This project aims to inhibit SRB (Sulfate-reducing bacteria) biofilm formation in the sewer through cocktail therapy. The secretion of antimicrobial peptides for targeted killing of SRB is an essential part of the project. Therefore, it is necessary to construct engineering bacteria that secrete antimicrobial peptides and culture SRB for antibacterial experimental verification.
We conducted two methods of culturing Desulfovibrio vulgaris, purchased from the Shanghai Conservation Biotechnology Center (SHBCC). The solid medium was Columbia blood agar medium, and we observed morphologically correct colonies on the plate. The liquid medium was CAMHB broth medium with 5% defibrated sheep blood, and we observed that the liquid became turbid after two days of cultivation. Cultivation protocol is available at experiments.
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Figure 1-1. SRB solid culture and liquid culture results
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Figure 1-2. a.Anaerobic workstation culture of SRB.
b. Anaerobic bag culture of SRB
We chose OmpA, a signaling peptide from E. coli, to assist in the secretion of our antimicrobial peptide. The OmpA signaling peptide contains 21 amino acid residues, which guide the secretion of OmpA protein into the outer membrane through SecB-dependent post-translational transport. The OmpA signaling peptide of E. coli has been proven to have sound effects on the expression and secretion of recombinant proteins.
We designed to add the OmpA signal peptide sequence to the front end of the antimicrobial peptide sequence and add three amino acids of ASA as the signal peptidase cleavage sites between the two sequences. This way, the complete antimicrobial peptide can be released extracellularly, and the functions of both the signal peptide and the antimicrobial peptide can be unaffected.Two AMPs from bovine, indolicidin(Ind) and bactenecin(Bac), have been demonstrated to have the ability to kill SRB, and we ligated them to OmpA-ASA and then to the pET28a vector.
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Figure 1-3. plasmids of pET28a-OmpA-Bac and pET28a-OmpA-Ind
Figure 1-4. Sequencing results of pET28a-OmpA-Bac.
The sequencing results of pET28a-OmpA-Ind and pET28a-OmpA-Bac showed that the sequencing results of the critical regions were utterly consistent with the standard gene sequence, and no mutations occurred. Plasmids pET28a-OmpA-Ind and pET28a-OmpA-Bac have been successfully constructed, and we retained the strains for functional verification.
To characterize the antibacterial effects of two antibacterial peptides secreted by engineered E. coli , Bactenecin and Indolicidin, we set three concentration gradients for experimental validation of the two antimicrobial peptides. By measuring the changes in OD600 absorbance, we analyzed the changes in SRB and E. coli DH5 α. At the same time, we verified the inhibitory effect on the growth of engineered E. coli BL21 itself to consider the rationality of antimicrobial peptide selection.
Figure 1-5.Comparison of inhibitory effects of two antimicrobial peptides against E. coli DH5 α
Positive control: ampicillin antibiotic treatment solution (1:1000); Negative control: supernatant of E. coli BL21 culture; Blank control: E. coli BL21 cultured in LB medium; Bac-1~3: supernatant of engineered E. coli BL21 secreting the antimicrobial peptide Bactenecin; Ind-1~3: engineered E. coli BL21 supernatant; where the ratio of the three concentration gradients numbered 1~3 was set as (1:2/3:1/3)
Treatment of E. coli DH5 with two antimicrobial peptides α In the antibacterial experiment, it can be seen that the two selected antimicrobial peptides are effective against E. coli DH5 α Both have a specific inhibitory effect, and the inhibitory effect increases with the increase of concentration. Overall, the treatment effect of the antibacterial peptide Indolicidin is slightly better than that of the antibacterial peptide Bactenecin. Still, both have much lower effects than the ampicillin antibiotic treatment group with a concentration of 1:1000.
To investigate the effect of antimicrobial peptides on the growth of engineered E. coli BL21, we conducted antibacterial experiments for characterization.
