iGEM 2020 — Team AHUT-ZJU-China

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# Team:AHUT-ZJU-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)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:AHUT-ZJU-China/Engineering

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# AHUT-ZJU-China

* [HOME](https://2020.igem.org/Team%3AAHUT-ZJU-China)
* TEAM
* PROJECT
* EXPERIMENTS
* PARTS
* HP
* [MODEL](https://2020.igem.org/Team%3AAHUT-ZJU-China/Model)
* [JUDGING FORM](https://igem.org/2020_Judging_Form?id=3656)

* [TEAM MEMBERS](https://2020.igem.org/Team%3AAHUT-ZJU-China/Team)
* [ATTRIBUTION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Attributions)
* [COLLABORATION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Collaborations)
* [PARTNERSHIP](https://2020.igem.org/Team%3AAHUT-ZJU-China/Partnership)

* [DESCRIPTION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Description)
* [DESIGN](https://2020.igem.org/Team%3AAHUT-ZJU-China/Design)
* [CONTRIBUTION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Contribution)
* [PROPOSED IMPLEMENTATION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Implementation)
* [PROOF OF CONCEPT](https://2020.igem.org/Team%3AAHUT-ZJU-China/Proof_Of_Concept)
* [EXCELLENCE IN ANOTHER AREA](https://2020.igem.org/Team%3AAHUT-ZJU-China/Excellence_in_another_Area)
* [ENGINEERING](https://2020.igem.org/Team%3AAHUT-ZJU-China/Engineering)

* [OVERVIEW](https://2020.igem.org/Team%3AAHUT-ZJU-China/Parts)
* [NEW PARTS](https://2020.igem.org/Team%3AAHUT-ZJU-China/New_parts)
* [IMPROVED PARTS](https://2020.igem.org/Team%3AAHUT-ZJU-China/Improved_parts)
* [CHARACTERIZATION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Characterization)

* [HP FOR SILVER](https://2020.igem.org/Team%3AAHUT-ZJU-China/Human_Practices)
* [INTEGRATED HP FOR GOLD](https://2020.igem.org/Team%3AAHUT-ZJU-China/Human_Practices)
* [EDUCATION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Education)

* [PROTOCOL](https://2020.igem.org/Team%3AAHUT-ZJU-China/Protocol)
* [RESULTS](https://2020.igem.org/Team%3AAHUT-ZJU-China/Results)
* [SAFETY](https://2020.igem.org/Team%3AAHUT-ZJU-China/Safety)

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# Engineering Success

##

READING TIME: 63mins

# Research

AHUT-ZJU-China team is committed to the study of CO2 capture. By consulting a large number of literature and analyzing its unique carbonic anhydrase from different organisms , we finally selected OT3-CA-WT as our project after comparative analysis. We found that OT3-CA-WT has good thermal stability and can maintain good catalytic activity even at higher temperatures, but its catalytic activity is very different from that of human beings.![img](https://static.igem.org/mediawiki/2020/0/0e/T--AHUT-ZJU-China--hsadkuashd.png)

Fig. 1 Three-dimensional structure of OT3-CA-WT protein

# Design

Therefore, we decided to use the method of synthetic biology to mutate OT3-CA-WT so that it can improve its catalytic activity on the basis of its original thermal stability.

![img](https://static.igem.org/mediawiki/2020/9/9e/T--AHUT-ZJU-China--hs21312d.png)

Fig. 2　 OT3-CA-WT’s active site

# Construction

We used Escherichia coli as engineering bacteria, constructed the vector with pET28 a(+), and successfully expressed OT3-CA-WT-His with high efficiency. At the same time, we also constructed the mutant vector and predicted its expression effect.

# Test

We used PyMOL, ADT and other software, and analyzed the original OT3-CA-WT amino acid sequence and three-dimensional structure. On this basis, we predicted the mutation site, and successfully simulated the change of its catalytic activity after a series of tests.

# Study

All of us have learned a lot through this project. First of all, we learned how to use some unique biological software. These software can help us to make reasonable inference and prediction before the formal experiment, reduce meaningless attempts, and save the time and cost of the experiment. At the same time, through the experiment, our understanding of synthetic biology has risen from theory to practice, and with the combination of theory and practice, we have deepened our understanding of synthetic biology.

Throughout the course of the project, all members are no longer confused about the concept of Greenhouse Effect. We realized that besides using the traditional physical and chemical methods to solve problems, it has advantages to use synthetic biology to transform organisms to achieve our goals. In the near future, we human beings will give full play to our imagination and use synthetic biology to solve more thorny problems.

