Team:CPU CHINA/Engineering

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ENGINEERING SUCCESS

ENGINEERING SUCCESS

• OVERVIEW
• ENGINEERING SUCCESS

OVERVIEW

In the project of CPU_CHINA 2021, We designed a multi-enzyme complex to degrade plastics and used synthetic biology tools to produce the expected results. 30 new BioBricks were designed for our project (See the Parts and Design details in Parts and Design.)

We have done a lot of amazing work to make sure they can realize the goals we set. Moreover, it is worth mentioning that BBa_K3853011, BBa_K3853010 and BBa_K3853008 are the representatives among them. These three BioBricks work well to perform their functions and occupations as expected. All the related data are recorded below. We hope they will make some contributions to the iGEM community.

ENGINEERING SUCCESS

1. dCas9-SpyCatcher (BBa_K3853011)

In our 2021 project, we fused deactivated CRISPR-associated protein 9 (dCas9) with SpyCatcher, turning it into a biological module that could fuse with various "standardized elements" link to SpyTag. T7 promoter was used to construct the express circuit, and the dCas9-SpyCatcher protein was expressed and purified to test its function.

1-1. Agarose Gel Electrophoresis

Method: We used PCR to obtain three homologous recombination fragments (dCas9; vector of pET-28a; G₃S*4-SpyCatcher) for the construction of expression plasmid. The successfully recombined plasmid was transformed into E.coli BL21(DE3), and the monoclonal colonies with positive transformation results were selected for subsequent sequencing verification.

Result: The three fragments used for homologous recombination were successfully obtained by PCR, and the result was shown in Fig. 1. The sequencing results of the E.coli BL21(DE3) monoclonal colonies (File 1) showed a successful transformation.

Fig. 1 Agarose gel electrophoresis of PCR products. Vector refers to plasmid pET-28a. G3S*4 refers to quadruple Ser/Gly link.

1-2. SDS-PAGE

Method: We used Ni-NTA affinity column to purify dCas9-SpyCatcher.

Result: Target bands could be observed at the position of about 176.2 kDa (Fig. 2), which means the protein of dCas9-SpyCatcher could be successfully expressed, and the related gene worked well.

Fig. 2 SDS-PAGE of purified products of dCas9-SpyCatcher. 50 mM imidazole and 500 mM imidazole represent corresponding eluates with different imidazole concentrations, and the binding buffer is generated by equilibrating the Ni-NTA affinity column after elution with 500 mM imidazole.

1-3. Western Blot

Method: Since we had introduced 6×his-tag at the N-terminus of dCas9-SpyCatcher, we used his-antibody as the primary antibody to perform Western Blot analysis to detect the expression status of the target protein.

Result: The protein band of dCas9-SpyCatcher could be observed clearly (Fig. 3), meaning that the related gene functioned well.

Fig. 3 Western Blot of dCas9-SpyCatcher. The concentration of dCas9-SpyCatcher: lane 1: 0.25 mg/ml; lane 2: 0.5 mg/ml; lane 3: 1 mg/ml.

1-4. SpyCatcher/SpyTag system combination

Method: We incubated SpyTag-MnP and dCas9-SpyCatcher together to verify the combination of SpyCatcher and SpyTag^[1]. SpyTag-MnP was mixed with dCas9-SpyCatcher in a ratio of 1 : 1, and they were allowed to conjugate for 1 h at 37℃. Followed by SDS-PAGE verification.

Result: As shown in Fig. 4. The band of the conjugated dCas9-MnP complex appeared at lane 3, with a slightly higher position than dCas9-SpyCatcher; Also, the original SpyTag-MnP band at lane 3 disappeared , meaning the two proteins had successfully conjugated.

Fig. 4 SDS-PAGE of the combination of SpyTag-MnP and dCas9-SpyCatcher. Lane 1: SpyTag-MnP; Lane 2: dCas9-SpyCatcher; Lane 3: SpyTag-MnP mixed with dCas9-SpyCatcher.

 

2. His-tag-SpyTag-HFB1 (BBa_K3853010)

Hydrophobin-1 (HFB1) is a kind of class Ⅱ HFBs derived from Trichoderma reesei, which can exert surface activity at the hydrophilic-hydrophobic interface. We introduced SpyTag at its N-terminus for the assembly of multi-enzyme complexes.