Figure 1-6. Comparison of the effects of two antimicrobial peptides on the growth of E. coli BL21
Blank control: BL21; Negative control: BL21-OmpA-Ind, BL21-OmpA-Bac without IPTG induction treatment; Positive control: BL21+Kana, Kanamycin was added to the LB culture of E. coli BL21 at a concentration of 1:1000. Experimental group: BL21-OmpA-Ind+IPTG, BL21-OmpA-Bac+IPTG, 1M IPTG was added to the culture system of engineered E. coli BL21 at 1:1000 for induction treatment.
The experimental results indicate that the expression and secretion of two antimicrobial peptides have a relatively low impact on the growth of engineered E. coli, and the effect of the antibacterial peptide Indolicidin is relatively small in both.
After completing the study on E. coli DH5α After conducting antibacterial experiments, we successfully verified the successful secretion and antibacterial function of two antimicrobial peptides. Subsequently, through experiments, we measured the impact of antimicrobial peptide secretion on the growth status of the engineering E. coli BL21 itself. We found that the selected two antimicrobial peptides had moderate antibacterial effects and had little impact on the engineering bacteria themselves. Subsequently, we conducted turbidimetric antibacterial experiments on the target bacteria SRB.
Figure 1-7. SRB growth curve
As shown in the SRB growth curve, we first completed the measurement of SRB growth characteristics, recorded relevant growth data, and plotted standard growth curves.
Figure 1-8. SRB turbidimetric antibacterial experiment
Positive control: treatment by adding ampicillin at 1:1000 to the SRB culture system. Experimental group: Ind: supernatant of engineered E. coli BL21 secreting the antimicrobial peptide Indolicidin; Bac: supernatant of engineered E. coli BL21 secreting the antimicrobial peptide Bactenecin; Concentrated Ind, Concentrated Bac: Ind and Bac treatment solution that was concentrated to twice the previous concentration. (Liquid concentration was accomplished using a vacuum centrifuge concentrator and freeze dryer)
The results of the antibacterial experiment showed that the antibacterial effect increased with the increase of the concentration of antibacterial peptides, among which the antibacterial effect of the antibacterial peptide Indolicidin was quite significant, and the antibacterial effect of its concentrated group even exceeded the treatment effect of ampicillin antibiotics. In contrast, the inhibitory effect of the antibacterial peptide Bactenecin on SRB is not ideal.
After completing the turbidimetric antibacterial experiment, we conducted the antibacterial zone method antibacterial experiment to provide a more intuitive and explicit characterization of the antibacterial effects of the two antimicrobial peptides.
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Figure 1-9. Culture environment for antibacterial zone experiment of E. coli DH5 α
Figure 1-10. Antibacterial zone experiment of E. coli DH5 α
BL21: E. coli BL21 culture fluid supernatant (20uL); A抗1X: normal working concentration of ampicillin antibiotic liquid (10uL);
BAC: Concentrated supernatant of engineered E. coli BL21 secreting the antimicrobial peptide Bactenecin; Ind: Concentrated supernatant of engineered E. coli BL21 secreting the antimicrobial peptide Indolicidin; (concentration of concentrated supernatant is double the initial concentration)
It can be seen that the experimental group showed a specific range of antibacterial circles. In contrast, the negative control group was covered with E. coli around the antibacterial circle paper, while the positive control group showed an antibacterial zone with a diameter much larger than the experimental group.
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Figure 1-11. SRB antibacterial zone experiment
The paper in the upper right corner shows the antibacterial peptide treatment group , and the other three antibacterial circles are the BL21 culture supernatant treatment group, which is the negative control group of the experimental group.It can be seen that a thick layer of E. coli BL21 growth circle grows around the antibacterial zone of the negative control group and less E. coli around the experimental group, but the inhibitory effect on SRB was also not obvious.
In contrast, the growth of E. coli and SRB in the experimental group treated with antimicrobial peptides is poorer compared to the control group. The reason why the antimicrobial peptide treatment group did not produce a significant inhibitory zone on SRB and only reduced the growth density of SRB around the inhibitory zone may be as follows:
The results of the experiment on the cultivation and inhibition of SRB shows that the two antimicrobial peptides we selected perform well in terms of antibacterial effect, especially for E. coli DH5 α It exhibits a certain degree of inhibition of growth rate. For the target bacteria SRB, it has a strong antibacterial effect, causing a significant reduction in the bacterial concentration of SRB in the short term.
