For the consideration of future application scenarios of our engineered bacteria, we will construct a hybrid colony, which will start from the two modules of defense and offense respectively. At the defense level, we will improve the defense ability of the colony by increasing the thickness of pod membrane polysaccharide and knocking out genes to make the hairs longer; at the offense level, we plan to overexpress remote offensive-related genes (mcmA, mchB and mcmI, mchI), enhance the aggressiveness of the CDI system, and we also try to activate the T6SS of EcN by introducing the tagh genes of Escherichia coli 042 into EcN to see if the T6SS of EcN can be activated. Escherichia coli 042 by introducing the tagh gene into EcN to see whether the aggressiveness of EcN was improved, and we also designed two lethal gene circuits to ensure the safety of our engineering experiments.
In podocarp studies, the 2023 XJTU-iGEM team has used pgmA and gal U gene overexpression to enable increased extracellular polysaccharide (EPS) production, based on which we additionally introduced overexpression of KpsE, KpsT, and FliC genes, which are capable of increasing the rate of podocarp polysaccharide translocation from intracellular to extracellular, thus optimizing our podocarp level of Defense.
Two expression vectors were constructed pETDuet-1-gal U-pgmA , pACYCDuet-1-KpsE-KpsT (inserthyperlinks), and the constructed plasmids and their empty vectors were introduced into Nissle 1917 (DE3) respectively.
We carried out bacterial pod polysaccharide extraction on the above modified bacteria and measured the content of the extracted polysaccharides, using the empty vector-imported bacteria as the control, as shown in Fig. 1, our constructed KpsE-KpsT genetically engineered bacterium showed a significant increase in pod production compared with the empty vector-imported bacterium. This is some placeholder content for a horizontal collapse. It's hidden by default and shown when triggered.
As mentioned above, KpsE and KpsT genes can effectively increase the pod yield, in the future, we will introduce pgmA and gal U together with KpsE and KpsT genes into EcN, meanwhile, considering the possible influence of expression vectors on the expression of exogenous genes by the chassis bacteria, we propose the idea of integrating the exogenous genes genetically into EcN genome in order to eliminate the influence of the expression vectors on the strain.
FliC protein overexpression can increase the amount of podoplanin polysaccharide attachment on the surface of the bacterium, and we took biofilm as a branch, from which we introduced FimH, NirC can all increase the bacterial biofilm area as a way of providing enough membrane sites for overexpressed FliC protein.
We constructed four plasmids, pACYCDuet-1-NirC, pACYCDuet-1-FimH, pETDuet-1-FimH, pETDuet-1-NirC, which were imported into Nissle 1917 and Nissle 1917(DE3), respectively, and tested.
Biofilm production was quantified by washing and crystal violet staining, and absorbance values were detected after solubilization with 95% ethanol under the same initial OD600 of 0.05 and incubation of the six-well plate for 12h.
The experiments related to biofilm systems were not carried out in depth as we failed to validate the function of FliC and we reflected and considered, but our preliminary experiments did provide some valuable conclusions.
Based on the fact that bacterial motility is affected by increased pod thickness, we designed the ΔfimE mutant to achieve gene knockout by homologous recombination method to overexpress the type 1 bacterial hairs of EcN , which in turn improves the motility and defense of the engineered bacteria.
The targeting vector (pCVD442- ΔfimE::Kn) was constructed, and the targeting vector was introduced into E. coli β 2155 strain by electrotransformation, and the β2155/pCVD442- Δ fimE::Kn bacterial fluid was mixed with the bacterial fluid of the recipient bacteria (Nissle1917) to carry out the splicing experiments, and after completion, the knockout strains were screened by using the kanal-resistant plate.
The upstream and downstream homologous recombination arms of the fimE gene were amplified and cloned from the genome of Escherichia coli (Nissle1917) using ultra-fidelity DNA polymerase, the Kn resistance gene was amplified from the pKD4 plasmid by a high-fidelity PCR reaction, and the complete targeting fragment Δ fimE ::Kn was obtained by connecting the fimE gene to the Kn resistance gene using a fusion PCR technique by referring to the interface (insert hyperlink).After performing splicing experiments, fimE knockout strains were screened and verified by PCR electrophoresis.
The homologous recombination method to construct the ΔfimE mutant strain was successful, and we screened the knockout bacteria for bacterial preservation.
E. coli Nissle 1917 (EcN) was utilized as a host and engineered to enable efficient production of micromycin, thereby enhancing its ability to fight pathogens. To this end, we focused on the overexpression of the endogenous mcmA and mchB genes of EcN, which encode key enzymes for micromycin synthesis. Also, to avoid the phenomenon of autotoxicity, whereby antimicrobial peptides may cause damage to their host cells, we also considered the overexpression of the corresponding immune proteins, mcmI and mchI, to protect the host cells from self-produced antimicrobial peptides. In addition, we verified the overexpression of EcN mcmA and mchB, as well as the related immunity proteins mcmI and mchI by bacterial antagonism assay to determine whether the tele-aggressiveness of EcN is enhanced.
