As described in project description (Description link), the goal of our project is to use optogenetically engineered bacteria to fight with P. aeruginosa infection and produce biological dressing simultaneously. The chassis we used is Gluconacetobacter hansenii, which can produce bacterial cellulose film and are widely used in food industry for coconut jelly production.

Using principle of synthetic biology, G. hansenii is engineered to produce antipseudomonal drugs. Moreover, the engineered G. hansenii can produce bacterial cellulose film and release the antipseudomonal drugs respectively through dual-light control. We hope to use the engineered G. hansenii for burn treatment by combining anti-infection and wound coverage. The architecture consists of three functional modules: antipseudomonal drug production modulec-di-GMP signal transduction and BC production modulesafety and drug release module. (To learn more, please click on the corresponding module.)
Figure 1. The overall scheme of project design (Click the red and blue light bulb)
● Antipseudomonal drug production module
Pyocin S2 is a soluble toxic protein produced by Pseudomonas aeruginosa that can kill P. aeruginosa specifically. But some P. aeruginosa strains contain the immunity protein IMMS2 which binds to the nuclease domain of pyocin S2 specifically and inhibits its activity. So, pyocin S2 can kill the immune-deficiency strains only. In order to construct a toxic protein that can kill P. aeruginosa specifically while having a wider antipseudomonal spectrum, we replaced the nuclease domain of pyocin S2 with that of colicin E3 to produce a chimeric bacteriocin termed SE. And the immunity protein IMME3 corresponding to the new SE protein is derived from the immunity protein corresponding to colicin E3, as shown in Figure 2.
Figure 2. Domains of SE protein and its immunity protein IMME3
SE protein can target P. aeruginosa by its Pseudomonas-specific receptor and translocase domains and kill the target cell by its nuclease domain originated from colicin E3. And our host cell G. hansenii is immune to SE as it expresses the immunity protein IMME3 that binds to nuclease domain of SE protein specifically and inhibits its activity.

As shown in Figure 3, in order to ensure the normal expression of antipseudomonal protein SE without damaging the growth of the host bacteria, we used different promoters and different RBSs for separate expressions of SE protein and its corresponding immunity protein IMME3.
Figure 3. Gene circuit of antipseudomonal drug synthesis module
● C-di-GMP signaling and BC film production module
Figure 4. Schematic for c-di-GMP signaling
Bacterial cellulose (BC) production in G. hansenii is regulated by the second messenger cyclic diguanylate (c-di-GMP). Therefore, we can control BC film production by regulating the concentration c-di-GMP. For effective and accurate regulation of c-di-GMP, we designed two sub-modules. One is the diguanylate cyclase (DGC) sub-module, which regulates the synthesis of c-di-GMP. The other is c-di-GMP phosphodiesterase (PDE) sub-module, which regulates the hydrolysis of c-di-GMP. We use bphS which is the coding gene of a photo-activated DGC named BphS and fcsR which is the coding gene of a c-di-GMP PDE in this module. Upon illumination with near-infrared light, BphS will be activated with changed protein conformation and start to synthesize c-di-GMP. When the synthesis rate of c-di-GMP is higher than its hydrolysis rate, the concentration of c-di-GMP will increases. And the bacteria will produce BC film accordingly.
Figure 5. Gene circuit of c-di-GMP signaling and BC film production module
● Safety and drug release module
A photo-induced lysis system is designed for this module. As a safety module, it can mechanistically reduce the consequences of environmental release of the engineered bacteria. At the same time, this module enables release of the antipseudomonal drugs described in the first module. This dual design ensures both safety control and drug release.

Here we use a blue light responsive system pDawn for light control. Upon illumination with blue light, the two-component system YF1/FixJ in pDawn represses the expression of the λ phage repressor cI, rendering the downstream gene expression photo-activated. We use X174 E which is the coding gene of the lysis protein in Escherichia phage PhiX174 for cell lysis. If this module is combined with the antipseudomonal drug production module, the engineered G. hansenii will release the antipseudomonal drugs to kill P. aeruginosa.
Figure 6. Gene circuit of safety and drug release module
● References
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