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

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

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

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.

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

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

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.

Build

We constructed as Design.

Test

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.

Build

We constructed as Design.

Test

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.

Build

We constructed as Design.

Test

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.

Build

We constructed as Design.

Test

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

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

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 S^2- in this group, and the degree of S^2- reduction was significantly greater than that in the wild-type control group.

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

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

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

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

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

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.