Result | BNUZH-China

Result
Degradation

Plasmid amplification and reconstruction

Strain construction

Expression

Effect verification

Plasmid amplification and reconstruction

In degradation module we constructed 4 main plasmids. Two PE degrading enzymes, CYPY96F-VHb and AlkB2-Rd45-AdhA, are designed to accelerate the degradation rate by improving the absorbing and hydroxylation of PE monomer. In order to express above two enzymes in our chassis, pAB1-cypY96F-vgb and pAB1-alkB₂-Rd45-adhA were constructed. pAB1-pS-PEBP-PEase was constructed to depolymerize PE microplastics. In addition, pAB1-PEBP-GFP was constructed to verify the adsorption capacity of PEBP to PE by fluorescence microscopy.

Figure 1 a. Map of plasmid pAB1-alkB₂-Rd45-adhA b. Map of plasmid pAB1-cypY96F-vgb c. Map of plasmif pAB1-pS-PEBP-GFP d. Map of pAB1-pS-PEBP-PEase

Figure 2 The PCR result of AlkB-Rd45-AdhA fragment. The band was identical to the expected length of 3033bp

Figure 3 The PCR result of CYPY96F-VHb fragment. The band was identical to the expected length of 2285bp

Figure 4 The PCR result of PEBP-GFP fragment. The band was identical to the expected length of 2108bp

Figure 5 The PCR result of PEBP-GFP fragment. The band was identical to the expected length of 3231bp

The bands of AlkB2-Rd45-AdhA, CYP-VHb, PEBP-GFP and PEBP-PEase, from PCR were identical to the theoretical lengths estimated by the designed primer locations, which could demonstrate that we successfully amplified our target fragments, we then used homologous recombination to link the vectors and fragments.

Strain construction

We transferred the constructed plasmids into Escherichia coli DH5αstrain and conducted colony PCR. After obtained the correct results we amplified and extracted the constructed plasmids. Then we transferred these plasmids into Pseudomonas aeruginosa PAO1 strain and E. coli BL21 strain and obtained correct colony PCR results, indicating that we successfully constructed strains containing these plasmids.

Figure 6 P.aeruginosa PAO1 colony PCR results of CYPY96F-VHb and AlkB2-Rd45-AdhA

Figure 7 P.aeruginosa PAO1 colony PCR result of PEBP-PEase and PEBP-GFP. In order to improve primer specificity, we change the location of primer and PEBP-GFP theoretical lengths estimated by the designed primer locations is 1875bp

Expression

In order to verify whether AlkB2-Rd45-AdhA, CYPY96F and VHb proteins were successfully expressed in the strains, SDS-PAGE was performed and the following results were obtained.

Figure 8 SDS-PAGE result of CYPY96F-VHb

The plasmid pAB1-cypY96F-vgb is expected to express CYPY96F and VHb. The molecular weight of CYPY96F is estimated to be 47.48kDa and that of VHb is estimated to be 16.73kDa. In the figure, it can be seen that the bands in the pAB1-cypY96F-vgb group were significantly deepened at around 45kDa and 15kDa compared with the control group, indicating that CYPY96F and VHb proteins were successfully expressed.

Figure 9 SDS-PAGE result of AlkB2-Rd45-AdhA

Plasmid pAB1-AlkB2-Rd45-AdhA is expected to express a large fusion protein AlkB2-Rd45-AdhA with a molecular weight of 94.59kDa. In the figure, it can be seen that there were an extra bands in the experimental group than in the control group at a position slightly less than 100kDa, suggesting that AlkB2-Rd45-AdhA protein was successfully expressed.

Figure 10 SDS-PAGE result of PEBP-GFP

The plasmid pAB1-pS-PEBP-GFP is expected to express a large fusion protein PEBP-GFP protein with a molecular weight of 67.73kDa. In the figure, it can be seen that compared with the control group, the experimental group had multiple marker bands in the middle of the marker bands slightly smaller than 75kDa and 60kDa, suggesting that the PEBP-GFP protein was successfully expressed.

