iGEM 2021 — Team BJU_China

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# Team:BJU China/Notebook

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![notebook](img/lazyload-ph.png)

**Our Lab Notebook**

Below is our lab notebook:

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# Team:BJU China/Engineering

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**Engineering**

**Overall Objective:**

In our project, we hope to convert tryptophan to halogenated tryptophan through halogenase, and then through TnaA proteolysis and MaFMO protein oxidation to become halogenated indigo, and finally synthesize a variety of indigo derivatives (Figure 1).

![](img/lazyload-ph.png)

Figure 1. Denovo synthesis indigo derivatives from tryptophan

**Our Process:**

In our engineering success section, we obtained the three proteins SttH, RedH, and PyrH through molecular cloning, and fused them with the Fre protein through L3 linker, thereby increasing the solubility of the three halogenated proteins in water and promoting the production of indigo derivatives. The figure below shows the relevant gene circuit diagram we constructed.

![](img/lazyload-ph.png)

Figure 2. Gene circuit diagram of Halogenase

We constructed halogenase plasmids using Gibson Assembly and verified by colony PCR and gene sequencing . According to the results in Figure 3, we can roughly identify the three genes (Frr-L3-SttH, Fre-L3-PyrH, Fre-L3-RebH) comparing with DNA Marker. And then the PCR product fragments were sent for sequencing and the results were also consistent with our target fragments.

![](img/lazyload-ph.png)

![](img/lazyload-ph.png)

![](img/lazyload-ph.png)

Figure 3. Molecular identification of Halogenase genes

Next, we started the protein level experiment to test gene expression. For better expression of enzymes, we optimize the induction condition of gene expression: three different temperatures (18℃, 30℃, 37℃) and inducer concentrations (0 mM, 0.1mM, 1mM) were set for test. Additionally, since the halogenases are fusion enzymes with higher solubility, we disrupted collected cells and tested both pellet and supernatant after induction (Figure 4-6).

From the results of three SDS-PAGE electrophoresis gels, it can be seen that compared with the control group induced by 0mM IPTG, there are obvious changes in the lanes induced by IPTG. Obvious bands can be seen in the precipitated protein gels of SttH, RebH and PyrH, which proves these halogenase can be effectively expressed under the induction of IPTG.

![](img/lazyload-ph.png)

![](img/lazyload-ph.png)

Figure 4. SDS-PAGE Gel of Fre-L3-SttH (Left is precipitation, Right is supernatant)

![](img/lazyload-ph.png)

![](img/lazyload-ph.png)

Figure 5. SDS-PAGE Gel of Fre-L3-PyrH (Left is precipitation, Right is supernatant)

![](img/lazyload-ph.png)

Figure 6. SDS-PAGE Gel of Fre-L3-RebH (Left is supernatant, Right is precipitation)

Through the expression of halogenase at different temperatures under the same IPTG concentration, we can get the following conclusions:

(1) There is a significant decrease in RebH when induced at 18℃ compared with the expression levels at 30℃ and 37℃.

(2) While PyrH protein expressed better and has obvious bands at 18℃ compared to the expression at 30℃ and 37℃, so we can conclude that 18°C is the most suitable induction temperature for PyrH.

(3) SttH induced by IPTG could express well at three different temperatures, and as the temperature increases, the expression level also gradually increases. There is no significant difference in the expression level of SttH at 37°C with 0.1, 1, and 5 mM IPTG, and the amount of protein is significantly more compared to the other two temperatures, so we can speculate that the optimal temperature for the expression of SttH is 37°C.

From the SDS-PAGE results of the three proteins, it is not difficult to see that the content of halogenase expressed in the supernatant solution is low. This may be due to low solublity of halogenases, even through Fre-L3 protein connection, the solubility is still not effectively improved, most enzymes are still exsit in the precipitation. At the same time, it may also be related to our parameter settings in the cell disruption. Because of the insufficient ultrasonication strength, the protein is wrapped in cytoplasm then forms precipitate.

Subsequently, we performed the protein expression induction experiment again with optimized induction conditions and ultrasonication strength, and better results were obtained (Figure 7).

![](img/lazyload-ph.png)

Figure 7. SDS-PAGE Gel of three halogenases (Left is supernatant, Right is precipitation)

Finally, we did a fermentation experiment with *E. coli* BL21 ΔTnaA containing Fre-L3-SttH/PyrH/RebH and the results were shown in Figure 8, from which we can see different indigo derivatives as expected.

