iGEM 2023 — Team scu-china

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## 1. Biofilm Cultivation Validation Experiments

Forming and removing biofilm has been a long-standing research problem in the biological field. However, during the initial stage of our project, our extensive literature review on laboratory extraction methods of biofilm for enzymatic function verification ended in vain. Eventually, we decided to cultivate biofilm ourselves to obtain purified biofilm and managed to work out a scheme to construct and verify the integrity of biofilm through multiple rounds of DBTL approach.

#### Round One

#### Design

Since SRB requires anaerobic cultivation and grows relatively slowly and with difficulty in laboratory settings, we chose *E.coli* DH5\(\alpha\) to replace SRB for biofilm cultivation and verification experiments. By observing the maturity of biofilms of *E.coli* with different treatments on LB plates without antibiotics, we aimed to determine the activity of glycosidase based on the growth of *E.coli*.

#### Build

Under the biological safety cabinet, 100 microliters of bacterial solution was respectively spread on LB plates without antibiotics. The plates were incubated at 37°C until biofilm formation. Then add LB liquid medium without antibiotics + bacterial lysate containing glycosidase to blank LB plate(blank control group), add LB liquid medium without antibiotics to a plate with biofilm (NC group), and add LB liquid medium without antibiotics + bacterial lysate containing glycosidase to other plates with biofilm. Finally, biofilm formation states in each group were observed. (The method of glycosidase treatment can be found in the function verification section of the experiment part.)

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/wly-1-1.png)

Fig 1-1. Bacteria grown on LB plates without antibiotics

The results showed bacterial contamination in the blank control group. The test group formed relatively intact biofilms. After spreading the bacterial solutions treated with different glycosidase, all groups formed biofilms to some degree.

#### Learn

During plating, it was found that the blank control also had quite some colony growth, which could be due to the following three reasons:

1. Non-standard aseptic operation during the procedure.
2. Environmental contamination of the plates due to prolonged plate culture time.
3. Presence of undegraded bacteria in the lysate containing glycosidase. Since the blank control was contaminated by mixed bacteria, the degradation effect in this experiment could not represent the activity of glycosidase. Further improvements to the experiment are needed.

#### Round Two

#### Design

In response to the contamination of the blank control in the first round, in order to make the blank control more meaningful as a reference, we will change the biofilm-forming bacteria to ampicillin-resistant *E.coli* DH5\(\alpha\) (PUC19; ampicillin resistance). At the same time, the lysate containing glycosidase will be sterilized.

#### Build

1. The PUC plasmid was chemically transformed into Escherichia coli DH5\(\alpha\) to obtain ampicillin-resistant *E.coli*. (The specific chemical transformation method is the same as in the molecular cloning experiment).
2. In a biosafety cabinet, the bacteriolytic solution containing AHL-degrading enzymes was filtered through a 0.45 μm filter membrane.
3. Under the biological safety cabinet, 100 microliters of bacterial solution was respectively spread on LB plates without antibiotics. The plates were incubated at 37°C until biofilm formation. Then add LB liquid medium without antibiotics + bacterial lysate containing glycosidase to blank LB plate(blank control group), add LB liquid medium to a plate with biofilm (NC group), and add LB liquid medium + bacterial lysate containing glycosidase to other plates with biofilm. Finally, biofilm formation states in each group were observed. (The method of glycosidase treatment can be found in the function verification section of the experiment part.)

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/wly-1-2.png)

Fig 1-2. Plates of round 2

No colonies formed in the blank control group, which aligns with our experimental design. However, due to the high bacterial concentration, only destroy effect of DspB on biofilm destroy could be observed.

#### Learn

1. Proper aseptic techniques and sterilization of liquids effectively avoided contamination, yielding more accurate results. However, biofilm formation on 90 mm plates depends on bacterial viability and incubation time, requiring prolonged incubation and introducing multiple confounding factors. Using smaller plates could minimize errors.
2. We roughly judged the two enzyme activities by qualitative means, but we would like to carry out further validation of the gap between the two enzyme activities by qualitative methods.

#### Round Three

#### Design

Biofilms were cultured in 96-well plates to shorten the culture time. Through literature review, we found that crystal violet can bind with lipid molecules on the biofilm through electrostatic interactions and hydrogen bonding, forming complexes and staining the lipids and proteins in the biofilm. Therefore, crystal violet staining can quantify biofilm content to some extent.

