Team:AHUT-ZJU-China/Poster

Poster: AHUT-ZJU-China

Poster Title
Carbon dioxide (CO2) mainly from the combustion of fossil fuels is the primary greenhouse gas associated with global warming and climate change. Therefore, reducing the amount of CO2 emitted into atmosphere is an urgent need, so CO2 capture has become an extensively investigated area of research. In this project, we decided to use the biomimetic method of enzyme (carbonic anhydrase, CA) for capturing CO2, which has the advantages of safety, high catalytic efficiency and environment-friendly compared with other methods. We designed an efficient and stable CA by improving the catalytic performance of CA from the OT3 strain of the hyperthermophilic archeon Pyrococcus horikoshii for capturing CO2. We use computer-aided analysis software to predict the ideal mutation site of the OT3-CA for improving its catalytic activity. In conclusion, we constructed a new part OT3-CA-WT and obtained its protein with higher thermostability, which is able to maintain its activity at 45°C and 70°C. Furthermore, we obtained the mutant OT3-CA via molecular simulation with enhanced catalytic activity. The thermostability and efficiency of OT3-CA make it a potential candidate for CO2 capture.
Problem
The global climate has undergone drastic changes and the greenhouse effect has become increasingly severe. Carbon dioxide is the main greenhouse gas. Therefore, reducing the amount of carbon dioxide released into the atmosphere is extremely urgent. Our team hopes to use synthetic biology to produce a new type of carbon dioxide capture device to reduce carbon dioxide emissions.
Project
In this project, we decided to use the biomimetic method of the enzyme (carbonic anhydrase, CA) for capturing CO2, which has the advantages of safety, high catalytic efficiency and environment-friendly compared with other methods. We designed an efficient and stable CA by improving the catalytic performance of CA from the OT3 strain of the hyperthermophilic archeon Pyrococcus horikoshii for capturing CO2. We use computer-aided analysis software to predict the ideal mutation site of the OT3-CA for improving its catalytic activity. In conclusion, we constructed a new part OT3-CA-WT and obtained its protein with higher thermostability, which is able to maintain its activity at 45°C and 70°C. Furthermore, we obtained the mutant OT3-CA via molecular simulation with enhanced catalytic activity. The thermostability and efficiency of OT3-CA make it a potential candidate for CO2 capture.
Inspiration
Recently,with the rapid development of science and technology, environmental protectionhas gradually attracted all circles globally, among which the "greenhouseeffect" is one of the most prominent problems. Thegreenhouse effect is the way in which heat is trapped close to the surface ofthe earth by “greenhouse gases”. These heat-trapping gases can be thought of asa blanket wrapped around the earth, which keeps it warmer than it would bewithout them.
Carbondioxide is the most abundant greenhouse gas, accounting for about 0.03 of thetotal atmospheric capacity. When the Sun’s energy reaches the earth’satmosphere, some of it is reflected back to space and the rest is absorbed andre-radiated by greenhouse gases. Without greenhouse gases, the surfacetemperature would drop by about 33 ℃ or more. Conversely, if the greenhouseeffect continues to strengthen, the global temperature will continue to riseyear by year. Therefore,reducing CO2 emissions is a top priority. Our project aims at designing a more heat-resistant and environmentally friendly CO2 capture method based onthe previous two years.
Idea
This year, we chose OT3-CA-WT as the research object. This carbonican hydrase can catalyze the hydration of CO2 to form HCO3-on the basis of high thermal stability, which has good thermal stability.We obtained the engineered bacteria expressing OT3-CA-WT through genetic engineering technology, purified the OT3-CA-WT protein and tested its thermalstability. At the same time, in order to further improve its catalyticactivity, we used molecular simulation technology to simulate its mutationsites, hoping to improve its catalytic activity, and finally obtained efficient and thermally stable OT3-CA-MU, laying a foundation for subsequent industrial applications.