# Improvement

We mutated six amino acids of OT3-CA-WT on the basis of the original: 122: P→K (the 122nd proline is mutated to lysine), position 123: P→K (the 123rd proline is mutated to Lysine), position 128: P→K (mutation of the 128th proline to lysine), position 132: L→K (mutation of 132nd leucine to lysine), position 134: L→K ( Leucine at position 134 was mutated to lysine), and position 137: P→K (leucine at position 137 was mutated to lysine).

After software simulation and prediction, we found that the OT3-CA-MU activity of the mutation was improved, which was consistent with our expected goal.

![img](https://static.igem.org/mediawiki/2020/d/dd/T--AHUT-ZJU-China--21312.png)

Fig. 3 Structure of OT3-CA-WT and OT3-CA-MU

# Research

Looking at the overall situation and through summary and reflection and re-review of the data, our project has been successful in terms of simulation and prediction this year. Due to the COVID-19 's pandemic situation, our laboratory was not open before, we cooperated with Zhejiang University in our experiment project.We also carried out a large number of simulated mutations on the computer using software such as PyMOL, ADT, analyzing the amino acid sequence and structure of the OT3-CA-WT, we predicted the simulated mutation sites, and finally got our results after a series of tests. The overall energy of the OT3-CA-MU was higher than that of the previous wild type, and the original binding site and Zn2+ catalytic site were not destroyed and could still achieve the original function. Therefore, our simulation prediction is successful from an engineering point of view. We have high hopes for the future. Even if the competition is over, we will continue to experiment and improve the simulation results. We firmly believe that eventually it can become a product that can really capture a large number of CO2, benefitting the mankind and the whole planet.

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Table of Content

Copyright 2020 © AHUT-ZJU-China All rights reserved.

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# Team:AHUT-ZJU-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

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:AHUT-ZJU-China/Results

![](https://static.igem.org/mediawiki/2020/7/74/T--AHUT-ZJU-China--logo%402x.png)

# AHUT-ZJU-China

* [HOME](https://2020.igem.org/Team%3AAHUT-ZJU-China)
* TEAM
* PROJECT
* EXPERIMENTS
* PARTS
* HP
* [MODEL](https://2020.igem.org/Team%3AAHUT-ZJU-China/Model)
* [JUDGING FORM](https://igem.org/2020_Judging_Form?id=3656)

* [TEAM MEMBERS](https://2020.igem.org/Team%3AAHUT-ZJU-China/Team)
* [ATTRIBUTION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Attributions)
* [COLLABORATION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Collaborations)
* [PARTNERSHIP](https://2020.igem.org/Team%3AAHUT-ZJU-China/Partnership)

* [DESCRIPTION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Description)
* [DESIGN](https://2020.igem.org/Team%3AAHUT-ZJU-China/Design)
* [CONTRIBUTION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Contribution)
* [PROPOSED IMPLEMENTATION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Implementation)
* [PROOF OF CONCEPT](https://2020.igem.org/Team%3AAHUT-ZJU-China/Proof_Of_Concept)
* [EXCELLENCE IN ANOTHER AREA](https://2020.igem.org/Team%3AAHUT-ZJU-China/Excellence_in_another_Area)
* [ENGINEERING](https://2020.igem.org/Team%3AAHUT-ZJU-China/Engineering)

* [OVERVIEW](https://2020.igem.org/Team%3AAHUT-ZJU-China/Parts)
* [NEW PARTS](https://2020.igem.org/Team%3AAHUT-ZJU-China/New_parts)
* [IMPROVED PARTS](https://2020.igem.org/Team%3AAHUT-ZJU-China/Improved_parts)
* [CHARACTERIZATION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Characterization)

* [HP FOR SILVER](https://2020.igem.org/Team%3AAHUT-ZJU-China/Human_Practices)
* [INTEGRATED HP FOR GOLD](https://2020.igem.org/Team%3AAHUT-ZJU-China/Human_Practices)
* [EDUCATION](https://2020.igem.org/Team%3AAHUT-ZJU-China/Education)

* [PROTOCOL](https://2020.igem.org/Team%3AAHUT-ZJU-China/Protocol)
* [RESULTS](https://2020.igem.org/Team%3AAHUT-ZJU-China/Results)
* [SAFETY](https://2020.igem.org/Team%3AAHUT-ZJU-China/Safety)