Two antimicrobial peptide expression plasmids, pACYCDuet-1-mCherry-mchI(6his)-EGFP-mchB(strep) and pETduet-1-mcmI-2(6his)-mcmA(strep), were constructed and introduced into the corresponding chassis bacteria.
To test whether our designed antimicrobial peptide expression plasmids functioned as expected, we
performed a zone of
inhibition (ZOI) assay. As detailed on the Experiments page, we inoculated 100 μL of concentrated
overnight cultures of
each predator strain on LB agar plates that were covered with a layer of soft LB agar containing
the prey strains. For
the mcmA and mchB antimicrobial peptide expression plasmids, the prey strains were E. coli DH5α.
the predator strains
were E. coli 1917 DE3 and BL21 DE3. in this experiment, the points of the first two plates ①
predators expressed mcmA,
mchB, respectively, and the prey were sensitive to this antimicrobial peptide.
After overnight incubation, we observed that significant repressive regions formed around the strains containing our designed mcmA and mchB expression plasmids. The discovery of these repressive regions not only confirmed the success of our plasmid constructs, but also showed that our strategy was effective. The results of these experiments demonstrate that our engineered strains are able to produce antimicrobial peptides as expected and that these antimicrobial peptides have the ability to inhibit the growth of other bacteria.
The modified engineered bacteria have both defensive and offensive capabilities, so we planned to design an inter-bacterial antagonism test to analyze the strength of bacterial antagonism. We compare the antagonistic strains to bactericidal cells and prey bacteria, and use the different resistance genes carried by the bacteria to design the experiment, and distinguish the strength of the inter-bacterial antagonism after antagonism by the antibiotic plate standoff method.
We constructed the following sets of inter-bacterial antagonism experiments as a way to verify whether the defense and offense ability of the engineered bacteria was improved.
| Bactericidal cells | Prey bacteria |
|---|---|
| Acinetobacter baylyi ADP1 | pACYCDuet-1-1917（DE3） |
| Acinetobacter baylyi ADP1 | E. coli strain Nissle 1917 (DE3)：pACYCDuet-1-kpsE-kpsT |
| Acinetobacter baylyi ADP1 | pETDuet-1-1917（DE3） |
| Acinetobacter baylyi ADP1 | pETDuet-1-galU-pgmA |
| Acinetobacter baylyi ADP1 | E. coli strain Nissle 1917 |
| Acinetobacter baylyi ADP1 | Knockout fimE of E. coli strain Nissle 1917 |
| pETDuet-1-1917（DE3） | Acinetobacter baylyi ADP1 |
| 1917（DE3）：pETduet-1-mcmI-2（6his）-mcmA(strep) | Acinetobacter baylyi ADP1 |
Bactericidal cells and prey bacteria bacterial fluids were inoculated into LB liquid medium and cultured in a certain proportion, respectively; after adding inducers and mixing them in a certain proportion, they were cultured; the mixed bacterial fluids were pipetted and coated on plates containing different resistances, respectively, and the survival rate of the strains was calculated. The survival rate of the strains was calculated and plotted as an ordinary one-way ANOVA algorithm (you can go to “Experiment” to see the detailed steps). The results of the antagonism experiments between several groups of bacteria in the table are shown below:
Through the above four groups of ordinary one-way ANOVA analysis, in the inter-bacterial antagonism experiment, compared with the control group, the modified strains have a significant increase in the defense and offense ability than the pre-modified strains. Figure 1 and 2: Increased pod production, which plays a big role in interbacterial antagonism, can improve the defense ability of the strain itself, thus resisting the attack of other bacteria. Figure 3: Higher survival of strains with knockdown of fimE, the ΔfimE mutant was able to lead to the formation of microcolonies, where target cells were protected from contact-dependent killing mediated by e.g. T6SS, leading to the survival of the engineered bacteria. Figure 4: The modified EcN producing micromycin enhances the long-range aggressiveness of the strain itself compared to the control, and the strain's own offensive system is thus greatly enhanced.
The ccdB gene was utilized to make suicide switches through synonymous codon mutations and the LapB/LpxC system in hopes of finding the genetic switch that kills our engineered bacteria within 20-24h. CONSTRUCTION: We introduced the involved lethal gene into Nissle 1917 (DE3) and screened it using resistance plates.
Nissle 1917 (DE3) was cultured using SOC medium and its growth was monitored by detecting the OD600 value, when the OD600 value reached 0.6, the lethal gene was induced to be overexpressed by adding 0.5mM IPTG, and the time of death of the engineered bacteria was determined by measuring the OD value at regular intervals. Figure: Growth curves of several ccdB synonymous codon lethal records
Due to the limited experimental time, we were not able to complete all the tests of the lethal gene circuits, but our experiments will not stop here, in the future we will continue to test and verify, so as to draw a perfect conclusion for our work.
Due to the limited experimental time, we were not able to complete all the tests of the lethal gene circuits, but our experiments will not stop here, in the future we will continue to test and verify, so as to draw a perfect conclusion for our work.