The plasmid PAB1-pS-PEBP-PEase was expected to express a protein with a size of 188.18kDa. Unfortunately, we did not see the corresponding bands, so the protein may have failed to be expressed in bacteria. We hypothesized that the protein could not be expressed correctly due to its large molecular weight. Therefore, we will verify the related effects of PEBP mainly by engineering bacteria with plasmid pAB1-pS-PEBP-GFP.For the part of the PEBP-PEase protein that are not verified, we used modeling methods to predict it.

Therefore, in the expression verification stage, we successfully verified the expression of AlkB2-Rd45-AdhA, CYPY96F, VHb and PEBP-GFP proteins, which laid a solid foundation for our subsequent effect verification.

Effect verification

In order to verify that the engineering bacteria did degrade the microplastics faster, a 7-day co-incubation experiment was conducted. When the co-culture was done, microplastics were carefully separated from the culture medium and weighed. Fourier infrared (FTIR) detection was performed on the incubated microplastics to check the property changes of the microplastics, and scanning electron microscopy was used to detect the morphologic change on the surface of the microplastics.

Weighing:

The results of the weighing were only slightly different. The remaining microplastics separated weighted 0.094g, 0.094g and 0.098g in PAO1::alkB2-Rd45-adhA, PAO1::cypY96F-vgb, wild type PAO1 group. This result may not be very convincing but it may also hint that our engineered bacteria can degrade microplastics more effectively.

Fourier infrared detection:

Figure 11 FTIR results of co-cultured PE microplastics *The peak around 2400 cm^-1 is caused by the instrument itself, that it lacks of argon

The FTIR results show that, the group without any bacteria have shown classic absorption peaks of PE. The characteristic PE absorbance peaks are located at 2921 cm^-1, 2851 cm^-1, 1456 cm^-1, and 722cm^-1, each standing for CH₂ asymmetric C-H stretch, CH₂ symmetric C-H stretch, CH₃ umbrella bending mode, and spilt CH₂ rock.

Compared to its line, the bacteria input group's peaks have decreased in the intensity level, indicating the decrease in chemical bonds. Especially, the engineered PAO1 have shown lower intensity than the wild type of PAO1. The decrease of chemical bonds indicates that the PAO1 is able to degradant PE, and our plasmids have improved its ability.

Scanning electron microscopy:

Figure 12 From left to right, PAO1::alkB2-Rd45-adhA, PAO1::cypY96F-vgb, wild type PAO1, blank control, respectively In the first row, the experimental group is magnified 900 times, the control group is magnified 300 times In the second row, the experimental group was magnified 5,000 times and the control group 2,500 times

It can be seen that the overall particle size of microplastics degraded by engineering bacteria is generally smaller than that of the control group and microplastics co-incubated with Engineering bacteria were broken into small fragments while control group maintains complete microplastic particles. In the enlarged images the microplastics co-incubated with the engineering bacteria displayed a rough surface, while the microplastic in the control group is rather smooth. All of these features suggest that engineering bacteria degrade plastics more efficiently.

Effect verification of PEBP:

Figure 13 From left to right are images of microplastics with 500mm diameter incubated with GFP with PE binding peptide, with GFP protein alone, and a blank control

The PEBP-GFP protein was extracted and purified. After being immersed in PBS and cleaned twice by the vortex shaker, the microplastics incubated with PEBP-GFP still generally showed obvious fluorescence. The microplastics incubated with GFP can see weak fluorescence of GFP residue after intense exposure, while the blank group can not see any fluorescence under the fluorescence microscope. It can be concluded that PEBP has strong binding ability with microplastics.

