Team:CPU CHINA/Engineering

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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_K3853009 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, 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 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, 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 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, 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 location 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. 1. Sequencing verification results (File 2) showed a successful transformation.

Fig. 5 Agarose gel electrophoresis of PCR products.

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, 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 hydrophobic angle, shape and flattening 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.

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

Video 1. Comparison of fluid movement status

3. His-tag-SpyTag-AAO (BBa_K3853009)

Aryl alcohol oxidase (AAO) is an enzyme containing flavin-adenine-dinucleotide (FAD). This enzyme was introduced as an H₂O₂-producing enzyme to assist MnP to function. Meanwhile, SpyTag was also introduced at its N-terminus for the assembly of multi-enzyme complexes.

3-1. Agarose Gel Electrophoresis

Method: The synthetic plasmid was linearized and electrotransformed into Pichia pastoris GS115. Colony PCR was applied to screen monoclonal colonies that had positive transformation results for subsequent gene sequencing verification.

Result: The target bands appeared in the normal position, and the result was shown in Fig. 1. Sequencing results (File 3) showed a successful transformation.

Fig. 8 Agarose gel electrophoresis of PCR products.

2-2. qRT-PCR

Method: For assaying the mRNA expression level of AAO, qRT-PCRs were performed. After methanol induction for 48h and 60h, samples containing AAO-expressing strain were taken.

We did data analysis using a variation of the Livak method. To determine the relative expression level of SpyTag-AAO vs. reference gene ACT1, total RNA was extracted from samples containing equal wet weight of recombinant P.pastoris. The C_T values for SpyTag-AAO and the reference gene ACT1 were then used to calculate the fold difference:

Result: The results were shown in Table 2, Fig. 9. The gene of SpyTag-AAO was normally transcribed in the cell.

Table 2. The C_t value of AAO and ACT1

Inducing time	$\Delta C_t$	$C_{t(ACT1)}$
60	-0.80195351269127	0.5735719937658

Fig. 9 RFU change curve with cycle number

2-3. Enzyme Activity

Method: Enzyme activity of AAO was determined using veratryl alcohol as substrate, and to continuously detect the formation of the product veratraldehyde (ε₃₁₀ = 9300 M^-1·cm^-1) at 310 nm ^[2] through a 96-well microplate reader. The enzyme activity test should begin immediately after preparing the reaction system.

Result: Purified SpyTag-AAO had high degrees of activity outside the living organism, which proves that AAO works well.

Fig. 10 The detection of veratraldehyde.

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)