2-1. Agarose Gel Electrophoresis

Method: The plasmid carrying the gene of SpyTag-HFB1 was transformed into E.coli Rosetta(DE3) for heterogenous expression. Colony PCR was applied to screen monoclonal colonies that had positive transformation results for subsequent gene sequencing verification.

Result: The bands of target gene appeared at the normal position, the result was shown in Fig. 5. Sequencing verification results (File 2) showed a successful transformation.

Fig. 5 Agarose gel electrophoresis of PCR products of monoclonal colonies of SpyTag-HFB1.

2-2. SDS-PAGE

Method: We used Ni-NTA affinity column to obtain purified SpyTag-HFB1.

Result: Target bands could be observed at the position of about 15 kDa (Fig. 6), which means the protein of SpyTag-HFB1 was successfully expressed, and the related gene worked well.

Fig. 6 SDS-PAGE of purification products of dCas9-SpyCatcher. flow-through is the liquid flowing out of the column when loading the sample, 50 mM imidazole and 500 mM imidazation are eluates of different  imidazole concentrations.

2-3. Functional Verification

Method: We dropped the liquid containing SpyTag-HFB1 and the liquid without SpyTag-HFB1 on a plastic dish, and observed the contact angle, shape and motion state of different liquids to evaluate the effect of HFB1.

Result: The results were shown in Table 1, Fig. 7, Video 1. The contact angle of the liquid containing SpyTag-HFB1 was smaller, and the droplets were dispersed. Compared to SpyTag-HFB1-free liquid, the droplets containing SpyTag-HFB1 were not easy to move on the plastic surface, which showed that HFB1 effectively improved the hydrophilicity of the plastic surface.

Table 1. Contact angle measurement among different samples.

Sample	Unmodified (°)	HFB1-modified (°)
Polypropylene (PP)	106.823 ± 1.888	74.033 ± 1.195****
Polyethylene (PE)	72.436 ± 1.600	51.642 ± 3.417***

Significant difference analysis: ***P < 0.001, ****P < 0.0001.

Fig. 7 Contact angle of two types of  liquid.A: Comparison between buffer and HFB1-buffer mixture after shaking on a PP surface. B: Contact angle on a PP surface . C: a HFB1-modified PP surface. D: Contact angle on a PE surface. E: a HFB1-modified PE surface .

Video 1. Comparison of fluid movement status

3. his-tag-SpyTag-MnP(BBa_K3853008)

Manganese peroxidase (MnP) is a highly glycosylated, heme-containing^[3] lignin peroxidase produced by the white-rot fungus Phanerochaete chrysosporium. Introduce SpyTag at its N-terminus for the assembly of multi-enzyme complexes.

3-1. Agarose Gel Electrophoresis

Method:  The synthetic pPIC9K plasmid was linearized and electrotransformed into Pichia pastoris strain GS115, and the monoclonal colonies were screened for colony PCR and sequencing verification.

Result: The target gene appears in the normal position, and the result is shown in Fig. 8. Sequencing results (File 3) showed successful transformation.

Fig. 8 Gel electrophoresis result of colony PCR to detect the insertion of BBa_K3853008 into Pichia pastoris strain GS115. Control refers to the wild-type Pichia pastoris strain GS115 without electrotransformation.

3-2. SDS-PAGE

Method: We set a series of concentration gradients of ammonium sulfate solution to determine the best salting-out concentration to roughly remove the impurity and our target bands were observed through SDS-PAGE.

Result: Target bands can be seen at SDS-PAGE, which means SpyTag-MnP gene is expressed successfully.

Fig. 9 SDS-PAGE analysis of SpyTag-MnP after ammonium sulfide salting out. Lane 1: SpyTag-MnP salting out with 40% (NH₄)₂SO₄ ; Lane 2: SpyTag-MnP salting out with 50% (NH₄)₂SO₄ ; Lane 3: SpyTag-MnP salting out with 60% (NH₄)₂SO₄ ; Lane 4: SpyTag-MnP salting out with 70% (NH₄)₂SO₄ . Control refers to the supernatant of wild-type Pichia pastoris strain GS115 without plasmid transfer. We finally chose 60% ammonium sulfate as our salting out concentration.