Comparing the comprehensive performance of two types of antimicrobial peptides, we plan to use the antibacterial peptide Indolicidin as the final practical exocrine antimicrobial peptide to target and kill the target bacteria SRB effectively. This antimicrobial peptide has the advantage of relatively less impact on engineered E. coli BL21 and a more substantial killing effect on target bacteria SRB.
Two glycosidases (DspB and DisH) and three N-acyl homoserine lactonase(Aiia, AidH, Moml) were selected. The glycosidases and N-acyl homoserine lactonase were screened from three aspects, namely, plasmid construction, protein expression, and enzyme function validation, to find out the glycosidases and N-acyl homoserine lactonase that were more efficient in eliminating biofilm and inhibiting the formation of biofilm in order to construct the SRBQuencher. In addition, this topic is strongly influenced by the dynamic changes in the concentration of AHLs, and we constructed four N-acyl homoserine lactonase by protein modeling to verify their efficiency from the three aspects mentioned above as well.
After reviewing the literature, we found the presence of a signal peptide segment upstream of the synthesized Moml enzyme gene that affects its secretion into the cytosol. Therefore, we excised the front-end signal peptide during PCR amplification to obtain del-Moml mutants. In addition, we designed four AHLs-degrading enzyme mutants with more stable and AHL-binding ability by protein modeling and obtained DNA fragments of these four mutants by point mutation. We amplified the DNA fragments of the three enzymes and five mutants by PCR, and then we ligated them into the pET28a vector, and the sequencing results showed the success of our plasmid construction.
Figure 2-1 Plasmids of glycosidase(DspB and DisH), N-acyl homoserine lactonase(Aiia, AidH, Moml)and five mutant
Figure 2-2 Sequencing results of glycosidase(DspB and DisH), N-acyl homoserine lactonase(Aiia, AidH, Moml)and five mutant
In our project, pET28a was chosen as a plasmid vector with lac manipulator and T7 promoter, which can be induced to overexpress exogenous gene clusters, which can reduce the harmful effects of exogenous genes on host cells. At the same time, the high concentration of expressed proteins facilitates our enzyme function detection.We transferred it into E. coli BL21 by chemical transformation and stored glycerol bacteria for subsequent protein purification and functional validation.
We added IPTG to induce the expression of six glycosidases and verified by SDS-PAGE that both DspB (41.8 kDa) and DisH (53.4 kDa) were expressed and secreted extracellularly. However, the amount of extracellular secretion was low, and the purification results showed that the expression of DspB was high in comparison to DisH.
Figure 2-3 a)Detection of secretion of the DspB, DisH and Aiia by SDS-PAGE ladder: Vazyme 180 kDa Prestained Protein Marker;1:protein supernate of E.coli BL21(pet28a-Aiia);2: LB culture medium of E.coli BL21(pet28a-Aiia);3:protein supernate of E.coli BL21(pet28a-DisH);4: LB culture medium of E.coli BL21(pet28a-DisH);5: protein supernate of E.coli BL21(pet28a-DspB);6: LB culture medium of E.coli BL21(pet28a-DspB)
b) Purification and validation of the DspB and DisH by SDS-PAGAE ladder: Vazyme 180 kDa Prestained Protein Marker;1:flow-through of E.coli BL21(pet28a-DspB);2:first wash buffer of E.coli BL21(pet28a-DspB);3:third wash buffer of E.coli BL21(pet28a-DspB);4:eluate of E.coli BL21(pet28a-DspB); 5: flow-through of E.coli BL21(pet28a-DisH);6:first wash buffer of E.coli BL21(pet28a-DisH);7: third wash buffer of E.coli BL21(pet28a-DisH);8:eluate of E.coli BL21(pet28a-DisH); 9:protein supernate of E.coli BL21(pet28a-sfGFP).
