Considering the future application scenarios of our engineered bacteria, we will construct mixed microbial communities, focusing on two main modules: defense and offense. In the defense aspect, we will enhance the microbial community's defensive capabilities by increasing the thickness of the capsule polysaccharide and elongating the pili through gene knockout. In the offensive aspect, we plan to overexpress genes related to remote aggression (mcmA, mchB, mcmI, and mchI) and enhance the competitiveness of the CDI system. Additionally, we will attempt to activate the T6SS in EcN by introducing the tagh gene from Escherichia coli 042 to observe any improvements in EcN's aggressiveness. We have also designed two lethal gene circuits to ensure the safety of our engineering experiments.
To enable co-expression with other genes, we selected pACYCDuet-1 and pETDuet-1 as our plasmid vectors. Through extracellular polysaccharide extraction experiments, both genes showed significant differences, successfully validating that our genes can promote the production of extracellular polysaccharides.
In addition, we performed qPCR detection on the constructed pACYCDuet-1-KpsE-KpsT, and found that the mRNA overexpression levels of KpsE and KpsT genes were high, suggesting the possibility of false positives. Currently, we are optimizing primer design and conducting further protein level detection. At the characterization level, overexpression of KspT and KspE has been shown to increase capsule yield.
Conclusion: At a 1% inoculation rate and a 5ml culture volume, incubated at 37°C and 220rpm for 12 hours, the genetically modified strains exhibited slower growth compared to unmodified strains, though no significant differences were observed.
In our biofilm validation, we tested the yield of the NirC and FimH genes in Nissle 1917 and Nissle 1917(DE3). With the same initial OD600 of 0.05 and a 12-hour incubation in a six-well plate, we analyzed the data using standard one-way ANOVA. In Nissle 1917, FimH expression was higher than in the original strain but showed no significant difference. In Nissle 1917(DE3), the pACYCDuet-1-NirC strain showed a significant difference, with biofilm production much higher than the other two strains.
We validated the defensive capability of the fimE knockout strain through bacterial antagonism tests. Data analysis showed that the ΔfimE mutant had a higher survival rate, indicating that knocking out the fimE gene leads to micro-colony formation, protecting target cells from T6SS-mediated contact-dependent killing, thus enhancing the survival of the engineered bacteria. Therefore, knocking out the fimE gene can improve the strain's resistance to bacterial attacks.
We inoculated 100 μL of each predator strain's overnight concentrated culture on LB agar plates, covered with soft LB agar containing prey strains. For the mcmA and mchB antimicrobial peptide expression plasmids, the prey strain was Escherichia coli DH5α, and the predator strains were E. coli Nissle 1917 DE3 and BL21 DE3. In this experiment, the first two plates showed predators expressing mcmA and mchB, with the prey sensitive to these antimicrobial peptides.
After overnight incubation, we observed that the strains containing our designed mcmA or mchB expression plasmids showed visible zones of inhibition. In addition, we verified the growth of these two antimicrobial peptide expression plasmid strains.
We also extracted the supernatant for fluorescence detection using the green fluorescence labeling gene, and the results were as follows.
Based on these results, we concluded that our designed mcmA and mchB expression plasmids were successful, and the overexpression of the corresponding immune proteins mcmI and mchI effectively protected host cells from the antimicrobial peptides produced, enhancing the aggressiveness of our engineered bacteria.
For safety considerations, we temporarily did not use actual pathogenic bacteria but chose Acinetobacter baylyi ADP1, which has both close-range and remote aggression through T6SS, as a substitute for the bacterial antagonism tests. The results indicated that compared to the unmodified strains, the engineered bacteria exhibited significant improvements in both defense and offense capabilities, with increased capsule polysaccharide yield, the fimE knockout mutant showing higher survival rates, and the secretion of antimicrobial substances.
We reviewed relevant literature and proposed two types of lethal switches: one through ccdB using synonymous codon mutations, designing 43 lethal switch tests; the other through intermembrane induction of lethality. We preliminarily explored the interaction between LapB and LpxC, where increased LapB promotes the degradation of LpxC, which negatively regulates lipopolysaccharide synthesis. YejM prevents LpxC degradation and can transfer substances from the inner membrane to the outer membrane. By mutating the LapB gene to increase its levels, LpxC levels decrease, leading to cell death at very low concentrations. Although we did not complete the lethal switch testing due to the project's full schedule, our experimental design is ongoing, and we will continue our journey in synthetic biology!
