OVERVIEW

To comprehensively prove the successful assembly and efficient work of our integrated system, there are four things we need to confirm:

• Q1: Dose the system has the ability to degrade PE?

  The most essential character of our whole system is the degradation ability on PE, thus it is the first character we need to confirm.

• Q2: Whether the SpyTag/SpyCatcher could assemble with each other and whether the combination of SpyTag/SpyCatcher would have negative impact on the enzyme?

  Our proteins are linked with SpyTag/SpyCatcher by a flexible peptide, thus there are possibilities that the combination of SpyTag/SpyCatcher would be influenced by our proteins, or in turn, the SpyTag/SpyCatcher would have adverse effect on the function of enzymes.

• Q3: Could the complex sustain its function under a complicated and changeable environment?

  The MnP is a relatively stable protein, which can maintain its enzyme activity at higher temperatures and a wider range of pH conditions. But linking to SpyTag might decrease its stability, and we shall evalute that.

• Q4: Whether the dCas9 would be influenced by SpyCatcher and loss its ability to bind sgRNA?

  dCas9 has been proved to combine with the sgRNA efficiently, but the dCas9-SpyCatcher's ability to interact with sgRNA needed to be measured.

A1: DETECTION OF SPYTAG-MNP DEGRADATION EFFECT ON POLYETHYLENE

1. Design

A series of experiments were designed to observe and assess the degradation ability of MnP on PE film. Polyethylene gloves were chosen as the experimental material, which is similar to the PE material used on express packaging thus can maximize the results of the experiment to match the real world effect, while avoiding the interference of additives in polyethylene products, and have quite easy access. They were cut into slices of 0.5 × 0.5 cm, cleaned three times with 75% ethanol and dried for 2h in an oven at 37°C, then reserved in clean pcr tubes for further incubation. Since the MnP can only degrade hydrocarbons with the assistant of Mn²⁺ and H₂O, all PE films were divided into two groups: one group were immersed in the degradation system (200 μl), containing 50 mM sodium malonate (pH 6.0), 0.2 mM MnSO₄, 0.1 mM H₂O₂, 150 mM NaCl, 0.1% Tween 80 and 1 U SpyTag-MnP, while the other group untreated. Then, the two groups were incubated in a shaker at 37°C and 200 rpm for 10 days.

After incubation, the PE films were collected and incubated with 1%SDS, then ultrasonicated for 10min. Soak them with 0.1m NaOH and discard the solution after 20min of ultrasonication. Finally, the films were rinsed with ddH₂O and 75% Ethanol, dried at 37℃ for 1 hour^[1]. After drying, PE film was observed by optical microscope and SEM to determine the degradation status of PE.

2. Optimization of design

Subsequently, we obtained corresponding suggestions through interviewing Professor Sun Chaomin from The Institute of Oceanology, Chinese Academy of Sciences, and further optimized and determined the details of our experimental scheme. We have made the following optimizations:

• Fourier Transform Infrared (FTIR) was applied to observe whether there were oxidized new functional groups, such as hydroxyl group and carbonyl group.
• High temperature gel permeation chromatography (HT-GPC) was added to measure the number average molecular weight(M_n) and weight average molecular weight(M_w), for evaluating the decrease of PE molecular weight after enzyme treatment, and judging whether the enzyme had broken the long carbon chains of PE.
• Also, we changed the incubation solution every 2 days to guarantee high enzyme activities in the system, avoiding poor PE degradation effect due to insufficient enzyme activity.

3. Result

We next used multiple techniques to further verify the PE degradation ability of SpyTag-MnP.

3.1 FTIR

Firstly, we used Fourier Transform Infrared (FTIR) imaging to analyze the changes of surface chemical components and functional groups. In SpyTag-MnP treated PE, FTIR spectra showed two distinct peaks (Fig. 1). One peak was observed in the vicinity of 1636 cm⁻¹, indicating carbonyl bonds (-C=O-), while the other peak was observed at a wave number of 3400 cm⁻¹ and was attributed to hydroxyl groups (Fig. 1). Overall, compared with the untreated PE, our FTIR spectra data suggested an oxidation reaction of PE films happened in SpyTag-MnP treated PE, including the formation of hydroxyl groups and carbonyl bonds.

Fig. 1 FTIR analysis of untreated PE films and PE films treated by SpyTag-MnP after 10 days incubation.

3.2 SEM

We then monitored the surface structure changes of the two groups by Scanning Electron Microscopy (SEM). As shown in Fig. 2, obvious fragments appeared in SpyTag-MnP treated PE, while the untreated PE seemed to have no change.

Fig. 2 SEM observation of untreated PE films and PE films treated by SpyTag-MnP after 10 days incubation. A-D: SEM observation of PE films treated by SpyTag-MnP after 10 days incubation. E-H: SEM observation of untreated PE films after 10 days.

