OUTLINE

In order to obtain all the proteins and nucleic acid for building the whole system, the gene sequences would be inserted into the yeast genome or transformed into E.coli. Then the proteins are purified and concentrated for further experiences.

The following table shows the current progress of the acquisition of protein and nucleic acids (the construction information could also be seen in the engineering success page) :

Table. 1 The current construction process of our proteins and nucleic acid. the label"√" means a successful construction; the label "×" means that we failed to accomplish this part.

construction progress	SpyTag-MnP	SpyTag-AAO	SpyTag-HFB1	dCas9-SpyCatcher	sgRNA	dsDNA
Design	√	√	√	√	√	√
Plasmid construction	√	√	√	√	√	√
Protein expression/purification of the nucleic acid	√	√	√	√	√	√
Protein purification	√	×	√	√	\	\
Characterization	√	√	√	√	\	\
Stability of the proteins	√	×	×	√	\	\

Also, we have applied semi-rational directed evolution towards Manganese Peroxidase for Stability Improvement, including thermo-stability, pH stability and organic solvents stability. 10 single point mutants were selected out by dry lab and 6 of them were successfully expressed and purified in Pichia pastoris. And we have evaluated their improvements in stability and activity compared to the original MnP.

Table 2 displays all the mutant library we built and the 6 mutants we obtained:

Table. 2 The information of our mutant library. the label"√" means a successful expression and purification; the ΔΔG refers to the energy difference before and after mutation.

Mutant No.	Position of amino acids	Mutation	ΔΔG(kcal/mol)	expression and purification
1#	74	E-P	-2.255	√
2#	74	E-M	-2.059	√
3#	182	D-I	-1.711
4#	182	D-V	-1.637
5#	182	D-T	-1.544	√
6#	232	S-P	-1.306	√
7#	74	E-L	-1.239	√
8#	78	S-P	-1.173	√
9#	183	Q-P	-1.079
10#	182	D-C	-0.9288

 

RESULTS

Detection of SpyTag-MnP degradation effect on polyethylene

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. 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.

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 detections:

• Optical microscope was used to roughly observe the attachment of MnP to the PE surface in the preliminary experiments.
• Fourier Transform Infrared (FTIR) was applied to observe whether new functional groups appeared, such as hydroxyl group and carbonyl group that can indicate a result of oxidation.
• 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.
• In order to obtain high resolution images, we applied Scanning Electron Microscopy (SEM) for better observing the the surface properties 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.

Optical microscope

In the preliminary experiments, we chose optical microscope to roughly evaluate the interaction between the SpyTag-MnP and PE film. As shown in Fig.1, after 10 days of incubation, several precipitated protein which were patchy and adhered to the PE surface, can be observed under the 400× field of view.

Fig. 1 Optical microscope observation of the SpyTag-MnP treated and untreated PE group after 10 days incubation. A,B: observation of untreated PE films after 10 days (A: 40×, B: 400×). C,D: observation of PE films treated by SpyTag-MnP after 10 days incubation (C: 40×, D: 400×).the red cycles highlight the attaching of SpyTag-MnP to the PE flim.

Thus, we can assume that the PE would probably be degraded after incubating for 10 days. And according to the positive results gotten from the preliminary experiments, we further taken the suggestions from Professor Sun Chaomin, applying more precise and persuasive detecting methods to certify the degradation of PE film.

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. 2). 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. 2 FTIR analysis of untreated PE films and PE films treated by SpyTag-MnP after 10 days incubation.

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.

SEM

We then monitored the surface structure changes of the two groups by Scanning Electron Microscopy (SEM). As shown in Fig. 3, obvious fragments appeared in SpyTag-MnP treated PE, while the untreated PE seemed to have no change.

Fig. 3 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.

The above results obviously indicate an interaction between SpyTag-MnP and the PE film: under the enzymatic oxidation, the surface smoothness of PE was significantly changed.

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 298046 Da to 249588 Da (Fig. 4 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. 4 A, B).