Modeling of PEBP-PEase:

Figure 14 Predicted structure of PEBP-PEase protein

For PEase, we tested its effect through moduling. We conducted structure predicting of PEBP-PEase. The predicting module clearly displayed a membrane spanning domain and an enzyme domain, which we exactly expected our bacteria to produce. After that molecular docking was conducted to study the interactions between the PEase enzyme and both large and small alkane molecules. It can be observed that the docking sites and the binding pockets are generally located within the same binding pocket, with multiple amino acid residues (such as LEU246, ILE250, VAL589, LEU622, etc.) surrounding the long-chain alkane. These amino acids are mostly hydrophobic residues, which likely stabilize the alkane molecules through hydrophobic interactions. Their affinity suggests that PEase may have the ability to bind to and degrade PE microplastics. (For detail information, please visit Modeling page)

Result
Extracellular Electron And CO₂ Transfer

Plasmid amplification and reconstruction

Strain construction

Expression

Validation

1.Calculation of NAD + / NADH amount

2. Flagella silver staining

3. MFC Testing

We engineered plasmids pAB1-pS-pilA and pAB1-pS-nqrf to boost electron transfer in P. aeruginosa and R. palustris, featuring a truncated pilA gene with mutations (E32Y, L51F, G57Y) and increased nqrf expression. Additionally, we developed plasmids pAB1-pS-PAO102 and pAB1-pS-acap to enhance CO2 transfer in P. aeruginosa and uptake in R. palustris.

Plasmid amplification and reconstruction

The introduction of the pAB1-pS-nqrf plasmid into P. aeruginosa induces the production of NADH dehydrogenase, which catalyzes the transformation of NADH into and electrons, increasing the amount of electrons in P. aeruginosa and thus promoting electron shuttles (PYO), which indirectly transmit electrons to R. palustris.

Figure 1 Plasmid construction of pAB1-pS-nqrf

Figure 2 Plasmid construction of pAB1-pS-pilA

Figure 3 Plasmid construction of pAB1-pS-PAO102

Figure 4 Plasmid construction of pAB1-pS-acap

Strain construction

Plasmids	Expected size(bp)	Fragments	Expected size(bp)
pAB1-pS-nqrf	9071	nqrf	1245
pAB1-pS-pilA	7810	pilA	201
pAB1-pS-PAO102	8381	PAO102	747
pAB1-pS-acap	5944	acap	837

Table 1 Plasmids constructed in the extracellular electron and carbon dioxide transfer module

We successfully constructed all the plasmids mentioned in the table and below are their colony PCR electrophoresis and gene line diagrams.

Figure 5 Agarose gel electrophoresis analysis of pAB1-pS-nqrf

Figure 6 Agarose gel electrophoresis analysis of pAB1-pS-pilA

Figure 7 Agarose gel electrophoresis analysis of pAB1-pS-PAO102

Figure 8 Agarose gel electrophoresis analysis of pAB1-pS-acap

Expression

SDS-PAGE analysis of the induced cells after the construction of the expression vectors showed that the Nqr(46.40 kDa) and pilA(12.08 kDa) proteins had the expected molecular weights.

Figure 9 SDS-PAGE analysis of the expression of pAB1-pS-nqrf,pAB1-pS-pilA in PAO1

Validation

1.Calculation of NAD⁺ / NADH amount

We used NADH Assay Kit with WST-8 to measure the amount of NAD⁺ / NADH in P. aeruginosa and P. aeruginosa/pAB-pS-nqrf. By comparing the proportion of NADH in the control group and the experimental group, we can find that NADH/(NADH + NAD⁺ ) in experimental group is lessen than control group, which means P. aeruginosa with pAB-pS-nqrf plasmid can product more NADH reductase and electronics. As shown in the Figure 10 and Table 2.

