Team:XMU-China/Engineering

Overview

This year XMU-China have tackled and solved one or more of our project's problems and use synthetic biology tools to generate expected results. Our project contains three systems: detection system, degradation system and kill switch systems.

After brainstorming and design improving through literature analysis and Human Practices(See the Human Practices details in Human Practices), we found several enzyme candidates. Totally, 101 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 that they can realize the goals we set. Moreover, it is worth mentioning that BBa_K3332006, BBa_K3332011 and BBa_K3332028 are typical among them. These three BioBricks works well to perform their functions and works as expected. All data related are recorded here. We hope they will make some contribution to the iGEM community.

Engineering Success

A. Detection System

1. GRHPR-his-tag (BBa_K3332006)

There is more than one enzyme with hydroxypyruvate reductase activity in humans. We choose one of these proteins which is found from human liver called GRHPR reduce glyoxylate when NADPH is used as cofactor. J23100-B0034 is used to construct the express circuit, and the GRHPR protein is purified and used to test the enzyme activity.

1-1. Agarose Gel Electrophoresis

Result: This target gene, GRHPR is designed successfully as the gragh shown below.

Fig. 1 DNA gel electrophoresis of restriction digest products of GRHPR-his-pSB1C3 (Xba I & Pst I sites)

1-2. SDS-PAGE

Result: GRHPR gene is expressed successfully, which means that the related gene worked well.

Fig. 2 SDS-PAGE analysis of protein in lysate of E. coli BL21 (DE3) cells and the eluant after AKTA by Coomassie blue staining. Target bands can be seen at about 35.7 kDa.

1-3. Enzyme Activity

Method: GRHPR is used to react with glyoxylate within the system which contains NADPH and buffer. Start testing immediately after preparing the reaction system (OD₃₄₀). Record the OD₃₄₀ value in real time.

Result: We can see the OD₃₄₀ of samples adding GRHPR decrease very quickly, while the OD₃₄₀ of control stay almost the same. Purified enzyme had high degrees of activity outside the living organism.

Fig. 3 Enzyme activity of GRHPR

We successfully got OD₃₄₀-Time curves of GRHPR in the presence of NADPH concentration and OD₃₄₀-Time curves of GRHPR in the presence of glyoxylic acid concentration. Then we calculated relevant enzyme activity and drew 1/V-1/[NADPH] and 1/V-1/[glyoxylic acid] curves, from which we can obtain relevant K_m and V_max.

The result is shown in figure. 4, figure. 5 and table 1.

Fig. 4 1/V-1/[NADPH] curve of purified GRHPR reacting with NADPH and glyoxylic acid

Fig. 5 1/V-1/[glyoxylic acid] curve of purified GRHPR reacting with NADPH and glyoxylic acid

Compound	K_m (mM)	k_cat (s^-1)
Glyoxylic acid	3.4298	11.0497
Glyoxylic acid	3.4298	11.0497

Table 1. Enzyme kinetic constants with two substrates.

2. GOX-his-tag (BBa_K3332006)

GOX, also known as EcAKR4-1, is found in Echinochloa Colona which can decompose glyphosate into AMPA and glyoxylic acid. His-tag makes it easy to purified via AKTA. J23100-B0034 is used to construct the express circuit, and the GOX protein is purified and used to test the enzyme activity.

2-1. Agarose Gel Electrophoresis

Result: This target gene, GOX is designed successfully as the gragh shown below.

Fig 6. DNA gel electrophoresis of restriction digest products of GOX-Histag-pSB1C3 (Xba I & Pst I sites)

2-2. SDS-PAGE

Result: GOX gene was expressed successfully, which meant that the related gene worked well.

Fig. 7 SDS-PAGE analysis of protein in lysate of E. coli BL21 (DE3) cells and the eluant after AKTA by Coomassie blue staining. Target bands can be seen at about 34.6 kDa.

2-3. Enzyme Activity

Method: Due to the lack of methods to detect glyphosate, GOX cannot be measured directly. GRHPR is used to co-react with GOX within the system which contains glyphosate, NADPH and buffer, which can react with the product of GOX and consumed NADPH added. Start testing immediately after preparing the reaction system (OD₃₄₀). Record OD₃₄₀ value in real time. Analyze the slope.

Result: The experiment group(GOX & GRHPR) Purified enzyme had high degrees of activity outside the living organism. The experimental result is shown on Fig. 8 When using GOX & GRHPR, we successfully found OD₃₄₀ decrease as time went on. And in negative control sample (only using GRHPR), we cannot get any decrease. The results prove that GOX can consume NADPH while converting glyphosate.

Fig. 9 OD₃₄₀-Time curve of GOX & GRHPR and negative control

B. Degradation System

1.phnO-his-tag (BBa_K3332028)

The phnO gene encodes the acyltransferase which catalyzes the transfer of acyl to AMPA by acetyl-coa. Through this one-step acyl transfer reaction, AMPA can be converted into glyphosate analogue, which can then be degraded by C-P lyase.

1-1. Agarose Gel Electrophoresis

Result: This target gene, phnO is designed successfully.

Fig. 10 DNA gel electrophoresis of restriction digest products of phnO-histag-pSB1C3 (EcoR I & Pst I sites).

1-2. SDS-PAGE

Result: PhnO gene is expressed successfully, which means that the related gene works well.

Fig. 11 SDS-PAGE analysis of protein in lysate of E. coli BL21 (DE3) cells and the eluant after AKTA by Coomassie blue staining. Target bands can be seen at about 16 kDa.

1-3. Enzyme Activity

Method: reaction rate of PhnO were determined using 4,4′-dithiodipyridine (DTDP) to continuously detect the formation of the product CoA at 324 nm (thiopyridone: ε= 19800 M^-1·cm^-1) at 25°C.(1)Enzyme activity was determined through enzyme-labeled instrument. K_m and k_cat of different substrates were determined with a concentration gradient. Start testing immediately after preparing the reaction system (OD₃₂₄). Record the light absorption value in real time. Analyze the slope.

Result:Purified enzyme had high degrees of activity outside the living organism, which proves that phnO works well. The related enzyme kinetic constants are shown below.

Fig. 12 The relationship of 1/Enzyme activity and 1/concentration of AcCoA. Used to determine the enzyme kinetic constants.

Fig. 13 The relationship of 1/Enzyme activity and 1/concentration of AMPA. Used to determine the enzyme kinetic constants.

Compound	K_m (mM)	k_cat (s^-1)
acetyl-CoA	0.1986	17.1394
aminomethylphosphonic acid	0.5637	3.2248

Table 2. Enzyme kinetic constants with two substrates.