iGEM 2021 — Team CPU_CHINA

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# Team:CPU CHINA/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:CPU CHINA/Engineering

![](https://static.igem.org/mediawiki/2021/c/c4/T--CPU_CHINA--loading.gif)

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* {{item}}

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# ENGINEERING SUCCESS

ENGINEERING SUCCESS

* [OVERVIEW](#section1 "OVERVIEW")
* [ENGINEERING SUCCESS](#section2 "ENGINEERING SUCCESS")
![](https://static.igem.org/mediawiki/2021/3/38/T--CPU_CHINA--flying.png)

## 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**](https://2021.igem.org/Team%3ACPU_CHINA/partsOverview)  and
 [**Design**](https://2021.igem.org/Team%3ACPU_CHINA/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](http://parts.igem.org/Part%3ABBa_K3853011), [BBa\_K3853010](http://parts.igem.org/Part%3ABBa_K3853010)** and **[BBa\_K3853008](http://parts.igem.org/Part%3ABBa_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](http://parts.igem.org/Part%3ABBa_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](https://static.igem.org/mediawiki/2021/b/b9/T--CPU_CHINA--dCas9-SpyCatcher_sequencing_results.zip)**) showed a successful transformation.

![](https://static.igem.org/mediawiki/2021/d/dd/T--CPU_CHINA--Engineering_success_fig_1.jpg)

**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.

![](https://static.igem.org/mediawiki/2021/5/5e/T--CPU_CHINA--Engineering_success_fig_2.png)

**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.

![](https://static.igem.org/mediawiki/2021/c/c6/T--CPU_CHINA--Engineering_success_fig_3.jpg)

**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.

![](https://static.igem.org/mediawiki/2021/7/75/T--CPU_CHINA--BBa_K3853008_fig_12.png)

**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](http://parts.igem.org/Part%3ABBa_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](https://static.igem.org/mediawiki/2021/8/8f/T--CPU_CHINA--SpyTag-HFB1_sequencing_results.zip)**) showed a successful transformation.

![](https://static.igem.org/mediawiki/2021/f/ff/T--CPU_CHINA--Engineering_success_fig_5.jpg)

**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.

![](https://static.igem.org/mediawiki/2021/f/f2/T--CPU_CHINA--Engineering_success_fig_6.png)

**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.

![](https://static.igem.org/mediawiki/2021/4/41/T--CPU_CHINA--Engineering_success_fig_7.jpg)

**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
.*

[

](https://static.igem.org/mediawiki/2021/c/c1/T--CPU_CHINA--Engineering_success_video_1.mp4)

**Video 1. Comparison of fluid movement status**

### 3. his-tag-SpyTag-MnP([BBa\_K3853008](http://parts.igem.org/Part%3ABBa_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](https://static.igem.org/mediawiki/2021/8/89/T--CPU_CHINA--MnP.zip)**) showed successful transformation.

![T--CPU_CHINA--BBa_K3853008_fig_2](https://static.igem.org/mediawiki/2021/0/0b/T--CPU_CHINA--BBa_K3853008_fig_2.jpg)

**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.

![T--CPU_CHINA--BBa_K3853008_fig_3](https://static.igem.org/mediawiki/2021/7/76/T--CPU_CHINA--BBa_K3853008_fig_3.png)

**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 CT values for the SpyTag-MnP and the reference gene *ACT1*
were then used to calculate the fold difference with the following equation:

![T--CPU_CHINA--BBa_K3853008_fig_A](https://static.igem.org/mediawiki/2021/9/91/T--CPU_CHINA--BBa_K3853008_fig_A.png)
![T--CPU_CHINA--BBa_K3853008_fig_4](https://static.igem.org/mediawiki/2021/4/44/T--CPU_CHINA--BBa_K3853008_fig_4.png)

**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)**.

![T--CPU_CHINA--BBa_K3853008_fig_5](https://static.igem.org/mediawiki/2021/5/53/T--CPU_CHINA--BBa_K3853008_fig_5.png)

**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.**

![T--CPU_CHINA--BBa_K3853008_fig_12](https://static.igem.org/mediawiki/2021/7/75/T--CPU_CHINA--BBa_K3853008_fig_12.png)

**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.

![T--CPU_CHINA--BBa_K3853008_fig_13](https://static.igem.org/mediawiki/2021/2/24/T--CPU_CHINA--BBa_K3853008_fig_13.png)

**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).

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# Team:CPU CHINA/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:CPU CHINA/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)