#### Build

1. By transferring the same amount of enzyme into 96-well plates and setting up multiple time gradients, crystal violet staining was performed at different times. After washing off the floating dye with ethanol, the staining was observed.
2. The specific construction of detecting glycosidase activity refers to the verification experiment in the experiment section.

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/wly-1-3.png)

Fig 1-3. Linear relationship between crystal violet and biofilm maturity

Staining biofilms grown for different times showed uneven purple liquid depths, with lighter colors indicating less biofilm and darker colors more biofilm. Crystal violet staining has a linear relationship with biofilm maturity, which can be quantified by detecting OD570nm.

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/wly-1-4.png)

Fig 1-4. a: Glycosidase (DspB and DisH) to *E.coli* DH5\(\alpha\) biofilm treatment. b: Glycosidase (DspB and DisH) to SRB biofilm treatment

#### Learn

We cultured biofilms for different times in 96-well plates and found a linear relationship between crystal violet staining and biofilm maturity, also proving that we cultured complete biofilms. In addition, through crystal violet staining, we quantified the activity differences between multiple AHLs degradation enzymes, which met our experimental expectations.

## 2. Turbidimetric bacteriostatic test

The turbidimetric bacteriostatic test reflects the growth of the test strain in the bacterial inhibition system by observing the numerical change of OD600 absorbance value, indicating the inhibition effect of the inhibitory substances and providing an index for the selection of antimicrobial peptides with better effect.

#### Round One

#### Design

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-t-1.png)

Table 2-1. Turbidimetric bacterial inhibition experiment first round design plan

The initial design configured the system by mixing the test bacterial solution (*E.coli* DH5\(\alpha\)) with the treatment solution (supernatant/lysate of engineered *E.coli* BL21 secreting antimicrobial peptides) and leveled with PBS buffer that inhibits bacterial growth. Two time points, 3h and 5h, were selected to measure. The supernatant was extracted by centrifugation at 12000 rpm, 4°C for 3 min, and the lysate was extracted by resuspending the bacterial precipitate from the supernatant group with 0.1 M PBS and ultrasonically crushing it (ultrasonication time of 5 s, gap time of 10 s, and number of times of operation of 20 times), and centrifuging the supernatant according to the above conditions again.

#### Build

We constructed as Design.

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-1-1.png)

Fig 2-1. Results of the first round of turbidimetric bacterial inhibition assay with the antimicrobial peptide Bactenecin

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-1-2.png)

Fig 2-2. Results of the first round of turbidimetric bacterial inhibition assay with the antimicrobial peptide Indolicidin

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-t-2.png)

Table 2-2. Data from the first round of bacterial inhibition experiments by turbidimetric method

#### Learn

1. The zeroing system should be configured for different experimental groups to zero the corresponding ratio of the system; otherwise, there will be some negative absorbance results and other unreasonable results as above.
2. Only two periods of valid data were obtained, so the OD600 absorbance should be measured continuously from 0h at 1h intervals, the sample volume should be increased, and the detection time should be prolonged to obtain the dynamic trend.
3. The experimental data showed that there was almost no difference between the data measured in the two time periods, indicating that 0.1M PBS inhibited the growth of bacteria, and it was impossible to detect the inhibition effect during the growth process continuously. And prolonged treatment with PBS will make the bacterial rupture, which will affect the experiment results.
4. he initial concentration of the test strain system was too low, requiring a higher concentration.
5. In the experiment, the lysis was ineffective and the supernatant after centrifugation was still turbid, so the lysis time and centrifugation conditions must be improved.

#### Round Two

#### Design

For the problems of the first round of experiments, the subsequent experiments were improved accordingly. The measurement timings of absorbance OD600 were adjusted 0h, 1h, 2h and 3h for three consecutive hours. Simultaneously, the volume of the test strain was increased to 2 ml and the precipitation of the system was resuspended to form a 3 ml experimental system. To continuously monitor the changes in the growth of the test strain, the test strain was changed to *E.coli* DH5\(\alpha\) strain with kanamycin resistance.

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-t-3.png)

Table 2-3. Turbidimetric bacterial inhibition experiment second round design plan

#### Build

We constructed as Design.