While induced expression of the three N-acyl homoserine lactonase and their mutants revealed that AidH (30 kDa) could be expressed and secreted extracellularly, dMoml (30 kDa) and Moml (32.5 kDa) could not be secreted extracellularly, and the expression of Aiia (27.8 kDa) was low. In addition, we used magnetic beads to protein purify Aiia, AidH, Moml, and their mutant. We demonstrated that AidH and its mutants were highly expressed in our engineered bacteria, Moml and Aiia were lowly expressed, and dMoml and AIIA mutants were very lowly expressed, which requires further enzyme function verification.
Figure 2-4 Figure 4 a) Detection of secretion of the Mmol, Aiia and AHL by SDS-PAGE ladder: Vazyme 180 kDa Prestained Protein Marker;1:LB culture medium nate of E.coli BL21(pet28a-sfGFP) induced by 0.5mM IPTG;2: LB culture medium of E.coli BL21(pet28a-sfGFP) without IPTG induction;3:LB culture medium of E.coli BL21(pet28a-Moml) induced by 0.5 mM IPTG;4: LB culture medium of E.coli BL21(pet28a-Moml) without IPTG induction;5:LB culture medium of E.coli BL21(pet28a-Aiia) induced by 0.5mM IPTG;6: LB culture medium of E.coli BL21(pet28a-Aiia) without IPTG induction;7:LB culture medium of E.coli BL21(pet28a-AHL) without IPTG induction; 8:LB culture medium of E.coli BL21(pet28a-AHL) induced by 0.5mM IPTG.
b) Purification and validation of the AidH by SDS-PAGE ladder: Vazyme 180 kDa Prestained Protein Marker;1:eluate of E.coli BL21(pet28a-AidH);2:Eluate of E.coli BL21(pet28a-sfGFP);3:flow-through of E.coli BL21(pet28a-AidH);4:flow-through of E.coli BL21(pet28a-sfGFP);5:first wash buffer of E.coli BL21(pet28a-AidH);6:first wash buffer of E.coli BL21(pet28a-sfGFP);7:third wash buffer of E.coli BL21(pet28a-AidH);8:third wash buffer of E.coli BL21(pet28a-sfGFP);8:protein supernate of E.coli BL21(pet28a-sfGFP).
c) Purification and validation of the Aiia by SDS-PAGAE ladder: Vazyme 180 kDa Prestained Protein Marker;1:flow-through of E.coli BL21(pet28a-Aiia);2:first wash buffer of E.coli BL21(pet28a-Aiia);3:third wash buffer of E.coli BL21(pet28a-Aiia);4:eluate of E.coli BL21(pet28a-Aiia); 5: flow-through of E.coli BL21(pet28a-sfGFP);6:first wash buffer of E.coli BL21(pet28a-sfGFP);7: third wash buffer of E.coli BL21(pet28a-sfGFP);8:eluate of E.coli BL21(pet28a-sfGFP); 9:protein supernate of E.coli BL21(pet28a-sfGFP).
d) Purification and validation of the Moml and dMoml by SDS-PAGAE ladder: Vazyme 180 kDa Prestained Protein Marker;1:flow-through of E.coli BL21(pet28a-Moml);2:first wash buffer of E.coli BL21(pet28a-Moml);3:third wash buffer of E.coli BL21(pet28a-Moml);4:eluate of E.coli BL21(pet28a-Moml); 5: flow-through of E.coli BL21(pet28a-dMoml);6:first wash buffer of E.coli BL21(pet28a-dMoml);7: third wash buffer of E.coli BL21(pet28a-dMoml);8:eluate of E.coli BL21(pet28a-dMoml); 9:protein supernate of E.coli BL21(pet28a-sfGFP).
Figure 2-5 a) Detection of secretion of the AidH and Aiia by SDS-PAGAE ladder: Vazyme 180 kDa Prestained Protein Marker;1: protein supernate of E.coli BL21(pet28a-AidH);2: protein supernate of E.coli BL21(pet28a-AidH147G); 3: protein supernate of E.coli BL21(pet28a-AidHN65K);4:protein supernate of E.coli BL21(pet28a-AiiaT195R.
