In synthetic biology, modularization is one of the most important steps to build a functional circuit. Also, to make the genetically engineered organisms under human’s control, we usually regard switches as the “faithful gatekeeper”. Various natural switches make it more possible and easier to achieve, which lays a solid foundation for microbial therapy.
However, natural functions don’t mean best effects. They all show the same defects:
The most directive way to optimize them is to give special restricts and evolve the parts to meet the requirements.
As for the trigging factors of the switch, temperature stands out for its 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.
In this case, we can use the temperature-sensitive switch to function and solve the problems in microbial therapy.
Figure 1. the expected effect of heat-inducible ON-switch and cold-inducible ON-switch is shown.
In order to enrich the function of our temperature-sensitive switch, we build two types: cold-inducible and heat-inducible switches. And also, we combine the cold-inducible with heat-inducible switches to create an on-off switch in order to achieve the goal of temperature controlling two circuits.
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)
To improve the effect of our switch, we add a degradation system mfLon. mfLon is a kind of protease used for protein degradation. Therefore, the response speed will be significantly increased. Without this degradation system, when the switch transforms from ON to OFF, although the expression is stopped, remained products only decrease according to the cell division. When adding this system, the bacteria can degrade the products which increase the threshold and fasten the process.
Figure 6. the expected results before and after we add the degradation system into our cold-inducible ON-switch are shown.
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
And 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 7. the orthogonality among four proteases represented by the fluorescence of sf-GFP is shown.
Figure 8. 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 9. 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 10. The induction curve of the heat-inducible ON-switches (Nissle 1917)
More: Double-status switches
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 11. 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 12, 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 12. The induction curve of the double-status switches (TOP10)
 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
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