Figure 10 NADH, NADH+NAD⁺amount of PAO1/pAB-pS-nqrf, PAO1

Concentration(μM) Group	Experimental Group	Control Group
NADH + NAD+	0.217	0.116
NADH	0.131	0.0917
NADH/(NADH + NAD⁺)	0.604	0.793

Table 2 NADH + NAD⁺ amount, NADH amount, NADH/(NADH + NAD⁺) of PAO1/pAB-PS-nqrf, PAO1

2. Flagella silver staining

We stained the flagella using silver staining, which was used to verify the validity of our plasmid pAB-pS-pilA. After staining, we found that the flagellum length of the experimental group was longer and larger than that of the control group, which was in line with our experimental expectations.

Figure 11 Light microscopy to visualize the bacterial surface flagella PAO1 and PAO1/pAB1-pS-pilA,magnification:40$\times$10.

3.MFC Testing

We measured the conductivity of the bacterial fluid by measuring the MFC experiment and found that the voltage of the bacterial fluid with the introduction of plasmid pilA was significantly higher than the voltage of the original bacteria over time. This proves that our plasmid is effective in enhancing the electron conduction function of P. aeruginosa, and this experiment met our expectations.

Therefore, compared to P. aeruginosa that did not import plasmids, plasmid-infiltrated P. aeruginosa had more advantages as electrical donator and were more conducive to extracellular transfer between R. palustris.

Figure 12 The out put voltage of PAO1,PAO1/nqrf+,PAO1/pilA+

Result
Carbon dioxide fixation

Plasmid amplification and reconstruction

Strain construction

1.pBBR1MCS2-bcsA-bcsB

2.pBBR1MCS2-nadM-nadK-pntA-pntB

Expression

Validation

1. Cellulose FT-IR Analysis

2. Congo red staining

Plasmid amplification and reconstruction

Plasmids	Expected size(bp)	Fragments	Expected size(bp)
pBBR1MCS2-bcsA-bcsB	10024	bcsA-bcsB	4892
pBBR1MCS2-nadM-nadK-pntA-pntB	7810	nadM	1044
		nadK	825
		pntA-pntB	2932

Table 1 Plasmids constructed in the Carbon Dioxide Fixation module

1.We constructed the plasmid pBBR1MCS2-bcsA-bcsB for cellulose production with the goal of carbon dioxide fixation.

Figure 1 Schematic diagram of the pBBR1MCS2-bcsA-bcsB plasmid

2.To increase the intracellular level of NADPH, we constructed a plasmid containing four exogenous genes, including nadM, nadK, pntA, and pntB. Here we successfully amplified the target gene.

Figure 2 Schematic diagram of the pBBR1MCS2-nadM-nadK-pntA-pntB plasmid

Strain construction

1.pBBR1MCS2-bcsA-bcsB

Figure 3 Agarose gel electrophoresis analysis of pBBR1MCS2-bcsA-bcsB(3226bp)

Due to the challenges associated with primer placement at the extremities of the target sequence, we designed primers for colony PCR to amplify a 3,226 base pair (bp) fragment. The resulting agarose gel electrophoresis pattern confirmed the accuracy of the colony PCR, indicating successful amplification of the intended DNA sequence.

2.pBBR1MCS2-nadM-nadK-pntA-pntB

Figure 4 Gene line diagram of pBBR1MCS2-nadM-nadN-pntA-pntB

Figure 5 Agarose gel electrophoresis of digested fragments of pBBR1MCS2

Figure 6 Electropherogram of nadM-nadK PCR gel

Figure 7 Electropherogram of pntA-pntB PCR gel

Figure 8 Electropherogram of nadM-nadK-pntA-pntB PCR gel

Since the efficiency of homologous recombination of multiple fragments was too low, we decided to ligate the two inserts first, and then ligate the inserts to the linearized vector.Here we have successfully concatenated the inserts.

As the deadline was already approaching, the time required to complete the experiment was insufficient. We will continue the subsequent experiments after the iGEM competition.

Expression

SDS-PAGE analysis of the induced cells after the construction of the expression vectors showed that the bcsA,bcsB proteins had the expected molecular weights.