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-1-3.png)

Fig 2-3. Overall results of the second round of turbidimetric bacterial inhibition experiments

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-1-4.png)

Fig 2-4. Results of the second round of turbidimetric bacterial inhibition assay with the antimicrobial peptide Indolicidin

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-1-5.png)

Fig 2-5. Results of the second round of turbidimetric bacterial inhibition assay for the antimicrobial peptide Bactenecin

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-t-4.png)

Table 2-4. Data from the second round of bacterial inhibition experiments by turbidimetric method

#### Learn

1. Since the experimental system was configured with only 3 ml, the 1 ml of liquid used for measurement was repeatedly drawn from the system into the cuvette, which was prone to contamination, resulting in abnormal experimental data. After being contaminated, the control group used for zeroing will cause the OD600 value of the experimental group to shift. Thus, it is necessary to increase the capacity of the experimental system and place the zeroing system at 4°C to inhibit the growth of hybrid bacteria.
2. It was observed that the result of the lysate-treated group was not good, and the preparation was complex, so it was considered to give up the lysate-treated group to reduce the experimental workload. Meanwhile, the positive and negative control groups should be added to reflect the inhibitory effect of the antimicrobial peptide.

#### Round Three

#### Design

The positive control group was the treatment group containing ampicillin antibiotic (1:1000), while the negative control was the group of *E.coli* BL21 culture supernatant. The concentration of the test strain was also further increased by resuspending a 3 ml system precipitation to a 6 ml system.

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-t-5.png)

Table 2-5. Turbidimetric bacterial inhibition experiment third round design plan

#### Build

We constructed as Design.

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-1-6.png)

Fig 2-6. Results of the third round of turbidimetric bacterial inhibition experiments with antimicrobial peptides

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-1-7.png)

Fig 2-7. Overall results of the third round of turbidimetric bacterial inhibition experiments

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-t-6.png)

Table 2-6. Antimicrobial peptide Indolicidin third round of turbidimetric bacteriostatic assay data

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-t-7.png)

Table 2-7. Antimicrobial peptide Bactenecin third round of turbidimetric bacteriostatic assay data

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-t-8.png)

Table 2-8. The third round of turbidimetric bacterial inhibition experiment control group experimental data

#### Learn

1. After the first two rounds of improvements, we successfully obtained reasonable data for the inhibition experiments, and to minimize random errors, we repeated the experiments three times for each data point.
2. The final results of the experiments suggested that both antimicrobial peptides had some lower degree of inhibitory effect on the growth of the tested strain *E.coli* DH5\(\alpha\).

## 3. Methods of SRB cultivation

SRB (sulfate reducing bacteria) are strictly anaerobic bacteria. We have conducted many experiments to find out the methods and conditions of SRB cultivation and finally succeeded in obtaining the culture formulas of the solid medium and liquid medium, as well as the culture environment conditions of SRB.

#### Round One

#### Design

In our initial conception, we proposed to use the AnaeroPouch anaerobic gas-producing bag kit (gas-producing bag, oxygen-concentration indicator, anaerobic bag) from MGC to culture SRBs at 37°C in a thermostat.

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-2-1-1.png)

Fig 3-1. Anaerobic bag usage & Oxygen indicator range(From MGC Company AnaeroPack Kit)

#### Build

We constructed as Design.

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-2-2.png)

Fig 3-2. SRB anaerobic bag culture

#### Learn

1. The oxygen concentration indicator indicated that the oxygen concentration in the anaerobic bag reached 0.1% or less. After overnight incubation in a 37°C thermostat, it was eventually found that there were no colonies, and it was considered that the inactivation of the oxygen concentration indicator had led to a misjudgment.
2. Since the inoculation process was done in an ultra-clean cabinet, it was possible that the SRBs may be too sensitive to oxygen and die after a short exposure to oxygen during inoculation. It should be ensured that the entire inoculation operation was done in a sterile and oxygen-free environment.

#### Round Two

#### Design

Based on the experience gained from the previous round of experiments, we used the E400 anaerobic workstation to complete a series of culture processes, such as inoculation, resuscitation and passage of SRBs. The anaerobic workstation was supplied with high purity nitrogen and gas mixture (10.2% CO2, 84.6% N2, 5.2% H2) at a pressure of 0.18 MPa, and the incubation temperature was guaranteed to be 37°C with an oxygen concentration of 0.0%. One of the plate operations was done using glass plating beads.

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-2-3.png)

Fig 3-3. SRB's E400 Anaerobic Workstation Culture

#### Build

We constructed as Design.

