Overview
To manage the problem of evolutionary instability, we carefully design a cheater-proof platform consisting of engineered workers and guards. Once the workers transform into cheaters that don’t produce the target protein, the mutation will be presented and then detected by guards, whose extermination system will therefore be activated to secrete cytotoxin to selectively target cheaters.
Workers Pathway
AraC Inhibitor Pathway
The workers, undertaking the responsibility of producing the target products, are designed to report their mutations of target products' genes in two alternative ways. If the expression of the protein is lost due to frameshift mutation, the AraC which labels the protein and serves as the inhibitor of extermination system, will not be expressed. Therefore, it can’t inhibit promoter pBAD, which initiates the expression of extermination system. Though closely linked by connection, the target protein and AraC are structurally and functionally independent. In our design, the protein complex can also be cut on linker, leaving the target protein for further extracellular purification.
mRNA Binding Pathway
As for genetic mutation caused by single-base substitution in key domain, the same extermination system is inhibited by a mRNA-complementary sequence of 10~30 bp. The short mRNA binding site, specifically designed based on the key domain sequence, is placed upstream of the extermination system. Transcribed mRNA of aimed gene with right sequence will bind to the transcribed mRNA of the extermination system. The complex will therefore hinder the translation of cytotoxin. Once mutated, the mRNA will fail to bind tight enough to prevent translation.
Extermination System
The extermination system in workers(cheaters) includes two parts.The first one is Agr system that activates our specifically-cytotoxic guards. AgrD combines AgrB on the membrane to generate autoactivating peptide (AIP) [1], which will activate cytotoxic guards. , which will activate cytotoxic guards.
The second one is a modified Pmr system that recognizes the killing signal from guards and triggers cellular death. Originally, PmrB receptor on cell membrane uses an iron(III)-binding motif to sense the environmental ferric level [1]. However, we replaced the iron(III)-binding motif with an anti-mCherry motif, which can specifically bind to mCherry protein and then facilitate PmrB to phosphorylate PmrA. PmrC promoter will be activated by phosphorylated PmrA protein, and then it will initiate the expression of ccdB cytotoxin. In a word, mCherry acts as the killing signal to trigger the cheaters’ death.
Guards Pathway
Toggle Switch
The guards play the role of workers when no AIP is present through our elaborately designed toggle switch. On the cellular membrane of guards lies the AIP receptor, called AgrC. In the absence of AIP, the guards work as ordinary workers producing the target protein with Ptrc-2 promoter. However, once bound to AIP, AgrC will phosphorylate AgrA, switching guards to the killer mode. Phosphorylated AgrA will activate P2 promoter [1] which will initiate the expression of LacI. Afterwards, LacI will inhibit the Ptrc-2 promoter in return[3], and eventually suppress the synthesis of target protein. [1] which will turn on the expression of LacI. Afterwards, LacI will inhibit the Ptrc-2 promote reversely[3], and eventually suppress the synthesis of target protein.
Guards can’t bear the metabolic burden of synthesizing proteins and killing signal at the same time. However, with this toggle switch, guards can not only contribute to the production of target product in the absence of cheaters, but also be relieved from the synthesis of target product and focus on exterminating cheaters.
Killing Signal
Activated by phosphorylated AgrA, P2 promoter also initiates the synthesis of OmpT-mCherry. Under the guidance of OmpT signal peptide, mCherry can be secreted out of guards and served as the cheater-killing signal molecule of the platform.
Future Design
Expression Burden Control Circuit
We designed a system to relieve the engineered workers from expression burden of the target proteins. When the synthetic burden increases to a certain level that threatens the proliferation of workers, the burden-inducible promoter PhtpG1[4] will activate the transcription of downstream genes, dCas9 and gene sgRNA designed for promoter pBAD and T7. Expressed dCas9 will bind to sgRNA and then be guided to corresponding promoter, inhibiting downstream transcription. When our engineered bacteria cannot afford the expression burden of the target genes, the expression of the target genes will be prohibited and the extermination system will not be activated mistakenly due to the inhibition on T7 promoter and pBAD. With this system, we can expect a dynamic balance between bacteria proliferation and the production of target proteins.
References
[1] Novick RP, Geisinger E. Quorum sensing in staphylococci. Annu Rev Genet. 2008;42:541-64.
[2] Liang H, Deng X, Bosscher M, Ji Q, Jensen MP, He C. Engineering bacterial two-component system PmrA/PmrB to sense lanthanide ions. J Am Chem Soc. 2013 Feb 13;135(6):2037-9.
[3] Gardner TS, Cantor CR, Collins JJ. Construction of a genetic toggle switch in Escherichia coli. Nature. 2000 Jan 20;403(6767):339-42.
[4] Lv Y, Qian S, Du G, Chen J, Zhou J, Xu P. Coupling feedback genetic circuits with growth phenotype for dynamic population control and intelligent bioproduction. Metab Eng. 2019 Jul;54:109-116.