Methology

1.Transformation

(1) Add the TB1 receptive state into the plasmid

(2) Put 42℃ in 90s

(3) Place on the ice

(4) LB medium was added

(5) Shaking table culture

(6) Evenly spread the bacterial solution on the KANA resistant plate for culture at 37℃

 

2.Induced expression

(1) A monoclonal colony was selected from the plate and incubated in liquid LB medium containing KANA resistance

(2) The bacterial solution was added to the culture medium containing KANA resistance to expand the culture

(3) Add IPTG

(4) Centrifuge and collect the bacteria

(5) Add lysis buffer and centrifuge to get supernatant

(6) Open peristaltic pump and wash the nickel column with distilled water

(7) Wash them in PBS once more

(8) 5x imidazole balanced nickel column

(9) Slowly pump the sample into the nickel column

(10) 50x imidazole washing can remove the heteroprotein

(11) 250x imidazole eluted target protein

(12) Add the target protein into the concentration tube and centrifuge it for concentration

(13) Add desalination solution and centrifuge

(14) Sds-page gel Coomassie bright blue staining was prepared

 

3.Measuring esterase enzyme activity

1) Prepare 0.1 M phosphate buffer solution

2) Preparation of 50mL p-nitrophenyl acetate (P-NPA) at a concentration of 3 mM

3) 100 mL p-nitrophenol (P-NP) was prepared at a concentration of 1mM.

4) Different concentrations of p-nitrophenol were prepared, as shown in Table 1:

1 2 3 4 5
Serial number 0.05 mM 0.1 mM 0.15 mM 0.2 mM 0.25 mM
p-NP（1 mM） 50μL 100μL 150μL 200μL 250μL
Water 950μL 900μL 850μL 800μL 750μL

5) The absorbance values of each concentration were measured with a microplate microscope at a wavelength of 400 nm, and the standard curve was drawn;

6) Different concentrations of p-nitrophenyl acetate were prepared. As shown in Table 2:

Number 1 2 3 4 5
Concentration 0.5 mM 1 mM 1.5 mM 2 mM 2.5 mM
p-NPA（3mM） 1mL 2mL 3mL 4mL 5mL
Water 5mL 4mL 3mL 2mL 1mL

7) 50 L phosphate buffer, 100 L different concentrations of P-NPA and 50 L enzyme solution (1 g/ mL) were added to the plate, and the absorbance was measured at 384nm for 6min.

8) Compare the standard curve and calculate the conversion rate.

Design
Based on the projects in the previous two years, this year, we design to construct an engineered E. coli stain expressing carbonic anhydrase (CA) from hyperthermophilic bacteria, which retains its activity in higher temperature. In PDB database, there are five different CAs from thermophilic bacteria with PDB code showed as follows: 1THJ, 1V3W, 1V67, 2FKO, 1QQ0. Therefore, we used molecular simulation technology and found that 2FKO has highest activity, so we selected the 2FKO to carry out our project.

Firstly, we establish an engineered TB1 strain expressing CA (2FKO) from OT3 strain of the hyperthermophilic archeon Pyrococcus horikoshii (OT3-CA-WT), and test its thermostability. At the same time, in order to further improve the catalytic activity of OT3 - CA - WT, we use the molecular simulation technique to do molecular docking of OT3 - CA - WT, hoping to keep its original thermostability and further improve its catalytic activity. At last, we obtain an efficient and thermostable OT3-CA for CO2 capture, which lays a foundation for subsequent industrial applications.
Modelling

Our aim with the use of modeling can be broken down into three parts:

  1. Explain the mathematical principle that the concentration and the reaction rate change exponentially under the condition of single enzyme.
  1. Analyze the multiple factors that influenced the reversible reaction behaviors.
  1. Analyze the influence of different strains on the results.

To achieve the above goals, we try to categorize our modelling into three parts:

  1. Michaelis-Menten Kinetics for predicting and explaining the phenomenon of changes of reaction rate in the lab experiment.
  1. Develop ODE equation for explaining the process principle of the final balance between the concentration of the substrate and the speed of the reverse reaction during the reversible reaction.
  1. Data analysis for exploring how the strain changes the relationship between enzyme activity.