b) Purification and validation of the AidH by SDS-PAGAE ladder: Vazyme 180 kDa Prestained Protein Marker;1:flow-through of E.coli BL21(pet28a-AidH147G);2:first wash buffer of E.coli BL21(pet28a-AidH147G);3:third wash buffer of E.coli BL21(pet28a-AidH147G);4:eluate of E.coli BL21(pet28a-AidH147G); 5: flow-through of E.coli BL21(pet28a-AidH147V);6:first wash buffer of E.coli BL21(pet28a-AidH147V);7: third wash buffer of E.coli BL21(pet28a-AidH147V);8:eluate of E.coli BL21(pet28a-AidH147V); 9:protein supernate of E.coli BL21(pet28a-sfGFP).
c) Purification and validation of the AiiaN65K and AiiaT195R by SDS-PAGAE ladder: Vazyme 180 kDa Prestained Protein Marker;1:flow-through of E.coli BL21(pet28a-AiiaN65K);2:first wash buffer of E.coli BL21(pet28a-AiiaN65K);3:third wash buffer of E.coli BL21(pet28a-AiiaN65K);4:eluate of E.coli BL21(pet28a-AiiaN65K); 5: flow-through of E.coli BL21(pet28a-AiiaT195R);6:first wash buffer of E.coli BL21(pet28a-AiiaT195R);7: third wash buffer of E.coli BL21(pet28a-AiiaT195R);8:eluate of E.coli BL21(pet28a-AiiaT195R); 9:protein supernate of E.coli BL21(pet28a-sfGFP).
We demonstrated the expression of glycosidases (DspB and DisH) by SDS-PAGE and protein purification, and then we verified the enzymatic activity and enzyme function of these two enzymes.
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Figure 2-6. Glycosidase (DspB and DisH) activity assay
First, we verified the enzymatic activities of DspB and DisH by using the α-glucosidase (α-GC) Activity Assay Kit of sengon biotech Co., Ltd. DspB and DisH can decompose p-nitrophenyl-α-D glucoside to produce p-nitrophenol which exhibits an absorption maximum at 400 nm. We first set up a standard curve and then tested our expressed DisH and DspB and found that the enzyme activities were not within the confidence interval of the standard curve and the activities were too low. We considered that the low enzyme inactivation was caused by the long time we took to detect the enzyme activity, the fast degradation of the enzyme after lysing the bacterial solution or the low sampling of the test samples, or the big change of the environment during the processing.
In order to minimize the loss during enzyme treatment, we simplified the validation steps and verified as soon as possible whether the expressed enzymes had the function of disrupting the biofilm. We cultured the biofilm in 96-well plates in advance and used crystal violet staining to observe the integrity of the biofilm before and after treatment with DisH and DspB, respectively. Because SRB culture is difficult, we temporarily used E. coli DH5α biofilm instead of SRB biofilm in the early stage and then switched to the validation of SRB biofilm in the later stage.
Figure 2-7. a Glycosidase (DspB and DisH) to E. coli DH5α biofilm treatment
b Glycosidase (DspB and DisH) to SRB biofilm treatment
Observation of the crystal violet staining results demonstrated that both our expressed DiSH and DspB had the ability to degrade the bio-permembrane, and DspB degraded the glycosidic bond of the biofilm more than DisH. For SRB biofilm, both glycosidases significantly disrupted the biofilm of SRB, and the degradation efficiency of DspB was still higher than that of DisH, suggesting that the DspB enzyme was more in line with our expectations.
During our validation of the function of glycosidases, we found that biofilms are constantly renewed and repaired, and we considered that quorum sensing of quenched sterilizers would inhibit the formation of new biofilms, We therefore detected by staining the biofilm maturation formed by the bacteria before and after treatment with N-acyl homoserine lactonase activity.
Figure 2-8 N-acyl homoserine lactonase to SRB biofilm treatment
By comparing the results of staining, we found that Aiia, AidH, Moml, and dMoml inhibited biofilm formation, which was in line with our expectations. In addition, we found that AidH and its mutant AidH A147G had a significant inhibitory effect on SRB biofilm formation, indicating that our mutants met the corresponding expectations.