#### Test

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-2-4-1.png) | ![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-2-4-2.png) |

Fig 3-4. SRB's E400 Anaerobic Workstation Culture Results

#### Learn

1. The results of the experiments showed that the use of plating beads to plate were not effective and the density of bacteria was too low to form a film.
2. Incubation conditions at 37°C were too intense for SRBs, resulting in an accelerated rate of nutrient depletion, and short-term denaturation of the medium. The incubation temperature and period should be adjusted.

#### Round Three

#### Design

The incubation temperature of the E400 anaerobic workstation was adjusted to 30°C ,and the incubation time was shortened to 10 h. The inoculation operation of SRB was changed to use an L-shaped applicator.

#### Build

We constructed as Design.

#### Test

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-2-5-1.png) | ![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/gs-2-5-2.png) |

Fig 3-5. Results of E400 anaerobic workstation cultures of SRBs after improved methods

#### Learn

After continuous adjustments to the experimental design, higher densities of SRB strains were successfully cultured on Columbia blood agar medium, and the film formed.

## 4. Thioredoxin oxidase SQR

#### Round One

#### Design

We wanted to optimize our chassis design by transferring the sqr gene into *E.coli* to degrade H2S in the environment. We decided to culture the bacteria in 50 mM Tris-HCl buffer to measure the enzyme activity. H2S is formed when Na2S is mixed with deoxygenated water. Na2S was chosen to mimic the H2S environment since it is more convenient for quantitative measurements. The ability of the engineered strain to degrade H2S can be measured by mixing the bacterial solution with the Na2S solution and determining the amount of H2S in the environment after a certain period. When H2S reacts with N,N-dimethyl-p-phenylenediamine in the color developing agent, the amount of material produced is proportional to the absorbance value of the mixture at 670 nm, and the absorbance value of the reaction system at 670 nm is measured to indicate the H2S content. In order to bring the absorbance value into the visible range, it is necessary to dilute the mixture to a certain extent to facilitate the measurement of H2S.

#### Build

The constructed engineered strain was cultured in LB medium to a certain concentration. Then, the LB medium in the bacterial solution was replaced with 50 mM Tris-HCl buffer to keep the bacteria in a resting state, which was convenient for us to measure the enzyme activity expressed by the bacteria. Three groups were designed separately: the test group was a mixture of bacterial solution and Na2S standard solution, the two control groups were a mixture of bacterial solution and distilled water, as well as a mixture of sodium sulfide and Tris-HCl buffer, and three replicate controls were designed for each group. After a certain time, the mixture of the three groups was diluted and then mixed with the color developing agent to measure the change of absorbance value at 670 nm.

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/2-1.png)

Fig 4-1. Echange of H2S content

The OD670 in the test group showed a decreasing trend but with large fluctuations, and the OD670 in the control group with a mixture of sodium sulfide and Tris-HCl buffer fluctuated too.

#### Learn

Possible reasons for large fluctuations in OD670 values:

1. To let the reaction proceed more fully, we mixed and diluted the reaction solution with distilled water before measuring the OD value. However, there may be differences in the degree of dilution each time, resulting in large fluctuations in the experimental data.
2. Bacteria were not removed from the reaction solution, and the number of bacteria in each sample may vary, which could have a large effect on the absorbance value.
3. The number of replicate groups was insufficient, resulting in significant errors in the experimental data.
4. We spiked samples at adjacent locations in 96-well plates and did not separate the experimental and control groups. The escaping H2S may enter the neighboring wells and affect the experimental results.

Because of the large fluctuation of this control group of H2S mixed with distilled water, it does not measure the degradation ability of the engineered strain to H2S, and further improvement of the experiment is still needed. In addition, in order to better simulate the ability of *E.coli* to respond to H2S in physiological condition, we considered using M9 medium to cultivate *E.coli*, because M9 medium is colorless and has a lower impact on the measurement of absorbance value.

#### Round Two

#### Design

We swapped 50 mM Tris-HCl buffer for a colorless M9 medium to mimic normal *E.coli* growth. Because H2S is a metabolite of *E.coli*, we hope to see that *E.coli* degrades H2S more than *E.coli* produces H2S. In addition, we have redesigned the experimental and control groups and further improved the treatment method of bacterial solutions to reduce errors.