3.3 HT-GPC

We eventually analyzed molecular weight distribution (MWD) changes using High Temperature Gel Permeation Chromatography (HT-GPC). We found the MWD of SpyTag-MnP treated PE film showed molecular weights decreasing from xxxx Da to 298046 Da (Fig. 3 C, D). And not only the peak molecular weight response value had decreased, but the proportion of hydrocarbon chain with a molecular weight of less than 10000 Da had declined from 1.37% to 0.70% after SpyTag-MnP treated (Fig. 3 A, B).

Fig. 3 HT-GPC analysis of untreated PE films and PE films treated by SpyTag-MnP after 10 days incubation. A: HT-GPC spectrum of untreated PE. B: HT-GPC spectrum of SpyTag-MnP treated PE. C: HT-GPC calculus curve graph of untreated PE. D: HT-GPC calculus curve graph of SpyTag-MnP treated PE.

A2: ASSEMBLABILITY OF SPYCATHER/SPYTAG

1. Design

The first step to assemble the whole complex is the combination of the SpyTag and SpyCather, and only if they two were able to integrate with each other can our system assembled. Since SpyTag-MnP is the key enzyme in our system to oxidize PE, whether it can bond to SpyCatcher is of great importance, thus we choose SpyTag-MnP and dCas9-SpyCatcher to conduct the following assembly experiments.

The complex were proved to be successfully assembled by 8% SDS-PAGE first. Then the enzyme activity of the complex were also measured and compared with the initial activity of SpyTag-MnP, to ensure that the assembly would not affect the original oxidization process of MnP.

2. Result

2.1 Assemblability

For assembling the dCas9-SpyCather/SpyTag-MnP complex, SpyTag-MnP was mixed with dCas9-SpyCather in a ratio of 1 : 1 and allowed to conjugate for 1 h at 37℃^[2]. As shown in Fig. 4, the band of the complex appeared, which was higher than that of dCas9-SpyCather, and the original SpyTag-MnP band had disappeared.

Fig.4 SDS-PAGE showing the conjugation of SpyTag-MnP to dCas9-SpyCatcher. Lane 1: SpyTag-MnP (0.3 μM); Lane 2: dCas9-SpyCatcher (0.3 μM); Lane 3: SpyTag-MnP (0.3 μM) mixed with dCas9-SpyCatcher (0.3 μM). Upon mixing the two components, the upward shift in the band corresponding to dCas9-SpyCatcher as well as the disappearance of the band corresponding to SpyTag-MnP were observed, indicating successful conjugation. Note that the conjugation is unaffected by the SDS-PAGE conditions due to covalent isopeptide bond formation.

2.2 Impact on enzyme activity

Then we compared the difference in MnP activity between SpyTag-MnP and the complex, and, as shown in Fig. 5, there was no significant change. This result suggested that the assembly of SpyTag-MnP and dCas9-SpyCather will not affect the enzyme activity.

Fig. 5 Comparision of MnP activity between SpyTag-MnP and dCas9-SpyCather/SpyTag-MnP complex. Complex refers to the dCas9-SpyCather/SpyTag-MnP complex. p > 0.05.

A3: THE STABILITY OF DCAS9-SPYCATHER/SPYTAG-MNP COMPLEX

1. Design

The stabilities of dCas9-SpyCather/SpyTag-MnP complex are another crucial characters to the operation of the whole system and would greatly impact its further application in the industrial world. A series of stability test were conducted including thermal stability, pH stability and organic solvents stability, and the experiments were set as the following^[3]:

• Thermal stability

  The complex were incubated in 20 mM sodium malonate buffer (pH 5.5) with 100 mM NaCl at different temperature for 6 h and the residual enzyme activity were measured and calculated every 2 h. The relative enzyme activity under different temperatures were calculated with the following equation:
  $$Relative\ Enzyme\ Activity(\%)=\frac{residual\ enzyme\ activity_{each\ time\ points}}{initial\ enzyme\ activity_{0\ h} }×100\%$$

• pH stability

  The complex were incubated in 20 mM sodium malonate buffer with 100 mM NaCl under pH 3-7 for 12 h at room temperature. The relative enzyme activity at different pH conditions were calculated with the following equation:
  $$Relative\ Enzyme\ Activity(\%)=\frac{residual\ enzyme\ activity_{12\ h} }{initial\ enzyme\ activity_{0\ h} }×100\%$$

• Organic solvents stability

  The complex were incubated in methanol and ethanol (10-30%) for 12 h at the room temperature, respectively. The incubation process was held in 20 mM sodium malonate buffer (pH 5.5) with 100 mM NaCl and the residual enzyme activity were measured and calculated after 12 h. The relative enzyme activity of different organic solvent at distinct concentrations were calculated with the following equation:
  $$Relative\ Enzyme\ Activity(\%)=\frac{residual\ enzyme\ activity_{12\ h} }{control\ enzyme\ activity_{12\ h} }×100\%$$

2. Results

2.1 Thermal stability of dCas9-SpyCather/SpyTag-MnP complex

As shown in Fig. 6, the relative enzyme activity of the complex would decline gradually after 6 h incubation in all temperture we set. However, compared with unassembled SpyTag-MnP, it shown an improve of thermostability at mild temperture (below 60 ℃) (Fig. 7).