Figure 9 SDS-PAGE analysis of the expression of bcsA (83 kd) and bcsB (95 kd) in E.coli BL21

Validation

1. Cellulose FT-IR Analysis

We used engineered and unengineered bacteria as experimental and control groups for a three-day cellulose production culture. After three days, we filtered, separated and dried the cellulose contained in 30 ml of the bacterial solution for FT-IR analysis.The characteristic peaks of the infrared Fourier spectrum (FTIR) of cellulose are mainly distributed in the range of 3300-3500 cm-¹ (-OH stretching vibration), 2900 cm-¹ (methyl or methylene stretching vibration), 1640-1650 cm-¹ (water molecule bending vibration), 1500-1600 cm-¹ (C-O stretching vibration), 1375-1425 cm-¹ (methylene bending vibration) 1500-1600 cm-¹ (C-O stretching vibration), 1375-1425 cm-¹ (methylene bending vibration), and the regions of 1160-1200 cm-¹ and 1100-1030 cm-¹ (C-O-C skeleton vibrations) and other regions.The characteristic peaks were evident in the experimental group, whereas in the control group, due to the problem of very small yields, the characteristic peaks were measured to be unclear.

Figure 10 Comparison of cellulose FTIR analysis between experimental and control groups

2. Congo red staining

When cellulose is present, Congo red forms a red complex with it, causing the medium to appear red.Since we have not been able to resuscitate Pseudomonas rubrum very well, we have temporarily tested it in E. coli.

Figure 11 Congo red staining of DH5α/pBBR1MCS2-bcsA-bcsB,DH5α,DH5α/pBBR1MCS2.

The engineered bacteria DH5α/pBBR1MCS2-bcsA-bcsB, as well as the individual E. coli DH5α and E. coli DH5α transformed with the empty vector pBBR1MCS2, were subjected to Congo red staining. The results showed that the engineering bacteria produced obvious red areas after staining, while E. coli DH5α alone did not show red, and there was a non-obvious red halo around the outer ring of E. coli transferred to the empty vector, thereby confirming the cellulose production capability of the engineered bacteria.

Result
Safety

Plasmid amplification and reconstruction

Strain construction

Validation

Plasmid amplification and reconstruction

We have constructed two plasmids. One is pAB1-P_opdH, which is aiming to validate the effect of citrate sensation of the promoter P_opdH; the other is pAB1-hok/sok, which is used to limit the activity range of engineered bacteria as well as the realization of plasmid anti-loss. And we obtain the corresponding vector fragments by the digestion of restriction endonuclease, which are separately used as the backbone of the vectors for reverse. We get the DNA sequences we need (hok/sok and P_opdH, both of them have homologous arms) through PCR.

Figure 1 Plasmid construction of pAB1-popDH-hok/sok

Strain construction

After having access to the elements that we use to construct the plasmids, we conduct the homologous recombination to form complete plasmids as well as plasmid transfection. Then we conduct colony PCR. The schemas of the DNA sequences and the results of colony PCR are attached below.

Figure 2 The schema of hok/sok and the result of corresponding colony PCR.

Figure 3 The schema of P_opdH and the result of corresponding colony PCR.

Validation

1. the system of plasmid anti-loss

We conduct the verification experiment of the plasmid anti-loss function of hok/sok system. We draw some squares on the LB-Amp plates and inoculate the bacteria with and without hok/sok system into the squares to examine whether the plasmid with Amp resistant gene still exists in cells or not.

Figure 4 (A) The photo of the plate of 17^th generation. (B) The result of the plasmid anti-loss experiment

2. the sensation of P_opdH to citrate

To verify the citrate-sensation function of P_opdH, we conduct bacteria-citrate coincubation, observing the fluorescence intensity in different gradients of concentration. Taking advantage of enzyme labeling apparatus, we examine the difference in fluorescence intensity of each group and create the diagram below.