3-3. qRT-PCR

Method: For assaying the mRNA expression of SpyTag-MnP, qRT-PCRs were performed. We did data analysis using a variation of the Livak method. To determine the relative expression of SpyTag-MnP vs. reference gene ACT1, total RNA was prepared from an equal volume of yeast solution.

Result: SpyTag-MnP reached its peak to a fold difference of 0.21 after 2% methanol inducing for 72 h. The C_T values for the SpyTag-MnP and the reference gene ACT1 were then used to calculate the fold difference with the following equation:

Fig. 10 qPCR results of SpyTag-MnP using the relative quantitative method.

3-4. Enzyme Activity

Method: MnP activity of SpyTag-MnP was measured by monitoring the oxidation of 2,6-dimethyloxyphenol (2,6-DMP) at 469 nm^[5]. The reaction mixtures contained 0.4 mM MnSO₄, 50 mM sodium malonate (pH 4.5), and 1 mM 2, 6-DMP. For a 96-well plate, 140 μl of the above reaction mixtures and 20 μl enzyme solution were mixed uniformly in advance and then 40 μl 0.1 mM H₂O₂ were added to initiate reaction. The concentration of 2, 6-DMP's oxidation products, 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone, were determined using ε469 = 49.6 mM-1 cm-1. One unit (U) of MnP activity is defined as the amount of enzyme required to convert 1 μM 2, 6-DMP to 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone in 1 minute.

Result: As shown in Fig. 11A, the absorbance of the reaction system with SpyTag-MnP continued to rise within 1 min, while the absorbance of the control group (without enzyme) did not change. Through UV-visible spectrum of the reaction system after 1 min, the characteristic absorption at 469 nm was observed (Fig. 11B). Besides, SpyTag-MnP showed the same characteristic as MnP and the enzyme activity of the former exhibited a slightly higher profile than the latter, which means that SpyTag-MnP retained the functions of the original MnP and had the potential to surpass the latter (Fig. 11).

Fig. 11 The detection of 2, 2', 6, 6'-tetramethoxydibenzo-1, 1'-diquinone. Control group refers to the reaction system without enzyme. A: The absorbance change at 469 nm in the reaction system within 1 min. B: UV-visible spectrum of the reaction system after 1 min.

3-5. the conjugation of SpyTag-MnP to dCas9-SpyCatcher

Method: 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℃^[6].

Result: As shown in Fig. 12, the band of the complex appeared, which was higher than that of dCas9-SpyCather and the original SpyTag-MnP band had disappeared. Then we compared the difference in MnP activity between SpyTag-MnP and the complex, and, as shown in Fig. 13, there was no significant change. This result suggested that the assembly of SpyTag-MnP and dCas9-SpyCather will not affect the enzyme activity.

Fig. 12 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.

Fig. 13 Comparison of MnP activity between SpyTag-MnP and dCas9-SpyCather/SpyTag-MnP complex. Complex refers to the dCas9-SpyCather/SpyTag-MnP complex. p > 0.05.

References

[1] Reddington, S. C. & Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Current opinion in chemical biology 29, 94-99, doi:10.1016/j.cbpa.2015.10.002 (2015).

[2] Jankowski, N., Koschorreck, K. & Urlacher, V. B. High-level expression of aryl-alcohol oxidase 2 from Pleurotus eryngii in Pichia pastoris for production of fragrances and bioactive precursors. Applied microbiology and biotechnology 104, 9205-9218, doi:10.1007/s00253-020-10878-4 (2020)

[3] Martínez, A. T. et al. Oxidoreductases on their way to industrial biotransformations. Biotechnol Adv 35, 815-831, doi:10.1016/j.biotechadv.2017.06.003 (2017).

[4] Sáez-Jiménez, V. et al. Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY. J Biol Chem 290, 23201-23213, doi:10.1074/jbc.M115.665919 (2015).

[5] Wariishi, H., Valli, K. & Gold, M. H. Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators. The Journal of biological chemistry 267, 23688-23695 (1992).

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