![](img/lazyload-ph.png)

Figure 8. Fermentation results of three halogenases

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#### Contact Us

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#### Schools

Tsinghua International School

Beijing No. 80 High School

Dulwich College Beijing

Beijing Royal School

RDFZ

Beijing World Youth Academy

Keystone Academy

Culver Academy

Western Reserve Academy

International School Manila

#### Support

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# Team:BJU 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)

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# Team:BJU China/Results

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**Results**

**1.Molecular Analysis**

To begin our experiment of producing 6BrIG from tryptophan in the proposed pathway, we have to first remove the TnaA already present in the genome of *E.coli* using CRISPR-Cas9 system. This action is carried out to prevent the endogenous TnaA expression and thus the reduction of 6BrIG production. However, due to a faulty design in the gRNA, the off-target effect disabled the ability of the Cas9 to remove TnaA. Through this process, the sgRNA is bounded to the targeted gene and lastly knock it out. After the redesignation of the gRNA, the TnaA was successfully removed and inserted onto the MaFMO plasmid. We then did a PCR of the plasmid and the results of the agarose gel electrophoresis (AGE) showed that the gene is completely removed (The length of the plasmid before removal is 2514bp, while the length of tnaA is 1334bp. Therefore after removal the length of the plasmid should be 1180bp, which is consistent with the results shown in the picture). The professional sequencing process yielded the same result.

![placeholder image](img/lazyload-ph.png)

Figure 1. Mutation result of TnaA

While doing the mutation experiment, we also underwent cloning and protein expression of our target genes.
 By using Gibson cloning, we infused Fre-L3-SttH, Fre-L3-PyrH, Fre-L3-RebH into pACYC plasmid. With IPTG induction, we analyze the protein expression of halogenase by SDS-PAGE method.

![placeholder image](img/lazyload-ph.png)

Figure 2. (a) SDS-PAGE of precipitated Stth protein. (b)SDS-PAGE of supernatant Stth protein. (c) SDS-PAGE electrophoresis of supernatant PyrH.(d) SDS-PAGE of precipitated PyrH. (e)SDS-PAGE of supernatant and precipitate of RebH protein. (f) SDS-PAGE of TnaA and MaFMO protein.

By changing the induct IPTG concentration and the induction temperature, we get the best induce condition for each halogenase. The best induce condition for SttH is 1mM 37℃(Figure 1a and Figure 1b), PyrH is 0.1mM 18℃(Figure 1c and Figure 1d), RebH is 0.1mM 37℃(Figure 1e) as shown by the arrow .
Additionally, we combined TnaA and MaFMO genes together and fused in pET28b plasmid. From Figure 1f, the induction of enzyme TnaA and MaFMO has no significant difference between IPTG concentration and temperature, so we decided to use 0.1mM IPTG and 18℃ to induce the protein for further fermentation analysis.

**2.Fermentation Analysis**

According to protocol 6 guidance on producing 6BrIG, we induced ΔTnaA E.coli with MaFMO gene transfered. We then centrifuged the induced bacteria and suspended with 0.6%(ω/V) glucose solution and added 6Br-indole to the final concentration of 1mM, 30℃ fermentation for 6h.

![placeholder image](img/lazyload-ph.png)

Figure 3. Solution after the reaction has a distinct purple color compared to the unreacted *E. coli* solution.

After 6h fermentation, we got the solution. Compared with the original solution(Figure 2. right EP tube), the final product has component with purple. In order to get a specific analysis of the final product, we extracted the component and sent to university to do MALDI-TOF spectra analysis.

![placeholder image](https://static.igem.org/mediawiki/2021/f/f0/T--BJU_China--result_fig5.jpeg)

Figure 4. The Extraction of fermented solution

We also produced 6BrIG from tryptophan as substrates. From the figure5 result, team 1 and 2 are the production result of 6BrIG comparing with control group. With the addition of Fre-L3-SttH reaction products, the final solution turns to purple.

![placeholder image](https://static.igem.org/mediawiki/2021/f/f0/T--BJU_China--result_fig5.jpeg)

Fig5. The fermentation of 6BrIG.

**3. Sensitized Solar Cell Analysis**

![placeholder image](img/lazyload-ph.png)

Figure 6. Fluorescence intensity of 6BrIG with the results of excitation photometry experiments

According to the measurement of 6BrIG fluorescence strength, the result is conformed with the statement of research report. We then applied hardware to measure its function. Here is the construction of the electrical circuit of sensitized solar cells with the combination of thermometers:

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And here is the construction of the electrical circuit of sensitized solar cells with the combination of ring:

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#### Contact Us

Our BiliBili channel

Our Zhihu

#### Schools

Tsinghua International School

Beijing No. 80 High School

Dulwich College Beijing

Beijing Royal School

RDFZ

Beijing World Youth Academy

Keystone Academy

Culver Academy

Western Reserve Academy

International School Manila

#### Support

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