The anhydrase 2 and this year's carbonic anhydrase 1 have both been confirmed to conform to the Michaelis equation, so this year we will expand on the data that has been verified.

SimBiology can be used to simulate the concentration of CO2 and HCO3− when the reaction reaches equilibrium. Even if we accelerate the positive reaction rate of HCO3− to H2CO3, the data in the figure has little connection with H2CO3 molecules. The model can be simplified to a single reversible reaction in the next model.

Then we establish a simplified set of ode equations to relate the effects of temperature and PH values on the rate.

Result

New Part

1.Construction of OT3-CA-WT expression plasmid

New part : BBa_K3656304

Map of OT3-CA-WT recombinant vector

Enzyme digestion of OT3-CA-WT recombinant plasmid

2.Induced OT3-CA-WT protein expression in E. coli TB1

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

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

3.OT3-CA-WT purification

Evaluation of OT3-CA-WT protein concentration via Bradford method

4.Enzyme activity assay of OT3-CA-WT

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

Improved part

1.Molecular simulation of Carbonic anhydrase 2 (L203K)-P247K

Improved new part:BBa_K3656310

Structure of CA2 (L203K)-P247K

2.Function analysis of improved and original parts

Name CA2（L203K） CA2 (L203K)-P247K
Part Number Original part BBa_K2547004 Improved new partBBa_K3656310
binding_energy -4.17 -4.59
ligand_efficiency -1.04 -1.15
inhib_constant 875.8 435.27
inhib_constant_units uM uM
intermol_energy -4.77 -5.18
vdw_hb_desolv_energy -1.7 -1.95
electrostatic_energy -3.07 -3.24
total_intermal 0.03 0.05
torsional_energy 0.6 0.6
unbound_energy 0.03 0.05

Docking Analysis results of CA2 (L203K) and CA2 (L203K)-P247K by Auto Dock software

Add information

1.Add information to an existing Part:BBa_K2547000(Carbonic anhydrase 2)

SDS-PAGE analysis for CA2 cloned in pET-30a(+) and expressed in TB1 strain

The sequence of BBa_K2547000 was synthesized and cloned it into the pET-30a(+) expression plasmid to obtain the recombinant expression vector, and then we characterized that this part could be expressed in another strain TB1

Reference

[1]Christopher K Savile,James J Lalonde. Biotechnology for the acceleration of carbon dioxide capture and sequestration[J]. Current Opinion in Biotechnology,2011,22(6).

[2]Guoping Hu,Kathryn H. Smith,Nathan J. Nicholas,Joel Yong,Sandra E. Kentish,Geoffrey W. Stevens. Enzymatic carbon dioxide capture using a thermally stable carbonic anhydrase as a promoter in potassium carbonate solvents[J]. Chemical Engineering Journal,2017,307.

[3]Jeyaraman Jeyakanthan,Sarani Rangarajan,P. Mridula,Shankar Prasad Kanaujia,Yoshitsugu Shiro,Seiki Kuramitsu,Shigeyuki Yokoyama,Kanagaraj Sekar. Observation of a calcium-binding site in the γ-class carbonic anhydrase from Pyrococcus horikoshii[J]. Acta Crystallogr D Biol Crystallogr,2008,64(Pt 10).

[4]Yong J K J , Stevens G W , Caruso F , et al. The use of carbonic anhydrase to accelerate carbon dioxide capture processes[J]. Journal of Chemical Technology & Biotechnology, 2015, 90(1).

[5]Wu Shenglan,Chen Jinrui,Ma Liang,Zhang Kai,Wang Xiaoxiao,Wei Yuping,Xu Jian,Xu Xia. Design of carbonic anhydrase with improved thermostability for CO2 capture via molecular simulations[J]. Journal of CO2 Utilization,2019,38.