Fig. 4 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.

Conclusion

Combined four different detecting methods together, we determined that the SpyTag-MnP could degrade the PE film with the assistant of H₂O₂ , Mn²⁺, sodium malonate and Tween 80. Also, according to the optical microscope results, the attachment of the enzyme would promote the degradation process, thus if the whole system could be assembled , with the assistant of HFB1, which could promote the interaction between SpyTag-MnP and PE film, the degradation process would have greater possibility to be faster and more thoroughly.

 

Assemble of SpyCather/SpyTag

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 chose SpyTag-MnP and dCas9-SpyCatcher to conduct the following assembly experiments.

To certify the combination of SpyTag-MnP and dCas9-SpyCatcher, we applied the 8% SDS-PAGE to determine whether the complex can run out a new strip whose molecular weight equals to the sum of SpyTag-MnP and the dCas9-SpyCatcher.

To ensure the combination would not interrupt the oxidizing process, the enzyme activity of the complex were also measured and compared with the initial activity of SpyTag-MnP.

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. 5, the band of the complex appeared, which was higher than that of dCas9-SpyCather, and the original SpyTag-MnP band had disappeared.

Fig.5 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.

Impact on enzyme activity

Then we compared the difference in MnP activity between SpyTag-MnP and the complex, and, as shown in Fig. 6, there was no significant change. This result suggested that the assembly of SpyTag-MnP and dCas9-SpyCather will not affect the enzyme activity. Thus, the worry that complex would decrease the enzyme activity was eliminated.

Fig. 6 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.

 

The stability of dCas9-SpyCather/SpyTag-MnP complex

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. Also, this results can provide us crucial information about the environment in which the system might be restricted. 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:

• 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:

• 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:

Thermal stability of dCas9-SpyCather/SpyTag-MnP complex

As shown in Fig. 7, 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. 8).

Fig. 7 Thermal stability of dCas9-SpyCather/SpyTag-MnP complex. The initial MnP activity before incubation was set as 100%.

Fig. 8 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.

pH stability of dCas9-SpyCather/SpyTag-MnP complex

As shown in Fig. 9, it didn't perform well at low pH range (pH 3-5) as both the complex and SpyTag-MnP had precipitated (Fig. 9), 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. 9 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.

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. 10). 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. 10).

Fig. 10 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.

Conclusion

According to the stability of complex, we can draw the conclusion that the integration would not have negative impact on all the three kinds of stabilities, indicating a broad usage in the industrial world. Also, it's worth noting that the Ethanol stability of complex is higher than the initial enzyme. Thus, when encounter conditions with higher ethanol concentration, the complex could be better qualified for the degradation.

 

Assemblability of CRISPR/dCas9 anchor system

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).

In Fig.11, by comparing the protein(Fig.11A) and nucleic acid(Fig.11B) 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. 11 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.

Since this CRISPR/dCas9 has been well characterized by researchers, we haven't waste our time on verifying the combination between dsDNA and dCas9-sgRNA. And if there is limited impact of SpyCather on the function of dCas9, and the dCas9-SpyCatcher could successfully interact with sgRNA, it could be assumed that the combination between dsDNA and sgRNA-dCas9-SpyCatcher was propitious.

 

Semi-rational directed evolution

As shown in Fig.9, either the SpyTag-MnP or the complex displays unsatisfied stability at low pH degree. Thus, we had applied semi-rational directed evolution to improve the stability of wild type MnP.(The details of the construction of the mutant library can be seen in the Molecular Modeling, and the construction of each mutant are displayed in the Parts Overview )

Stability of each mutants

Thermal stability and pH stability of the 6 mutants were evaluated and displayed in Fig. 12 & 13. Most of them kept the same pace with wtMnP, but mutant 2^# possessed outstanding thermal stability and sustained the same level of pH stability compared to wtMnP.(detailed information of mutant 2^# can be found in the improvement part)

Fig.12 The thermal stability of mutants.

Fig.13 The pH stability of mutants.