#### Build

1. After the constructed engineering strain was cultured with OD600 to 0.8 in LB, the LB medium in the bacterial solution was replaced with M9 medium.
2. The bacterial solution and Na2S mixture were first diluted with M9 medium, and the diluted solution was directly taken for measurement.
3. Before the measurement, a 0.45 μm filter was used to filter the bacterial solution, and then the absorbance value in the filtrate was measured.
4. The experimental group and control group were redesigned: the experimental group was the mixture of bacteria and Na2S of engineered strains, the control group was the mixture of bacteria and Na2S of wild-type strains, and the mix of engineered strains and distilled water, and the blank control group was M9 medium and Na2S mixture. By adding a control group, the possibility of H2S oxidation by other oxidants was ruled out, and the H2S content of the engineered strain was measured separately; the ability of *E.coli* to produce H2S was characterized.
5. Different experimental groups were placed in different plates for testing, and PBS solution was added to the blank wells to maintain a constant pH.

#### Test

By detecting the H2S content in the environment, the H2S content in the engineered strain and Na2S mixture decreased rapidly within 30 min, indicating that SQR can rapidly degrade H2S in the environment within a short period. There was a clear trend of decreasing S2- in this group, and the degree of S2- reduction was significantly greater than that in the wild-type control group.

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/2-2.png)

Fig 4-2. Changes in hydrogen sulfide content

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/2-3.png)

Fig 4-3. Hydrogen sulfide content after 120 min

#### Learn

By measuring the absorbance of the reaction solution at 670 nm, we successfully detected a decrease in H2S content in the environment under the action of SQR, which was in line with our experimental expectations, indicating we successfully cloned and characterized the SQR product in *E.coli* DH5\(\alpha\) and experimentally verified that *E.coli* can rapidly and effectively reduce environmental H2S.

## 5. Suicide System Validation Experiments

The suicide system is an essential module of this project, related to the self-clearance of the engineered bacteria and the biosafety of the project. Due to the toxicity of CcdB to prokaryotes, ensuring the stable existence of the ccdB gene on the plasmid became a tricky thing. In the early experiments, we were always puzzled by the strange loss of the ccdB gene. Eventually, we verified our guess through a series of experiments, kept the ccdB gene on the plasmid, and were able to further advance the experiments.

#### Round One

#### Design

At the initial stage of the experiment, we referred to the experimental methods of BNU-China in 2019. By adding different volumes of 1 mol/L IPTG solution to the BL21 star competent cells containing the pet28a-ccdB plasmid, a series of experimental groups with IPTG concentration gradients were formed. The OD600 was measured every 6 hours, and 10 μl of the bacterial solution was plated. After 24 hours, the number of single colonies was counted. In our expectations, the higher the IPTG concentration, the more the growth of the engineered bacteria would be inhibited.

#### Build

1. The pet-28a-ccdB plasmid was transformed into BL21 star competent cells using the heat shock method. After recovery, the cells were plated, and single colonies were picked next day and inoculated into K+ LB liquid medium for amplification.
2. The bacterial culture was aliquoted into 96 1.5mL EP tubes and divided into three experimental groups with 0mg/ml, 0.5mg/ml, and 1mg/ml IPTG concentrations. For each group, 8 tubes of bacterial culture were taken every 6h, with 4 tubes plated and the other 4 tubes measured for OD600 absorbance values.
3. Line charts were plotted according to the experimental results.

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/1-1.png)

Fig 5-1. Liquid Culture Absorbance Curve

The experimental groups with IPTG addition only exhibited relatively lower absorbance values compared to the control group without IPTG in the first 6h or so. This result shows that CcdB did not demonstrate significant bacterial inhibition effects, which is highly unusual. Therefore, we attempted to analyze by Sanger sequencing and found that only the ccdB fragment was deleted in the plasmid.

#### Learn

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/1-2.png)

Fig 5-2. Sequencing results of *ccdB*

The sequencing result showing ccdB gene deletion leads us to speculate the following reasons:

1. The terminal structures of the *ccdB* gene are unstable.
2. The expression of the *ccdB* gene produces the cytotoxic CcdB protein, creating tremendous survival pressure. After culturing for a period of time, mutant strains that have escaped begin to dominate in the bacterial culture and propagate extensively.

Since the results of this experiment failed to demonstrate the function of the ccdB gene, further improvements are still needed for the experiment. This experiment also found that the engineered bacteria lost the *ccdB* gene even without IPTG induction after prolonged culture, presumably due to leaky expression from the lac operator.