Fig. 6 Thermal stability of dCas9-SpyCather/SpyTag-MnP complex. The initial MnP activity before incubation was set as 100%.

Fig. 7 Effect of temperature on the stability of the complex and SpyTag-MnP after 6 h incubation. The initial MnP activity before incubation was set as 100%. The complex refers to dCas9-SpyCather/SpyTag-MnP complex. ^*P < 0.05, ^**P < 0.01.

2.2 pH stability of dCas9-SpyCather/SpyTag-MnP complex

As shown in Fig. 8, it didn't perform well at low pH range (pH 3-5) as both the complex and SpyTag-MnP had precipitated (Fig. 8), which probably because the 100 mM NaCl was not enough for the complex as its molecular weight became about 5 times than the unassembled SpyTag-MnP. However, it would perform better at high pH range (pH 6-7), although its relative enzyme activity was lower than SpyTag-MnP at pH 7.

Fig. 8 Effect of pH on the stability of the complex and SpyTag-MnP after 12 h incubation. The initial MnP activity before incubation was set as 100%. The complex refers to dCas9-SpyCather/SpyTag-MnP complex. ^*P < 0.05.

2.3 Organic solvents stability of dCas9-SpyCather/SpyTag-MnP complex

As for organic solvent stability, we would be happy to say that our assembly could tolerate methanol and ethanol while concentration was less than 30% (Fig. 9). This may means that our assemblies would more adaptable to industrial environments, as methanol and ethanol were commonly used in industry. Besides, its organic solvent stability was significantly higher than the unassembled SpyTag-MnP (Fig. 9).

Fig. 9 Effect of different concentrations of different organic solvents on the stability of the complex and SpyTag-MnP after 12 h incubation. The MnP activity without adding any organic solvent was set to 100% as the control. The complex refers to dCas9-SpyCather/SpyTag-MnP complex. A: The effect of different concentrations of methanol on MnP activity. B: The effect of different concentrations of ethanol on MnP activity. ^*P < 0.05, ^***P < 0.001.

A4: ASSEMBLABILITY OF CRISPR/DCAS9 ANCHOR SYSTEM

1. Design

To certify that the CRISPR/dCas9 anchor system can work efficiently, the combination of protein and nucleic acid were tested by Electrophoretic Mobility Shift Assay (EMSA). If the combination between dCas9-SpyCatcher and sgRNA could normally proceed, it means that the SpyCatcher would have little impact on the function of dCas9.

2. Result

In Fig.10, by comparing the protein(Fig.10A) and nucleic acid(Fig.10B) results, it could be found that the sgRNA band overlaped with the protein band(Lane 5,6,7), suggesting that the dCas9-Spycatcher was well combined with sgRNA.


Fig. 10 The EMSA results showing the combination of dCas9 -SpyCatcher and sgRNA. Lane1: dCas9-SpyCather, Lane2: sgRNA-1, Lane3: sgRNA-2, Lane4: sgRNA-3, Lane5: dCas9-SpyCatcher+sgRNA-1, Lane6: dCas9-SpyCatcher+sgRNA-2, Lane7: dCas9-SpyCatcher+sgRNA-3. A: the gel was stained by Coomassie brilliant blueG250. B: the gel was stained by gel GelRed® Nucleic Acid Gel Stain.

DISCUSSION

According to the results we obtained, the four questions are answered to determine that the whole integrated system can be assembled. Also, SpyTag-MnP can exert PE degradation ability in the presence of Tween 80 and H₂O₂, and significant changes on PE films can be observed within 10 days.

After iGEM, for further optimization of the system, the SpyTag-AAO and SpyTag-HFB1 would be applied into this system:

• AAO is able to produce H2O2 in a low but steady rate, therefore, the inhibition of MnP due to an excess of H2O2 concentration can be effectively prevented when applying AAO as the source of H2O2.
• HFB1 can enhance the affinity between hydrophilic proteins and hydrophobic materials PE, thus facilitating its contact with aqueous environment, thereby facilitating MnP to degrade PE.

Joint the three proteins together, the degradation process would be faster and more thoroughly.

Reference

[1] Zhang J, Gao D, Li Q, et al. Biodegradation of polyethylene microplastic particles by the fungus Aspergillus flavus from the guts of wax moth Galleria mellonella. Sci Total Environ. 2020;704:135931. doi:10.1016/j.scitotenv.2019.135931

[2] Lim S, Kim J, Kim Y, Xu D, Clark DS. CRISPR/Cas-directed programmable assembly of multi-enzyme complexes. Chem Commun (Camb). 2020;56(36):4950-4953. doi:10.1039/d0cc01174f

[3] Qin X, Zhang J, Zhang X, Yang Y. Induction, purification and characterization of a novel manganese peroxidase from Irpex lacteus CD2 and its application in the decolorization of different types of dye. PLoS One. 2014;9(11):e113282. Published 2014 Nov 20. doi:10.1371/journal.pone.0113282