Figure 2-9 Difference effect on AHLs by applying AidH(Left) and AiiA(Right)
In order to further verify the degradation efficiency of our degradation enzymes on AHLs, we selected Aiia and AidH, which have more obvious differences in degradation efficiency, to degrade different species (oxo-C6-HSL,C6-HSL,C8-HSL,C10-HSL,C12-HSL), but due to the lack of high performance liquid chromatography (HPLC) columns we could not directly detect the changes in the concentration of AHLs. However, we have shown that the fluorescence expression of our AHLs induction validation system (p15A-lux-sfGFP) is positively correlated with the concentration of AHLs, so we induced AHLs before and after treatment with N-acyl homoserine lactonase into the system and found that the fluorescence expression was much lower than that of the control group after the treatment, which demonstrated that AidH and Aiia were able to degrade a wide range of AHLs.
In summary, the expressed glycosidases and N-acyl homoserine lactonase were able to eliminate SRB biofilms and inhibit their production, and validation of multiple enzymes showed that the mutant AidH A147G of DspB and AidH was more suitable for use in the construction of our SRBioQuencher.
we expressed SQR in SRBioQuencher, in order to rapidly convert hydrogen sulfide generated by E. coli and improve the efficiency of SRBioQuencher.
The vector P15A was linearized by reverse PCR and the P15A backbone was amplified. The sqr gene was ligated after PJ23110 by Gibson assembly. We transformed the plasmid into E. coli DH5α.Sequencing results showed that the construction was successful.
Figure 3-1 Plasmid of SQR
Figure 3-2 Sequence results of SQR
When \(Na_2S\) is mixed with the solution, \(S^{2-}\) will quickly react with water to form \(H_2S\), \(H_2S\) is difficult to quantitatively configure, while the standard solution of \(Na_2S\) is easier to obtain, so we chose the standard solution of \(Na_2S\) to simulate the \(H_2S\) in theenvironment.
Figure 3-3 Efficiency Verification of SQR
Although \(H_2S\) is a metabolite of E. coli, the recombinant strain did not produce high levels of \(H_2S\) when cultured under normal conditions, suggesting that the production of \(H_2S\) by E. coli does not have a noticeable impact on the environment. The experimental group was p15A-cpSQR+\(Na_2S\), and the \(H_2S\) content in the recombinant strain and \(Na_2S\) mixture decreased rapidly within 30 min, indicating that SQR can rapidly degrade \(H_2S\) in the environment within a short period of time.
The \(H_2S\) content of group of M9 medium + \(Na_2S\) also decreased substantially, probably because, at the time of the shock culture, \(H_2S\) and oxygen in the environment mixed together, resulting in oxidization of \(H_2S\).
Figure 3-4 content of \(H_2S\) after 120 min
After mixing the bacterial solution with \(Na_2S\) for 120 min, the \(H_2S\) in the p15A-cpSQR+\(Na_2S\) group was significantly lower than that in the two groups, WT+\(Na_2S\) and M9+\(Na_2S\). After expressing SQR, a thioredoxin oxidoreductase, our engineered bacteria can effectively reduce \(H_2S\) in the environment.
In general, we successfully cloned and characterized the SQR product in E. coli DH5alpha and experimentally verified that E. coli can rapidly and effectively reduce environmental \(H_2S\).
In order to improve the safety of SRBioQuencher, we enabled SRBioQuencher to regulate its population according to the change in the number of SRBs in the environment by adding a suicide regulation system. We, therefore, coupled the AHL regulatory system with the lactose manipulation system and verified its function.
By reading the literature, we screened several control systems that could affect the change in AHL concentration. After comparing the expression difficulty and effect sensitivity, we improved the pluxR control system to construct SRBioQuencher kill switch.
Using sfGFP as a reporter gene, we constructed a plasmid (p15A-luxR-sfGFP) to validate our manipulation system for AHLs. We cloned this manipulation system into the p15A vector and used the J23100 promoter constitutively to express LuxR, which binds to the upstream sequence of PluxR and regulates the expression of the downstream marker gene sfGFP. Sequencing results proved the success of our plasmid construction.