#### Round Two

#### Design

To verify our speculation, we conducted a preliminary experiment using IPTG gradient plates (with IPTG concentration distributed in a gradient from one side to the other on the medium). After transforming the pET-28a-*ccdB* plasmid into the BL21 star competent cells and recovery, the cells were immediately spread onto the IPTG gradient plates for induction, allowing the transformed colonies to form single colonies. We expected that within a shorter culture time, colonies located in the higher IPTG concentration areas of the plate would be induced to produce CcdB, creating greater survival pressure, exhibited as smaller colonies or death, while colonies in the lower IPTG concentration areas would form larger single colonies.

#### Build

1. LB solid medium was prepared, sterilized by autoclaving at 121°C for 20 min, then cooled to around 50°C. Kanamycin solution was added at a ratio of 1:1000, then poured into plates with one end propped up by 5mm. After solidification, LB K+ medium containing 10 μmol/L IPTG solution was added to create IPTG gradient plates.
2. *E.coli* BL21 star competent cells were transformed with pet-28a-*ccdB* plasmid, recovered for 30 min, then 50 μl of culture was evenly spread onto the IPTG gradient plates. The plates were incubated in a constant temperature incubator, observed after 24h, and the number of single colonies was counted.

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/1-3.png)

Fig 5-3. Results of IPTG gradient plate induction assay

With increasing IPTG concentration, the number of single colonies continuously decreased. This is consistent with our hypothesis. However, since the IPTG concentration could not be quantitatively analyzed, this result can only initially verify our speculation, and more rigorous experiments are still needed for confirmation.

#### Learn

Compared to IPTG gradient liquid culture, we have successfully obtained the expected results, which initially verified our hypothesis and demonstrated the effectiveness of this part.

#### Round Three

#### Design

To conduct a more quantitative analysis, induction experiments were performed using plates with different concentrations of IPTG. We expected that the number of single colonies would continuously decrease with increasing IPTG concentration.

#### Build

1. *E.coli* BL21 star competent cells were transformed with pet-28a-*ccdB* plasmid, recovered for 30 min, then 50 μl of culture was evenly spread onto a series of plates with different IPTG concentrations. The plates were incubated in a constant temperature incubator.
2. After 24h, observations were made, the number of single colonies was counted, and a line graph was plotted.

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/engineering/1-4.png)

Fig 5-4. Induction Experiments Using Plates with Different IPTG Concentrations

With increasing IPTG concentration, the number of single colonies on the plates showed a clear downward trend. When IPTG concentration reached 0.02 mg/ml, even low expression of CcdB began to exhibit bactericidal effects.

#### Learn

The results of this experiment demonstrated the bactericidal function of CcdB and the high expression efficiency of the *ccdB* gene under the control of the T7 promoter. However, this system could not stably exist. Even without IPTG induction, engineered bacteria sequencing showed that the pet-28a-*ccdB* plasmid was missing *ccdB* fragments, this illuminated that low leaky expression from the lactose promoter would also lead to self-cleavage of *ccdB*. Therefore, we urgently need to find a solution to allow this system to stably persist.

#### Round Four

#### Design

To improve the original suicide system, the *ccdA* antidote gene controlled by the low expression J23105 promoter was introduced into the pET-28a-*ccdB* plasmid to construct pET-28a-*ccdA*-*ccdB* plasmid.

#### Build

1. The linearized vector and target fragments were obtained by PCR, and then Gibson assembly was used to construct the pET-28a-*ccdA*-*ccdB* plasmid.
2. *E.coli* BL21 star competent cells were transformed with pET-28a-*ccdA*-*ccdB* plasmid, recovered for 30 min, then 50 μl of culture was evenly spread onto plates with a series of IPTG concentrations. The plates were incubated in a constant temperature incubator.
3. After 24h, observations were made, the number of single colonies was counted, and a line graph was plotted.

#### Test

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/12.jpg)

Fig 5-5. Experimental results of inducing the CcdA-CcdB system on plates with different IPTG concentrations

Compared to the original system, this curve demonstrates a clear threshold effect. It proves that under conditions of low IPTG induction and leaky expression, low expression of CcdA can completely neutralize the toxic CcdB protein.

#### Learn

The results of this experiment demonstrate that CcdA controlled by the J23105 promoter can prevent accumulation of CcdB during culture growth, avoid loss of *ccdB* due to survival pressure, and play a protective role.

