Team:UCAS-China/Design

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Design

Classis Organism

For classis organism, we chose the strain GIM1.321 of Lactobacillus acidophilus as carrier. This strain can be colonized in the greater curvature of stomach and pylorus. Its characteristics of autogenous bacteria will not induce immune response of human body, and its acid resistance and genetic operability can meet our requirements.

Drug Production

LL-37 is a human cathelicidin with broad-spectrum antimicrobial, lipopolysaccharide binding, and chemotactic activities. It is named as LL-37 because it is a 37-residue-peptide and has a characteristic double-L structure on its N-head. It is also called hCAP18[1], which is the abbreviation of “human cationic antimicrobial peptide 18”. Several studies have suggested a potential application of a biological H. pylori reproduction regulating system based on LL-37 because of its antimicrobial activity against H. pylori.

Some research has revealed that LL-37 can be expressed in prokaryotes such as E. coli, which shows the possibility of LL-37 expression in gastric flora bacteria.[2]

Because the oxygen concentration in the stomach is generally low and the target H. pylori is anaerobic, so we connected the gene of LL-37 behind the anaerobic induced promoter, PfnrS, to ensure its expression is only near the H. pylori.

Ammonia Sensor

H. pylori has a special marker urease, which can decompose urea into ammonia, neutralize gastric acid, and form a buffer barrier of ammonium salt. Therefore, as long as the high concentration of ammonium salt is detected in the gastric mucosa, we can determine the colonization site of H. pylori. Considering the special trait, we designed a switch triggered by ammonia concentration.

Fig.1 Ammonia Sensor

NtrC is a NOT gate protein which is inactivated and does not bind to glnAp2 when the concentration of ammonium is high, thus inhibiting the expression of downstream genes (TetR). The downstream TetR is also a NOT gate as an inhibitory transcription factor, when it expresses, the function of tetR/tetA promoters is inhibited. The synthesized effect of the two NOT gate is positive, that is, when the concentration of ammonium ion exceeds the threshold, the promoter glnAp2 is inhibited, in consequence TetR cannot express, ultimately the inhibition of lysis gene is removed, after the expression of the lysis gene, the antimicrobial peptide is released into the gastric body environment by L. acidophilus.

Biosafety

We add the biosafety module to protect natural ecosystems also our patients for microbial therapies and address public concern. Cold-inducible switch was constructed based on the temperature-sensitive parts TEVts and CI434ts. TEV protease (TEVp) from tobacco etch virus recognizes specific amino-acid sequence ENLYFQG/S and cleaves between Q and G/S.TEV protease gene is a kind of temperature sensitive promoter, which can be activated by sensing low temperature (leaving the human body). The cold-inactivated cI434 variant (cI434ts) exhibited approximately 3-fold induction when the temperature was shifted from 37 to 25°C. Both of them compose the cold-inducible on-switch. The mutual inhibition between protease and transcription factor creates a balance, once there is a small temperature disturbance, the balance will lean to one side to act, and the downstream DOC gene can be normally transcribed and translated, killing the leaked Lactobacillus acidophilus.

The cold-inactivated cI434 variant (cI434ts) exhibited approximately 3-fold induction when the temperature was shifted from 37 to 25°C.

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. 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.

Reference

[1] Hase, K. , Murakami, M. , Iimura, M. , Cole, S. P. , Horibe, Y. , & Ohtake, T. , et al. (2003). Expression of ll-37 by human gastric epithelial cells as a potential host defense mechanism against helicobacter pylori. Gastroenterology, 125(6), 1613-1625.

[2] Ding Jing, Shen Juan, Zhu Jiayong, Jin Xiaobao, Lu Xuemei, Mei Hanfang & Li Xiaobo. (2012). Prokaryotic expression and activity identification of human antimicrobial peptide LL-37. Biotechnology (01), 18-22