Figure 4-1 Plasmid of p15A-luxR-sfGFP
Figure 4-2 Sequence results of p15A-luxR-sfGFP
Figure 4-3 Growth curve
We verified whether our manipulation system could sense the multiple AHLs produced by SRB, and we used different AHLs to induce green fluorescence in the validation plasmid for expression. First we first tested the growth curves of our bacteria in 96-well plates after transferring to the manipulation system, and found that the number of bacteria was stable at an initial OD600 value of 0.6, so our validation of the manipulation system began with testing at an OD600 value of 0.6.
Figure 4-4 Preferability on Different AHLs of our system.
When we added AHLs to our validation system (p15A-luxR-sfGFP), AHLs could bind to LuxR and detach it from the upstream of PluxR, releasing the inhibitory effect of LuxR on PluxR and thus activating the downstream expression of sfGFP. By measuring the fluorescence intensity induced by the same concentration of different kinds of AHLs, we can determine the preference of the AHLs-induced expression system in response to induction by different types of AHLs. Through the preference test, we can find that our manipulation system can respond to the induction of oxo-C6-HSL, C6-HSL, C8-HSL, C10-HSL, C12-HSL, and our manipulation system is most responsive to oxo-C6-HSL. It indicates that our manipulation system can respond to the induction of multiple AHLs expressed by SRBs, which aligns with our expectations.
Figure 4-5 Effects on Different AHLs Concentration of our system.
To adjust the threshold at which our suicide system turns on, we must verify the AHL threshold to which the manipulation system responds. Our validation system (p15A-luxR-sfGFP) was induced by adding oxo-C6-HSL at concentrations of 0, 10e-10, 10e-9, 10e-8, 10e-7, 10e-6, 10e-5, and 10e-4 M, respectively. Finally, it was found that the control system could respond to the AHL of 10e-10M, which is consistent with the threshold we set for the suicide system. At the same time, we calculated the concentration of AHLs that induced the suicide system to turn on and off by mathematical modeling Under a similar design, the modeling results showed that 10e-9MAHLs could turn on the expression of the suicide system, indicating that our system met the expectation.
Unfortunately, a certain amount of fluorescent expression was always detected in the control group during our assay, and we guessed that there was a weak leakage of expression in the AHLs-LuxR inducible system, and thus we need to optimize this system in our subsequent work.
CcdB is a toxic protein in the CcdB/CcdA toxin-antitoxin system which promotes the breakage of chassis plasmid and chromosomal DNA, and ultimately leads to chassis division defects. We used CcdB protein as the toxin of the suicide system in our design, and in order to verify the feasibility and effectiveness of the suicide system, we evaluated the function of CcdB protein by designing a validation gene circuit to characterize it.
The expression of CcdB is regulated using IPTG induction in the prophase and metaphase of characterization validation. pET-28a-ccdB plasmid within the validation gene circuits consists of the LacO/LacI system, pT7, and ccdB. We constructed pET-28a-ccdB plasmid to verify the expression of CcdB toxic protein in the chassis and the characterization of its bactericidal function through the IPTG induction experiment.
Figure 4-6 Plasmid of pET-28a-ccdB
Figure 4-7 Sequence of pET-28a-ccdB
Figure 4-8 Results of IPTG gradient plate induction experiments
We verify the function of CcdB by IPTG gradient plates test. The initial minimum induced concentrations of IPTG on plates with different gradients were 0.08mg/ml, 0.10mg/ml (0.42mM) and 0.12mg/ml, respectively. The experimental results showed that, with the increase of IPTG concentration on plates with different gradients, The number of single colonies showed a decreasing trend, indicating that IPTG-induced CcdB could play a bactericidal role.
Figure 4-9 Results of induction experiments with different concentrations of IPTG plates
In order to further understand the ability of CcdB protein to exert bactericidal effects under the induction of different IPTG concentrations, we performed different concentrations of IPTG plate induction experiments. With the increase in IPTG concentration, the number of single colonies in the chassis showed an apparent decreasing trend. When the IPTG concentration was 0.02 mg/ml, the low amount of expressed CcdB began to play a more obvious bactericidal effect.
In order to minimize the survival stress of ccdB on chassis organism, we refined the suicide system by adding the ccdA antitoxin gene to the pET-28a-ccdB plasmid to construct the pET-28a-ccdA-ccdB plasmid.