We can easily know from the fig.6(B) that the death rate of bacteria with hok/sok system is lower than the control group with the increasing generation, which means that the hok/sok system we design can exactly prevent plasmid loss.

Figure 5 The fluorescence intensity variability analysis chart

From the chart we can know from the result that the group without citrate activation has essentially no fluorescence compared to pure broth. However, the groups with citrate activation fluoresce to varying degrees, in which the group of 5mM citrate concentration has the strongest fluorescence. We can make a conclusion that citrate is actually able to activate the function of P_opdH, and we achieve our goal of regulating genes based on the concentration of citrate in the environment.

the system of suicide when there is no citrate (or activators) in the environment

Given that Pseudomonas aeruginosa is able to take use of citrate as a kind of carbon source, we introduce pLac in front of sok for research convenience. We separately cultivate engineered bacteria with the plasmid pAB-hok/sok in LB-Amp broth with and without IPTG in the broth and examine OD600 of both groups every 16h using the enzyme labeling apparatus. The corresponding diagram is attached below. The concentration of IPTG we use is 0.6mM.

Figure 6 The different OD600 of groups with and without IPTG

We can know from the diagram that the OD600 of the group with IPTG is much higher than the other group, which means that IPTG is able to induce the expression of sok and secure the living of bacteria. Therefore, the system of conditional suicide is effective.

Result
Co-culture

Co-culture experiments with P. aeruginosa and R. palustris

Co-culture power generation experiments

Co-culture experiments with P. aeruginosa and R. palustris

The ultimate requirement for the system we constructed is that the two bacteria are able to work together, so it is important to explore the optimal co-culture conditions. We could not find any examples of co-culture of related bacteria, so we set up our own co-culture medium with a volume ratio of LB medium: ATYP medium of 1:1 for the growth of the two bacteria and performed a growth curve test for 72 hours. In addition to the choice of medium, we also considered other factors such as temperature, whether to oscillate the culture or not, whether to light or not, and finally chose the culture conditions of 27 degrees Celsius, light, anaerobic for the R. palustris and aerobic for the P. aeruginosa in the static culture program.

The final growth curve results show that both bacteria can grow effectively in the co-culture medium, and the growth rate of P. aeruginosa is faster than that of R. palustris, which is in line with the reality, so it can be proved that the results of the co-culture experiment have reached the experimental expectations, which lays the foundation for our work of testing the voltage later.

Figure 1 Co-culture growth curves for 72 hours

Co-culture power generation experiments

In order to test whether the extracellular electron transfer between the two bacteria can be efficiently carried out, we prepared engineered P. aeruginosa possessing genes that promote electron transfer for voltage determination. For the first short period of time at the beginning of the experiment, we added only the P. aeruginosa, and when the voltage was stabilized, we added the R. palustris in an attempt to observe the changes produced by this manipulation of the voltage. The experimental data revealed that the engineered P. aeruginosa strain harboring the nqrf gene initially generated a substantial voltage, indicative of robust metabolic activity. However, upon the introduction of R. palustris, there was a notable decrease in voltage by approximately 5000 mV, followed by a fluctuating voltage distribution that stabilized around a constant mean value. This observation suggests that the voltage shift post-addition of R. palustris might be indicative of inter-bacterial electron transfer, leading to a diminished multimeter response.

In a parallel experiment, the R. palustris strain engineered to contain the pilA gene exhibited stronger voltages compared to the control. Nevertheless, no significant alteration in solution conductivity was detected after the addition of the R. palustris culture. We speculate that the primary reason for this discrepancy is the prolonged soaking of the cation-exchange membrane, which may have compromised its integrity. However, due to the limited availability and the high value of the experimental material, coupled with time constraints, we have not been able to replicate this series of experiments. We intend to address this in future studies to further elucidate the observed phenomena.

Figure 2 Output voltage of co-culture R. palustris solution