Team:UCAS-China/Results

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Demonstrate

Microbial Therapeutics (demonstrate of drug production module)

Because the oxygen concentration in gastric environment is generally low, we connected the gene of LL-37 cathelicidin behind the anaerobic induced promoter, PfnrS, to ensure its expression is only in gastric environment.

glnAp2+riboJ+RBS (demonstrate of ammonia sensor)

According to the work done by team UCAS in 2017 and the reference [1], we have faith in the availability of NtrC-regulated promoter glnAp2, their work gives valid evidence that glnAp2 is a reliable promotor regulated by NtrC.

The figure below shows that when in high-nitrogen circumstance (ammonium as the nitrogen substance), the promoter will work after an obvious delay time therefore suppress the transcription, while in low-nitrogen circumstance, the promoter will start the transcription rather sensitive.

Since the behavior of the promoter glnAp2 changes linearly with the variation of concentration of ammonium, we believe it will work as a predictable way we expected in the design part.

The figure is from Part: BBa_K2287001

Hence the downstream of glnAp2 of the genetic circuit in the ammonia concentration switch is orthogonal to the original genes of L. acidophilus, the last question is whether the lysis part will work.

The table (from [2]) above shows antimicrobial activity of endolysin P28 against several Gram-positive bacteria. It is a strong evidence that the endolysin will work in L. acidophilus as a Gram-positive bacterium.

Shown is SDS-PAGE (15%) of the cellular proteins from 80 μL cultures of bacteria. The gel was stained by Coomassie brilliant blue R-250 to visualize the bands. E. coli BL21 (DE3; pET-26b-ORF8) before induction (lane 1) and 6 h after induction (1 mM IPTG; lane 2). The overexpressed oligohistidine-tagged endolysin P28 was purified (lane 3). M, molecular masses of reference proteins (Thermo Scientific; in kilodaltons).[2]

In addition, the figure above shows the overproduction of endolysin P28 in E. coli BL21 (DE3) cells, which validate that the gene expresses P28 has good compatibility in different kinds of bacteria.

In one word, we testified not only the circuit will work as expectation, but also the production protein will carry out its function.

Doc-SsrA (demonstrate of biosafety part)

Toxin-antitoxin system is an ingenious and effective strategy for biosafety, an appropriate toxin protein can effectively control the escape of engineering bacteria. The robustness of the system depends on the effectiveness of the toxin-antitoxin and the sensitivity of the upstream switch.

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. In 2019, UCAS's iGEM team designed a cold-inducible on-switch on the base of the evolved transcription factor and the evolved protease, which can be activated below 37℃, to control the expression of toxin protein (Fig. 1). We characterized the tendency of fluorescence change of a series of cold-inducible ON-switches using different TEVts mutants under different temperatures and chose two kinds of switches consisting of TEVts#6 and TEVts#18 (Fig. 2). Then we measure their temperature response curve more precisely by flow cytometer in TOP10 strain. From Figure 3 we can see that two groups both show narrow transition ranges and have about 100-fold induction within the range of ten degrees.

Figure 1. The cold-inducible ON-switch is encoded on two plasmids. The circuit is shown with the genetic parts and relationships among them.

Figure 2. The tendency of fluorescence change of a series of TEVts mutants under different temperatures (measured by ELIASA)

Figure 3. The induction curve of the cold-inducible ON-switches (TOP10)

Through the above experiments, we have proved that the cold induced switch has high sensitivity. We also measured the escape rate of the bacteria with DOC gene in solid and liquid medium (Fig4, 5).

In solid medium, we measured the escape rate, and the 20h average solid escape frequency is 2.318×10^(-2). In liquid medium, we measured the growth curve of the strains with DOC gene and strains without it at different temperatures, the different growth of the two strains at 37℃ showed that the leakage of the system exists.