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# Result

READING TIME: 63mins

As we have explained in the background, traditional CO2 capture technologies are still in their infancy,and these capture technologies tend to have high energy consumption but low efficiency. Therefore, our project goal is to develop a new type of low-energy, environmentally friendly, and efficient carbon dioxide capture method by using synthetic biology methods. Based on this goal, we intend to use Pyrococcus horikoshii OT3 carbonic anhydrase (OT3-CA-WT) as the research object because this carbonic anhydrase (CA) can catalyze the hydration of CO2 to HCO3- based on high thermal stability. (Fig. 1). This year, based on the previous two years of projects, we obtained engineered bacteria expressing OT3-CA-WT through genetic engineering technology and then purified OT3-CA-WT protein to test its thermal stability. Simultaneously, to further improve the catalytic activity of OT3-CA-WT, we used molecular simulation technology to do molecular docking of OT3-CA-WT, hoping to improve its catalytic activity while maintaining its original thermal stability, and finally obtained efficient thermally stable carbonic anhydrase, laying the foundation for subsequent industrial applications.

![img](https://static.igem.org/mediawiki/2020/d/d2/T--AHUT-ZJU-China--juzehao1.jpg)

Fig. 1 The catalytic mechanism of carbonic anhydrase

# Engineered E. coli TB1 (DE3) with high OT3-CA-WT expression

## Construction of OT3-CA-WT expression plasmids

The coding sequences of OT3-CA-WT ([BBa\_K3656304](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3656304)) were both synthesized , then cloned into the expression vector pET-28a(+) (Fig.2). The length of digested OT3-CA-WT was 546 bp, confirmed by the band above 500 bp in electrophoresis as indicated in Fig. 3, which indicated that the pET-28a(+) OT3-CA-WT recombinant plasmid was successfully constructed.

![](https://static.igem.org/mediawiki/2020/f/f1/T--AHUT-ZJU-China--juzehao2.jpg)

Fig. 2 Map of OT3-CA-WT recombinant plasmid

![](https://static.igem.org/mediawiki/2020/a/a0/T--AHUT-ZJU-China--jzh3.jpg)

Fig. 3 Enzyme digestion of OT3-CA-WT recombinant plasmid.

Lane M: DL marker; Lane 1: OT3-CA-WT recombinant plasmid; Lane 2: the enzyme digested OT3-CA-WT recombinant plasmid, the length was 546 bp (the arrow indicated).

## Induced expression of OT3-CA-WT protein

The OT3-CA-WT expression plasmid was transformed into E. coli TB1, and positive clones were screened for kanamycin resistance. The recombinant bacteria were expanded and cultured, and isopropyl-1-thio-β-Dgalactopyrasonide (IPTG) was used to induce the expression of OT3-CA-WT in E. coli TB1. Then the bacteria were lysed to extract the total protein, and SDS-PAGE and Western Blot were applied to verify the expression of OT3-CA-WT. As shown in Fig. 4 and Fig. 5, it showed that after induction by IPTG, a band about 19.5 KDa with a molecular weight similar to CA was appeared, as indicated by the arrow. This indicates that we have successfully constructed an engineered strain of E. coli TB1 expressing OT3-CA-WT.

![](https://static.igem.org/mediawiki/2020/0/04/T--AHUT-ZJU-China--jzh4.png)

Fig. 4 Induced expression of OT3-CA-WT in TB1 strain verified by SDS-PAGE

![](https://static.igem.org/mediawiki/2020/0/00/T--AHUT-ZJU-China--jzh5.png)

Fig. 5 Induced expression of OT3-CA-WT in TB1 strain verified by Western blot

## OT3-CA-WT purification

After confirming that OT3-CA-WT can be expressed in E. coli TB1, due to the nickel chloride in the nickel column can bind to the protein with His (histidine) tag, we used nickel column to further purify the OT3-CA-WT,then we used the Bradford method to test the concentration of the purified OT3-CA-WT protein. The analysis showed that the purified protein concentration of OT3-CA-WT was 7.7 mg·mL-1(Fig. 6).

![](https://static.igem.org/mediawiki/2020/8/81/T--AHUT-ZJU-China--jzh6.png)

Fig.6 Evaluation of OT3-CA-WT protein concentration via Bradford method

# Enzyme activity assay of OT3-CA-WT

We measured the enzymatic activity of OT3-CA-WT protein at 37°C, 45°C and 70°C by esterase activity assay.As shown in Fig. 7, the result showed that the enzyme activity at 45°C is almost or slightly higher than that at 37°C. Although the enzyme activity of OT3-CA-WT at a higher temperature of 70°C slightly decreases, it still has a higher activity. This shows that we have obtained carbonic anhydrase with specific high-temperature resistance and
good thermal stability, which is also in line with our previous assumptions.