### Acknowledgement

* ![](https://static.igem.wiki/teams/4817/wiki/wiki/footer/t-scu-china-scu-footerlogo5.png)
* ![](https://static.igem.wiki/teams/4817/wiki/wiki/footer/t-scu-china-scu-footerlogo6.png)
* ![](https://static.igem.wiki/teams/4817/wiki/wiki/footer/scu-chian2023.png)

### Contact Us

* +86 13308049545
* scu\_igem@163.com
* College of Life Science in Sichuan University,
  NO.29 Wangjiang Road, Chengdu, Sichuan, China

© 2023 - Content on this site is licensed under a
[Creative
Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).
The repository used to create this website is available at
[gitlab.igem.org/2023/scu-china](https://gitlab.igem.org/2023/scu-china).

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### Experiments

* [Home](index.html)
* Project

Here, we provide the protocols for the various experiments involved in our project for future use by other teams.

## Molecular Cloning

Molecular cloning was applied to the construction of all plasmids in our project.

## Protein Expression

We verified the expression and roughly determined the content of AHLs degrading enzymes and their mutants, glycosidases degrading biological periplasm in our project by protein induced expression and purification, in order to initially select the more efficient enzymes for the next validation experiments.

## Engineering and Validation

### Protein efficiency verification

We designed multiple characterization experiments by observing the results of enzyme action to verify that the exogenous proteins expressed in our project functioned as expected, and selected superior proteins to construct our SRBioQuencher.

### Operon efficiency verification

In order to better design the suicide system, we determined the effect of our regulatory gene cluster on the growth of *E. coli* and verified the sensitivity and preference of our regulatory system to AHLs.

## SRB Culture

We construct engineering bacteria that secrete antimicrobial peptides and culture SRB for antibacterial experimental verification.

### Acknowledgement

* ![](https://static.igem.wiki/teams/4817/wiki/wiki/footer/t-scu-china-scu-footerlogo5.png)
* ![](https://static.igem.wiki/teams/4817/wiki/wiki/footer/t-scu-china-scu-footerlogo6.png)
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### Contact Us

* +86 13308049545
* scu\_igem@163.com
* College of Life Science in Sichuan University,
  NO.29 Wangjiang Road, Chengdu, Sichuan, China

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### Results

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### Tasks

1. screen and design more efficient, stable and specific glycosidases, AHLs degrading enzymes and antimicrobial peptides
2. validate the efficiency of SQR in converting hydrogen sulfide, reduce harmful substances produced by *E. coli*, and optimize the chassis
3. Construct a suicide system based on luxR-lac-ccdB to regulate the growth of engineered bacteria using dynamic changes in the environment.

## 1. Cultivation and clearance of SRB

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.

#### 1.1 SRB cultivation

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/4-1.jpg) | ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/4-2.jpg) |

Figure 1-1. SRB solid culture and liquid culture results

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| ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/1-1.jpg) | ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/2.jpg) |

Figure 1-2. a.Anaerobic workstation culture of SRB.
b. Anaerobic bag culture of SRB

#### 1.2 Construction antimicrobial peptide plasmids

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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| ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/fig1-3-1.png) | ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/fig1-3.png) |

Figure 1-3. plasmids of pET28a-OmpA-Bac and pET28a-OmpA-Ind

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/fig4-1.png)

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/fig4-2.png)

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.

#### 1.3 Turbidimetric Antibacterial Experiment

##### 1.3.1 Antimicrobial peptide secretion and inhibition validation

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/13.jpg)

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.

##### 1.3.2 Antimicrobial peptide host toxicity validation

To investigate the effect of antimicrobial peptides on the growth of engineered *E. coli* BL21, we conducted antibacterial experiments for characterization.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/16.jpg)

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.

##### 1.3.3 SRB turbidimetric antibacterial experiment

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/17.jpg)

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/18.jpg)

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.

#### 1.4 Antibacterial zone experiments

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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| ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/19-1.jpg) | ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/19-2.jpg) |

Figure 1-9. Culture environment for antibacterial zone experiment of *E. coli* DH5 α

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/20-3.jpg)

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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| ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/21-1.jpg) | ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/21-2.jpg) |

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:

1. SRB culture did not form a biofilm, and it was difficult to determine the effect of bacterial inhibition
2. The concentration of the antibacterial peptide treatment solution needs to be higher, and further concentration treatment is required.
3. Antibacterial peptides undergo significant degradation in the treatment solution, resulting in a lower final working concentration.
4. Poor purification of antimicrobial peptide

We will further improve our experimental program in the follow-up work.

