Design
Overall introduction
To achieve tumour-targeting and drug delivery in EcN, Peking 2023 has designed three genetic circuits.
Considering the strain has to express multiple additional proteins and carry exogenous plasmids, Peking 2023 attempts to design a low-load bioswitch compared to conventional AND-gate. After an investigation of the structure of pPepT and pLldR promoters, we designed a hybrid promoter to realize TME-targeting.
Targeting module
Hypoxia-induced promoter
A pivotal aspect of our innovative project involves constructing a hybrid promoter. TME, characterized by hypoxia and elevated lactate levels, remains relatively stable despite genetic mutations. Thus, we establish an AND-gate-equivalent part, driving the expression of a knock-out essential gene (asd) on a loop vector Fig.1. Consequently, the engineered Escherichia coli strain Nissle 1917 (or EcN, a safe probiotic strain widely used in the clinic) can only thrive and multiply in a hypoxia and lactate-abundant environment, achieving precise targeting of pancreatic cancer.
Figure 1 | Design of the AND-gate. We have introduced a simple and delicate component that can realize the relatively complex AND-gate function, sensing hypoxia and high lactic acid.
In Escherichia coli and Salmonella typhimurium, the Fumarate and Nitrate Reduction regulator (FNR) is a key regulator of hypoxia-induced promoters. Under hypoxic conditions, FNR maintains its hold on an oxygen-labile 4Fe-4S cluster, triggering its own homodimerization and binding to a specific DNA site termed “FNR box”, located in -35 region. Upon its binding, the homodimer interacts with the RNA polymerase binding to TATA box (with a conserved sequence of TATAAT) at -10 region, thus activating transcription Fig.2.
Figure 2 | Mechanism of pPepT. FNR homodimer binds to FNR binding site as it interacts with RNA polymerase to activate transcription. FNR is prohibited when oxygen concentration is high.
Lactate-induced promoter
The LldPRD operon in Escherichia coli mediates aerobic L-lactate metabolism. This operon comprises three genes organized into a single transcriptional unit, inducible by L-lactate. Specifically, LldD codes for the dehydrogenase, LldP for the permease facilitating L-lactate uptake in vivo, and LldR for a regulatory protein. The operon contains two LldR binding sites: one at -90 and another at +40. LldR dimers form at these sites, potentially repressing the promoter through DNA loop formation, which inhibits RNA polymerase binding. Lactate can alleviate this repression. At high L-lactate concentrations, the LldR dimer disassociates, permitting transcription. Fig.3
Figure 3 | Mechanism of pLldR. When lactate concentration is low, two LldR homodimers are attached to LldR binding sites O1 and O2 and form a loop structure, blocking the binding of RNA polymerase. When lactate concentration is high, the loop structure deteriorates and LldR detaches from O2 site. Therefore, RNA polymerase can attach to the promoter region, while LldR combined with O1 facilitates expression.
Concept of hybrid promoter
After investigation, we found it theoretically possible to integrate two promoters in one sequence. pLldR functions by forming steric hindrance, whereas pPepT operates through the combination of FNR. We can replace the constitutive promoter in BBa_K1847008 with pPepT. Thus, the promoter can function like an AND-gate while not producing additional peptides.Fig.4
Figure 4 | Concept of hybrid promoter. When lactate concentration is low, the loop prohibits the combination of RNA polymerase and FNR homodimer. When lactate concentration elevates, the loop structure breaks down and the sequence acts as a pPepT promoter.
Vesicle expressing
A paper published in Nature in 2018 (Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts, Raymond W. Bourdeau, Audrey Lee-Gosselin.) caught our attention. According to the report, the research team genetically modified the vesicular protein from a photosynthetic autotrophic bacterium cyanobacterium Anabaena flos-aquae and transformed it into E. coli BL21(AI) to successfully express the vesicular protein. They named this class of vesicles ARGs (Acoustic Reporter Genes). Such vesicular proteins can be detected by medical ultrasound probes. Therefore, the research team put forward the preliminary idea of using engineered bacteria to indicate tumour tissue.
We hypothesized that ARG could be introduced into the engineered bacteria containing and induce its expression vesicles. Since only the engineered bacteria colonized in the tumour tissue could survive due to our hybrid promoter, the signal would only be detected in the tumour tissue when detected by a medical ultrasound probe.
Figure 5 | Ultrasound approach tracks engineered bacteria16. Bourdeau et al. 15 genetically engineered bacteria to express what they term acoustic response genes (ARG), which encode the components of hollow structures called gas vesicles that scatter sound waves and generate an echo that can be detected by ultrasound. Pressure-pulse application causes gas-vesicle collapse and disappearance of the ultrasound signal, which can be used to improve signal detection when tracking the location of cells containing gas vesicles. This approach enables in vivo monitoring of a cell population that light microscopy cannot track.
In addition, in our design, the expression of vesicles is not controlled by the hybrid promoter. Therefore, we can induce its expression in vitro and the vesicles may be a promising carrier of anti-cancer molecules in future.
Controllable encapsulation
To achieve controlled immunogenicity of engineered bacteria with minimal harm to the human body, we introduce a dynamic circuit17 into Escherichia coli, allowing it to dynamically alter the thickness of its cell wall. Prior to ultrasound exposure, a thicker cell surface is induced through arabinose to evade host immune responses. This surface gradually degrades and becomes thinner over time, enabling rapid clearance from the body after ultrasound.
Figure 6 | The schematic diagram of dynamic circuit17. By regulating the intensity and duration of arabinose induction, we strive to replicate the desired effect as "iCAP" shown in the figure, with the goal of minimizing toxic reactions in the body.
Figure 7 | A glimpse of our design. Engineered EcN has the capability to detect hypoxic environments and high lactic acid levels. Surviving bacteria could express microvesicles, generating appreciably echogenic when sonicated. This innovative method offers comprehensive and precise spatial localization of tumours, aiding clinicians in making informed medical decisions and ensuring affordability for a wider range of patients' medical needs.