![img](https://static.igem.org/mediawiki/2020/8/87/T--AHUT-ZJU-China--jzh7.png)

Fig. 7 Esterase activity analysis of OT3-CA-WT protein at different temperatures

# Obtain of mutant OT3-CA with enhanced catalytic activity via molecular simulation

The obtained OT3-CA-WT protein has high thermal stability, but its catalytic activity is not as good as human carbonic anhydrase. Therefore, we decided to mutate the original OT3-CA-WT by the molecular simulation toimprove its catalytic activity.

###### 3.1

Through searching a large number of references about carbonic anhydrase, especially literature about OT3-CA-WT (Fig.8) and human carbonic anhydrase 2. We found that lysine at some sites can significantly enhance the activity of OT3-CA-WT, while proline and leucine may reduce the activity of OT3-CA-WT. Therefore, we designed the gene sequence of the mutant OT3-CA-MU via molecular simulation. The specific process is as follows:

1) Maintain the 3D structure of enzyme;

2) Modify the interactions between residues around active sites;

3) Improve the rigidity of active sites;

4) Shorten the distance of proton transfer.

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|  | ![img](https://static.igem.org/mediawiki/2020/e/e4/T--AHUT-ZJU-China--jzh8.png) |

Fig. 8 Pyrococcus horikoshii carbonic anhydrase (OT3-CA-WT)

###### 3.2 Perform enzyme-substrate molecular docking.

Using the software Autodock for molecular docking, the docking conformation of the substrate at the catalytic site was studied, and the interaction between the residues at the catalytic site and the substrate was analyzed. We used Autodock and PyMOL to further study the effect of the catalytic site's secondary and tertiary structure on the catalytic process. After simulation, we set the mutation site and substitution residue, carry out molecular docking of the recombinase, and then compare the enzyme-substrate docking conformation before and after the recombination. Finally, we selected suitable mutation sites and substitution residues to simulate, which improved its catalytic performance in theory.

###### 3.3

Based on the above simulation analysis, we finally determined that the appropriate mutation sites are: position 122: P→K (the 122nd proline is mutated to lysine), position 123: P→K (the 123rd proline is mutated to Lysine), position 128: P→K (mutation of the 128th proline to lysine), position 132: L→K (mutation of 132nd leucine to lysine), position 134: L→K ( Leucine at position 134 was mutated to lysine), and position 137: P→K (leucine at position 137 was mutated to lysine), and finally a mutant OT3-CA (OT3-CA-MU, [BBa\_K3656307](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3656307), Fig.9 and 10) to enhance its catalytic activity under high-temperature conditions was obtained (Table 1).

![](https://static.igem.org/mediawiki/2020/6/66/T--AHUT-ZJU-China--jzh9.png)

Fig.9 and 10 Structure of Wild-type and Mutant OT3-CA

![](https://static.igem.org/mediawiki/2020/4/4d/T--AHUT-ZJU-China--jzh11.png)

Fig. 11 Map of OT3-CA-MU

Table 1 Molecular docking results of OT3-CA-WT and OT3-CA-MU

| Name | OT3-CA-WT | OT3-CA-MU |
| --- | --- | --- |
| Part Number | [BBa\_K3656305](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3656305) | [BBa\_K3656309](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3656309) |
| binding\_energy | -5.08 | -5.37 |
| ligand\_efficiency | -1.27 | -1.34 |
| inhib\_constant | 187.83 | 116.58 |
| inhib\_constant\_units | uM | uM |
| intermol\_energy | -5.68 | -5.96 |
| vdw\_hb\_desolv\_energy | -1.76 | -1.92 |
| electrostatic\_energy | -3.92 | -4.04 |
| total\_intermal | 0.05 | 0.02 |
| torsional\_energy | 0.6 | 0.6 |
| unbound\_energy | 0.05 | 0.02 |

# Outlook

In the future, based on the mutation results of molecular simulations, we will continue to express and purify the OT3-CA-MU protein and test the activity, to obtain an environmentally friendly CO2 capture device that will benefit humanity and the earth.

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Copyright 2020 © AHUT-ZJU-China All rights reserved.