Figure 4-10 pET-28a-ccdA-ccdB plasmid
Figure 4-11 Correct result of Colony PCR. 1-4 : fragments of CcdA
Different concentration IPTG plate experiments were still carried out. pET28a-ccdA-ccdB plasmid was transformed BL21 Star, which was spread onto different concentration IPTG plates respectively, and the number of single colonies was counted.
Figure 4-12 Result of the complete suicide system
As can be seen, constitutively low expression of CcdA made the IPTG-induced inhibition appear as an obvious concentration threshold ( 0.04 mg/ml), and the problem of CcdB leakage expression was solved to some extent.Still, the effects of CcdA antitoxin on CcdB toxin were almost the same between the highest IPTG-inducing concentration and the lowest IPTG-inducing concentration. This suggests that CcdA antitoxin has a good antagonistic effect on CcdB toxin and that the presence of CcdA does not affect the maximum inhibitory effect of CcdB when the CcdB-induced expression reaches a specific value. We believe that the complete CcdA/CcdB antitoxin-toxin system should be introduced into the suicide system.
Figure 4-13 Correct result of Colony PCR. 1 : fragment of CcdB, 2 : fragment of CcdA
Primers were designed for both ends of CcdA and CcdB fragments of pET28a-ccdA-ccdB, respectively. The single colonies induced by IPTG were subjected to colony PCR. It was found that the single colonies induced by IPTG had the CcdB gene, which proved that CcdA could inhibit the CcdB leakage to some extent.
We introduced the quorum sensing system into the pET-28a-ccdA-ccdB plasmid and constructed the pET-28a-LuxR-ccdA-ccdB plasmid for complete logical circuit validation of quorum sensing to induce suicide in engineered E. coli.
Figure 4-14 Plasmid of CcdA-CcdB-LuxR
Figure 4-15 Colony PCR showing the correct size of luxR gene fragments
The stress caused by CcdB to the engineering bacteria will lead to the self-cutting of the ccdB gene fragment. Based on this particularity, we design short-time liquid culture induction experiments according to the characteristics of AHL, which can not tolerate high temperature for a long time and is not applicable to be induced in the solid medium, and we spread the plates every 1h and draw growth curves by the number of colonies coated in the plates in different periods for the verification of the logical line.
Figure 4-16 Final Logic Line Verification
Our suicide system inhibited bacterial growth in the absence of AHLs during the first two hours, and the number of colonies in the control group induced by adding AHLs was higher than that in the experimental group without AHLs throughout the incubation process.
However, we found the growth of the experimental group and the control group converged to the same after 2-3h of AHL-induced expression, which indicated that the stress caused by CcdB on the engineered bacteria, as well as the excision of the ccdB fragments from the engineered bacteria, occurred in advance. We suspect that the leakage expression of CcdB intensified after the introduction of the quorum-sensing promoter. Therefore, we will continue to explore options to reduce the stress of CcdB on the host bacterium during the subsequent improvement of the project.
We have successfully expressed DspB, a glycosidase that degrades SRB biofilm, AidH 147V, an AHLs-degrading enzyme that inhibits SRB population sensing, and an antimicrobial peptide that degrades SRBs in SRBioQuencher. We will design fusion of glycosidase, N-acetylhomoserine lactonase and antimicrobial peptide by modeling, and set up linker degradation mechanism and targeting mechanism according to the environmental conditions of SRB, hoping to reduce the harm to our chasis bacterium.
We are considering integrating some of the functions into the genome of our engineered bacterium and adjusting the energy distribution in E. coli to reduce the metabolic pressure in our engineered bacterium. However, it involves the modification of genome and endogenous metabolism, which will be our long-term work.
In addition to this, when we validated the AHLs-luxR manipulation system, we found that there is leaky expression in this system, and we are searching for other ways to solve this problem.
Finally, it is not yet known whether our SRBioQuencher can grow properly and perform the desired functions in sewers with more complex environments with more bacterial colonies. so we need to further optimize the stability of each of our solution strategies, and design protection hardware and delivery strategies in conjunction with the actual environment of the sewers in order to improve the efficacy of our engineered bacteria.
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