[6]Alexandra Giatromanolaki,Adrian L. Harris,Alison H. Banham,Constantinos A. Contrafouris,Michael I. Koukourakis. Carbonic anhydrase 9 (CA9) expression in non-small-cell lung cancer: correlation with regulatory FOXP3+T-cell tumour stroma infiltration[J]. British Journal of Cancer,2020,122(8).

[7]J R Chen. Thermal stability of carbonic anhydrase based on molecular simulation [D]. Anhui University of Technology,2019. (in Chinese)

[8]Chunxiu Li, Xiaochen Jiang, Yongjun Qiu, Jianhe Xu. The physiological function and diversity of carbonic anhydrase and its application in CO2 concentration [J]. Bioprocessing,2013,11(01):94-103.

[9]Zhaohui Zhang, Xiaozhou Ma. Cloning expression and enzymatic properties of a thermophilic carbonic anhydrase gene [J]. Industrial microbiology,2015,45(04):1-6.

[10]Y Y Huang, H X Chen, L M ZHAO. Study on simulated carbonic anhydrase activity of three zinc-containing metal complexes [J]. Journal of guangdong pharmaceutical university,2019,35(03):337-341.

[11]Lixi Cai, Yunmeng Chu, Guangya Zhang. Mining and modification of microbial carbonic anhydrase for carbon dioxide capture [J]. Chinese journal of bioengineering,2019,35(01):1-12.

Humanpractise

In this year's human practice, our team wants more people to rethink the importance of protecting the environment while thinking about the disasters brought to us by COVID-19. During the preparation of this year's competition, we completed many Human Practices, which all have distinct subjects, such as child education, expert interviews, business visits and so on.

·HP—Creative Art Course for Primary School Students

·We discussed with the AHUT's model airplane team

·Promote iGEM through couriers and takeout industry

·Two visits to Ma'anshan Iron and Steel Plant

·Hold a Maxiang-Road Promotion

At the same time, we have worked with ZJU-China Team this year, and we have given each other a lot of help, covering everything from competition preparation, project planning, to wiki style, experiment design and so on. We are very grateful for their help.

Acknowledgement

Our team consists of 8 instructors, including Xiangrong Xu, Hao Xu, Liang Ma, Zi Liu , Ruilan Cao, She Huili, Xu Xia, Xu Jianqin, Tingxuan Yan. Our PI is Xiangrong Xu and Secondary PI is Hao Xu & Liang Ma. We are so grateful to the instructors and advisors for their advice on our work. They provided us with laboratories and professional guidance. Prof.Xiangrong Xu provided some information of Human practice. Prof.Xu Xia, Prof.Xu Jian, Dr.Liang Ma and Dr.Zi Liu are responsible for the guidance of the wet lab. Our experiment mainly carried out in Dr.Liang Ma'slaboratory. At the same time, we carried out experiments with the help of some instruments in Biochemical Engineering Research Center, BERC-AHUT by Prof.Xu Xia and Prof.Xu Jian. Prof. Ruilan Cao gave us some suggestions on English translation and English presentation Dr.She Huili gave us some advice on art design. Dr.Hao Xu guides us the programming, mathematical modeling and Dry lab.



Special thanks:

ZJU-China team



Wiki:

Prof. Xiangrong Xu

Hao Xu

Pengcheng Tan

Feng Jiang

Zehao Ju

Jingxian Hu



Logo:

Prof. Xiangrong Xu

Huili She

Hao Xu

Jiang Yu

Yajie Deng

Presentation video:

Prof. Xiangrong Xu

Zi Liu

Tao Tao

Liang Ma

Jiajun Li

Qicheng Sun

Yuexin Xie

Yu Chan

Jinchong Yan



Experiment:

Liang Ma

Zi Liu

Jinchong Yan

Rongmei Chen

Yuxi Sun

Feiyu Huang

Human practice:

Prof. Xiangrong Xu

Hao Xu

Jiajun Li

Qicheng Sun

Yuexin Xie



Promotion video:

Qiyu Liang

Jiajun Lee



Sponsors:

Ma Steel

Triplan

Anhui University of Technology