Valid Contribution & Proof of Concept
Concept 1: Cold-inducible ON-switch and heat-inducible ON-switch
Temperature has unique advantages in microbial therapy: non-invasive nature, good penetrability and reversibility. We can control the function gene being expressed in some temperature range and being closed in other ranges so that easily changing the temperature can switch on or off the expression of targeted gene.
Figure 1 shows the expected effect of heat-inducible ON-switch and cold-inducible ON-switch.
Figure 1. the expected effect of heat-inducible ON-switch and cold-inducible ON-switch is shown.
Proof of concept: The function of cold-inducible ON-switches
Figure 2. The cold-inducible ON-switch is encoded on two plasmids. The circuit is shown with the genetic parts and relationships among them.
Within these screening circuits, the expression of GOI (goal of interest) was inhibited by the evolved transcription factor and the evolved protease could activate the reporter gene by cleaving the sensitive repressor.
We got the temperature-sensitive parts from our adviser, which are TEVts and CI434ts-TEVsite.
TEVts means TEV protease from Tobacco Etch Virus which can recognize special amino acids sequence (ENLYFQ) and cleave the sequence at the site between F and Q. Its activity is triggered by coldness.
Table 1. the thermo-sensitive mutants of TEV and their mutation sites
CI434ts-TEVsite is a thermo-sensitive transcription repressor. We insert the TEV recognition sequence into the link part of transcription factor so that TEV protease can cut the transcription factor to make it inactive.
Meanwhile, we put the TEV protease coding gene under the promoter of corresponding CI434 so that TEV and CI434 inhibit each other. Once there is a small disturbance, the balance will lean to one side to act. We put our target gene under the promoter of the corresponding CI434, so that its expression is controlled by the transcriptional factor side. When coldness comes, the TEV protease is active and cut the transcription factor to relieve the inhibition of promoter so that the target gene can be express.
The cold-inducible TEV protease mutants and heat-inducible transcription factors mutants are all evolved through a suitable restriction, which creates many different switches with a wide range of transition temperature.
We characterized the performance of this series of switch combinations, and the results are as follows.
We firstly used ELIASA to characterized the tendency of fluorescence change of a series of cold-inducible ON-switches using different TEVts mutants under different temperatures. As shown in Figure 3, all five switches show high fluorescence under low temperature, while their fluorescence all decreases when temperature rises. The groups of TEVts#11, TEVts#17 and TEVts#18 began to inhibit the expression of fluorescence at 37℃ and nearly inhibit the expression of fluorescence completely at 42℃. While the groups of TEVts#6 and TEVts#7 begin inhibition at 30℃ and achieve complete inhibition at 37℃.
Figure 3. The tendency of fluorescence change of a series of TEVts mutants under different temperatures (measured by ELIASA)
Then we chose two kinds of cold-inducible ON-switches, consisting of TEVts#6 and TEVts#18 correspondingly, to measure their temperature response curve more precisely by flow cytometer in TOP10 strain. From Figure 4 we can see that two group both show narrow transition ranges and have ∼100-fold induction, which was achieved within less than ten degrees. Thus, our cold-inducible ON-switches show high performance and versatility, which ensures the potential for basic research, as well as industrial and biomedical applications, and truly makes engineered bacteria precisely controlled.
Figure 4. The induction curve of the cold-inducible ON-switches (TOP10)
Considering our platform is going to serve microbial therapeutics, we found a more suitable E.coil strain, Nissle 1917, a probiotic with more than 100 years of medical application. We also measured the best one, consisting of TEVts#18, in Nissle 1917. It also shows high performance similar with it in TOP10 strain.
Figure 5. The induction curve of the cold-inducible ON-switches (Nissle 1917)
Proof of concept: The function of heat-inducible ON-switches
Figure 6. The heat-inducible ON-switch is encoded on one plasmid. The circuit is shown with the genetic parts and relationships among them.
In this circuit we just make full use of the cold-inducible transcription repressors. We found TCI transcription factor family and TlpA family whose activity will be lost under high temperature. The structure of the corresponding transcription factor changes with the temperature’s change.
When it is above threshold temperature, TF will be allosteric, losing its activity. Then the inhibition of target gene is relieved while the target gene is expressed. We characterized the performance of this series of switch combinations, and the results are as follows.
We first characterized the performance of 6 members from the two family in Top10 strain, the result shows that most of the transcription repressors show sharp thermal transitions, especially TCI and TCI42, with more than 100-fold induction within 10 degrees Celsius. Their impressive performances make them candidate parts for our further circuit design.
Figure 7. The induction curve of the heat-inducible ON-switches (TOP10)
Also, we tested these heat-inducible ON-switch in the chassis E.coli Nissle 1917, a probiotic with more than 100 years of medical application, their robustness give us more confidence in the stability and preciseness of our ark.
Figure 8. The induction curve of the heat-inducible ON-switches (Nissle 1917)
Concept 2: Double-status switch
The temperature-dependent on-off switch consists of both the two types of switches mentioned above. We can choose different transition temperature to meet different environments and goals. Also, by combining the heat-inducible with the cold-inducible ON-switch, we can get a double-status switch to realize more functions in one creature.
Figure 9. The expected effect of double-status switches
Proof of concept: The function of double-status switch
Figure 10. The double-status switch is encoded on three plasmids. The circuit is shown with the genetic parts and relationships among them.
Based on the temperature response curve of cold-inducible ON-switches and heat-inducible ON-switches, we chose the best mutant, TEVts#6 and TCI42 to build our double-status switch. But actually, users can choose other thermo-sensitive protease or transcription factors to build other more suitable double-status switches based on their needs.
