli to make a proof of concept.

     To let the metabolism of E. coli pause without ultralow temperature, we design to make dry bacteria powder by 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). 3% glucose solution was used as lyoprotectant.


Fig. 1

     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

     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

     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.


Fig. 4

     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.


Fig. 5

     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.


Fig. 6



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).


Fig. 7



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).


Fig. 8

     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 SDS-page experiment, we confirmed that AAV tag extremely promoted the degradation (Fig. 9B).


Fig. 9

     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. As what is shown in Fig. 9B, After adding AAV tag, we could hardly see the expression of CAHS 106094. 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. 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 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), which again meant trehalose was not a suitable protective agent in our system.


Fig. 10

     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