#### Conclusion

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.

## 2. Biofilm removal and inhibition

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.

#### 2.1 plasmids construction of glycosidase(DspB and DisH), N-acyl homoserine lactonase(Aiia, AidH, Moml)and five mutant

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part2/1.png)

Figure 2-1 Plasmids of glycosidase(DspB and DisH), N-acyl homoserine lactonase(Aiia, AidH, Moml)and five mutant

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part2/2.png)

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.

#### 2.2 Expression of glycosidases (DspB and DisH), N-acyl homoserine lactonase (Aiia, AidH, Moml), and five mutant protein

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part2/3.png)

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part2/4.png)

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).

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part2/5.png)

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).

#### 2.3 Validation of Glycosidase (DspB and DisH) Efficiency

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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| ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part2/6-1.png) | ![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part2/6-2.png) |

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part2/7.png)

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.

#### 2.4 Validation of N-acyl homoserine lactonase (Aiia, AidH, Moml) and five mutants

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part2/8-revise.png)

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part2/9.png)

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.

## 3. *E. coli* hydrogen sulfide oxidation

we expressed SQR in SRBioQuencher, in order to rapidly convert hydrogen sulfide generated by E. coli and improve the efficiency of SRBioQuencher.

#### 3.1 construction of SQR plasmid

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part3/1.png)

Figure 3-1 Plasmid of SQR

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part3/2.jpg)

Figure 3-2 Sequence results of SQR

#### 3.2 Efficiency Verification 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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part3/3-1.png)

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 \(H\_2S\).

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part3/4-1.png)

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 \(H\_2S\).

## 4. Quorum Sensing Induced Suicide Systems(Low AHLs)

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.

#### 4.1 AHLs induction system

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.

##### 4.1.1 Plasmid construction

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/1.jpg)

Figure 4-1 Plasmid of p15A-luxR-sfGFP

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/2.jpg)

Figure 4-2 Sequence results of p15A-luxR-sfGFP

##### 4.1.2 AHLs Preference testing

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/3.png)

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/4.png)

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.

##### 4.1.3 AHLs sensitivity testing

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/5.png)

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27gene_layer_model%27%29%20%7D%7D) 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.

#### 4.2 CcdB toxin proteins

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.

##### 4.2.1 pET-28a-ccdB Plasmid Construction

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/6.jpg)

Figure 4-6 Plasmid of *pET-28a-ccdB*

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/7.jpg)

Figure 4-7 Sequence of *pET-28a-ccdB*

##### 4.2.2 CcdB Functional Validation

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/8.jpg)

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/9-1.jpg)

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.

##### 4.2.3 Antagonism of CcdB by CcdA

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/10.jpg)

Figure 4-10 *pET-28a-ccdA-ccdB* plasmid

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/11.jpg)

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/12.jpg)

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/13.jpg)

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.

#### 4.3 Logic Circuit Verification

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*.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/14.jpg)

Figure 4-14 Plasmid of CcdA-CcdB-LuxR

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/15.jpg)

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.

![](https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/result-part4-16.png)

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.

## 5. Future work

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.

Reference

Li W, Zheng T, Ma Y, Liu J. Current status and future prospects of sewer biofilms: Their structure, influencing factors, and substance transformations. Sci Total Environ. 2019 Dec 10;695:133815.

Wang Y, Bian Z, Wang Y. Biofilm formation and inhibition mediated by bacterial quorum sensing. Appl Microbiol Biotechnol. 2022 Oct;106(19-20):6365-6381.

Yan X, Sun J, Wang Y, Zhang Z, Zhang C, Li W, Xu J, Dai X, Ni BJ. Low-rate ferrate dosing damages the microbial biofilm structure through humic substances destruction and facilitates the sewer biofilm control. Water Res. 2023 May 15;235:119834.

Pagsuyoin SA, Luo J, Chain FJ. Effects of sewer biofilm on the degradation of drugs in sewage: A microcosm study. J Hazard Mater. 2022 Feb 15;424(Pt D):127666.

Ran, M. et al. Rhodaneses minimize the accumulation of cellular sulfane sulfur to avoid disulfide stress during sulfide oxidation in bacteria. Redox Biol 53, 102345 (2022).

### Acknowledgement

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