AS shown in Figure 11, our cold-inducible ON-switch works when the temperature is low and then express green fluorescence. When the temperature rises, the activity of cold-inducible ON-switch is inhibited and the heat-inducible ON-switch works. Therefore, we can turn on different genes’ expression at different temperatures. Similar with our single thermo-sensitive parts, this double-status switch also shows high performance and versatility.
Figure 11. The induction curve of the double-status switches (TOP10)
Concept 3：Construction of orthogonal protease system
According to resent research, we can find many similar proteases to build similar cold-inducible switches such as TVMV protease (cleavage site TVRFQS), SUMMV protease (cleavage site EEIHLQ), HRV3C (cleavage site LEVLFQGP).
Table 2. the proteases and their cleavage sites
We can use them to construct orthogonal protease system if they have no much cross-talking.
Proof of concept: Orthogonality between basic parts--protease
We can combine the two different cold-inducible switches to achieve the function of sequence switches. However, we should make sure that there two combinations have no influence on each other. Thus, we design experiments to verify the orthogonality of different cold-inducible switches. That is the orthogonality of different proteases and their cleavage sites. We inserted all the four cleavage sites into the transcription factor CI434 to build the relation between protease and transcription factor. Then we transform all the 16 kinds of combination of protease and CI434 into E.coli using sf-GFP as the reporter gene (GOI). Only the right pair can erase the inhibition of CI434 and trigger the expression of sf-gfp. Through the results of the fluorescence expression, we can find whether there are interactions between one protease and one cleavage site. The results below demonstrate good orthogonality between these proteases and their cleavage sites.
Figure 12. the orthogonality among four proteases represented by the fluorescence of sf-GFP is shown.
Concept 4: Incorporation non-canonical amino acids to target protein
Currently, another biocontainment strategy is establishing auxotrophies for essential compounds, however, metabolic auxotrophies can be circumvented by scavenging essential metabolites from nearby decayed cells or cross-feeding from established ecological niches. By establishing ‘synthetic auxotrophy’ for a non-canonical amino acid, we would like to introduce a robust and effective strategy against environmental supplementation, which is considered as the major problem of current auxotrophy strategy.
Proof of concept: Incorporation efficiency and selectivity of L-dopa and CL2Y
We assigned the TAG stop codon to incorporate a non-canonical amino acid and tried to redesign an essential gene to develop corresponding dependent bacteria.
Figure 13. The general design of establishing our ‘synthetic auxotrophy’. pEVOL plasmid is a vector with aaRS and tRNACUA, it can help incorporate the ncAA into proteins in Escherichia coli with increased yields and stability. We assigned the TAG stop codon into an essential gene so that only when the bacteria are provided with the ncAA, the essential enzyme can be synthesized and support the survival of the engineered bacteria.
Getting a high-performance aminoacyl-tRNA synthetase (aaRS) system from our PI’s laboratory, we chose 3,5-dichloro-L-tyrosine (Cl2Y) as the non-canonical amino acid. First, we proved the concept of our design using GFP as the target gene. We constructed mutant GFP with 9TAG and 17TAG as the potential incorporating site. When additionally providing Cl2Y, we could see a prominent increase(~100 folds) of the fluorescence, indicating the high efficiency of incorporation.
Figure 14. When provided Cl2Y, the fluorescence of GFP can be increased almost 100 folds, indicated the high efficiency of incorporation.
MS result further proved that Cl2Y was incorporated into the target site with high selectivity and efficiency.
Figure 15. Mass spectrum result of 9Cl2Y GFP. 27824 indicates the molecular weight of interested protein.
Figure 16. Mass spectrum result of 17Cl2Y GFP. 27794 indicates the molecular weight of interested protein.
Concept 5: Appropriate heating speed and region using our hardware
In our model part, we simulate the temperature distribution of intestine in 1000 seconds using by our hardware:
Figure 17 The temperature of heating area in 1000s
The heating range is about 4-5 centimeters in 1000 seconds, which is not too large, effecting colonized bacteria in intestine, not too small, causing little drug effect or target miss, proving that the design of heating capsule is feasible.
Proof of concept: Heating curve by our hardware
In the heating system of our hardware, we used PTC thermostatic heating element as the heat source. Due to the limitation of its physical properties, the temperature of PTC heating element is constant under a certain voltage, which can well meet the requirements of constant temperature heating, safe and simple.
Figure 18. 5V50℃ PTC we use
Figure 19. PTC inside the capsule.
 Zheng, Y., Meng, F., Zhu, Z., Wei, W., Sun, Z., Chen, J., . . . Chen, G.-Q. (2019). A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts. Nucleic Acids Research. doi:10.1093/nar/gkz785
 Guet C , L. C . Combinatorial Synthesis of Genetic Networks[J]. Science, 2002, 296(5572):1466-1470.
 Zong Y , Zhang H M , Lyu C , et al. Insulated transcriptional elements enable precise design of genetic circuits[J]. Nature Communications, 2017, 8(1):52.
 Piraner D I , Abedi M H , Moser B A , et al. Tunable thermal bioswitches for in vivo control of microbial therapeutics[J]. Nature Chemical Biology, 2016.
 Mandell, D. J. et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55-60, (2015).
 Liu, X. et al. Significant expansion of fluorescent protein sensing ability through the genetic incorporation of superior photo-induced electron-transfer quenchers. J Am Chem Soc 136, 13094-13097, (2014).