Figure 4. The escape rate of bacteria on solid LB plate after 20h. The escape rate is calculated using the formula per Escape Frequency=(Colonies on nonpermissive plate×dilution)/(Colonies on permissive plate×dilution)[6] , the escape rate of 3 groups are (1×10^7)/(2×10^8 )=5×10^(-2) , (11×10^7)/(6×10^10 )=1.83×10^(-2) and (2×10^7)/(16×10^9 )=1.25×10^(-3) respectively(from above to below). And the Escape Frequency=Average Escape Frequency±standard deviation[6]=2.318 ×10^(−2) ± 0.122.

Figure 5. The growth curve of bacteria on liquid LB. Incubated in 4 mL volume in 24-deep-well plate.

It can be seen that the leakage of DOC toxin protein is the cause of high escape rate. To improve the performance of cold-inducible kill switch, we chose to reduce the leaky expression of Doc toxin by fusing a hybrid tevS/ssrA tag to the C-terminal domain of Doc toxin (Figure 6c). The ssrA tag is used for proteasome-dependent degradation of Doc toxin by endogenous proteases ClpAP and ClpXP42, while tevS for the proteolytic removal of ssrA tag. When the temperature is lower than 34°C, TEVts, as a component of cold-inducible switch, will be highly expressed, and cleave tevS to release ssrA tag from Doc protein. By contrast, as the expression of TEVts is repressed by cI434ts at 37°C, the ssrA tag cannot be removed and Doc toxin is degraded.

We characterized this advanced version of kill switch (Doc-SsrA) in E. coli TOP10 and EcN at different temperatures. The escape frequency of TOP10 Doc-SsrA and EcN Doc-SsrA at 25°C were (2.68±1.56)×10^(-6) and (1.47±0.78)×10^(-6) respectively, which were comparable to the lowest escape frequency achieved by previously reported thermal sensitive kill switch, much lower than Doc without SsrA. Similarly, the escape frequency of TOP10 Doc-SsrA and EcN Doc-SsrA at 30°C were (1.13±0.68)×10^(-5) and (9.78±7.34)×10^(-6), two to three orders of magnitude lower than 10^(-3) described in previous research by Stirling et al(Fig. 6). Growth curves in liquid culture also showed that TOP10 Doc-SsrA and EcN Doc-SsrA suffered significant growth arrest at both 25°C and 30°C (Fig. 7).

Figure 6. The construction and characterization of cold-inducible kill switch. (a) Genetic circuit design of cold-inducible kill switch ver1.0. The protease tevts is cloned under pR to a p15A plasmid. The transcription factor is cloned under a strong promoter J23119 with toxin doc cloned under pR downstream to a pSC101 plasmid. (b) The escape rate of bacteria on solid LB plate after 20h. The escape rate is calculated as below: per Escape Frequency = (Colonies on nonpermissive plate ∙ dilution) / (Colonies on permissive plate ∙ dilution) 19. The escape rate of three groups are (1×107)/(2×108)=5×10-2 (i) ,(11×107)/(6×1010)=1.83×10-2 (ii) and (2×107)/(16×109)=1.25×10-3 (iii) respectively. And the Escape Frequency = Average Escape Frequency ± standard deviation = 2.318 ×10-2 ± 0.122.

Figure 7.The construction and characterization of double-status switch. (a) Genetic circuit design of the double-status switch. The heat-inducible switch that has mRFP as the output is placed on the same p15A1 plasmid as the protease TEVts.

References

[1] Reitzer, Lawrence J., and Boris Magasanik. Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell 45.6 (1986): 785-792.

[2] Dong, H., Zhu, C., Chen, J., Ye, X., & Huang, Y. P. (2015). Antibacterial Activity of Stenotrophomonas maltophilia Endolysin P28 against both Gram-positive and Gram-negative Bacteria. Frontiers in microbiology, 6, 1299. https://doi.org/10.3389/fmicb.2015.01299

[3] Huo, Yi‐Xin, et al. "Protein induced DNA bending clarifies the architectural organization of the σ54‐dependent glnAp2 promoter." Molecular microbiology 59.1 (2006): 168-180.