iGEM 2020 — Team XMU-China

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

# Team:XMU-China/Notebook

[![](https://static.igem.org/mediawiki/2020/c/cb/T--XMU-China--logo.png)](https://2020.igem.org/Team%3AXMU-China)

* [Home](https://2020.igem.org/Team%3AXMU-China)
* Team
  [+ Members](https://2020.igem.org/Team%3AXMU-China/Team)
  [+ Collaborations](https://2020.igem.org/Team%3AXMU-China/Collaborations)
  [+ Partnership](https://2020.igem.org/Team%3AXMU-China/Partnership)
  [+ Attributions](https://2020.igem.org/Team%3AXMU-China/Attributions)
  [+ Notebook](https://2020.igem.org/Team%3AXMU-China/Notebook)
  [+ Experiments](https://2020.igem.org/Team%3AXMU-China/Experiments)
  [+ Safety](https://2020.igem.org/Team%3AXMU-China/Safety)
* Project
  [+ Description](https://2020.igem.org/Team%3AXMU-China/Description)
  [+ Design](https://2020.igem.org/Team%3AXMU-China/Design)
  [+ Models](https://2020.igem.org/Team%3AXMU-China/Model)
  [+ Result](https://2020.igem.org/Team%3AXMU-China/Results)
  [+ Proof of Concept](https://2020.igem.org/Team%3AXMU-China/Proof_Of_Concept)
  [+ Hardware](https://2020.igem.org/Team%3AXMU-China/Hardware)
  [+ Proposed Implementation](https://2020.igem.org/Team%3AXMU-China/Implementation)
* Parts
  [+ Parts](https://2020.igem.org/Team%3AXMU-China/Parts)
  [+ Contributions](https://2020.igem.org/Team%3AXMU-China/Contribution)
  [+ Engineering Success](https://2020.igem.org/Team%3AXMU-China/Engineering)
  [+ Improve](https://2020.igem.org/Team%3AXMU-China/Improve)
* Human Practices
  [+ Overview](https://2020.igem.org/Team%3AXMU-China/Human_Practices)
  [+ Silver](https://2020.igem.org/Team%3AXMU-China/Human_Practices#Silver)
  [+ Gold](https://2020.igem.org/Team%3AXMU-China/Human_Practices#Gold)
  [+ Science Communication](https://2020.igem.org/Team%3AXMU-China/Education)
  [+ Supporting Entrepreneurship](https://2020.igem.org/Team%3AXMU-China/Entrepreneurship)
* Awards
  [+ Awards](https://2020.igem.org/Team%3AXMU-China/Awards)
  [+ Judging](https://2020.igem.org/Team%3AXMU-China/Judging)
  [+ Best Integrated Human Practices](https://2020.igem.org/Team%3AXMU-China/Human_Practices)
  [+ Best Education](https://2020.igem.org/Team%3AXMU-China/Education)
  [+ Best Supporting Entrepreneurship](https://2020.igem.org/Team%3AXMU-China/Entrepreneurship)
  [+ Best Model](https://2020.igem.org/Team%3AXMU-China/Model)
  [+ Best Hardware](https://2020.igem.org/Team%3AXMU-China/Hardware)
  [+ Best Basic Parts](https://2020.igem.org/Team%3AXMU-China/Entrepreneurship#Basic)
  [+ Best Composite Parts](https://2020.igem.org/Team%3AXMU-China/Entrepreneurship#Composite)
  [+ Best Part Collection](https://2020.igem.org/Team%3AXMU-China/Entrepreneurship#Collection)

* February
* Mars、April、May
* June
* 7.13
* 7.16
* 7.17
* 7.18
* 7.21
* 7.24
* 7.26
* 7.27
* 7.30
* 8.1
* 8.6
* 8.9
* 8.10
* 8.12
* 8.14
* 8.16
* 8.20
* 8.23
* 8.25
* 9.3
* 9.13
* 9.15
* 9.18
* 9.19
* 9.20
* 9.23
* 9.25
* 9.28
* 10.1
* 10.7
* 10.8
* 10.10
* 10.11
* 10.15

![](https://static.igem.org/mediawiki/2020/5/59/T--XMU-China--timeline_left.png)
![](https://static.igem.org/mediawiki/2020/0/01/T--XMU-China--timeline_right.png)

# February

- Our team was formally founded.
- All student members learned the fundamental knowledge of molecular cloning and iGEM.
- All student members looked through the previous excellent projects and learned the basic notion of synthetic biology and iGEM.
-We decided our project theme.

# Mars、April、May

- We were divided into three groups and polish our project separately (and we reported each’s progress every weekend).
- Our team was formally accepted by iGEM.

# June

-All student members came back to school and learned laboratory fundamental qualities and received molecular cloning operation training.

# 7.13

-[BBa\_E0030](http://parts.igem.org/Part%3ABBa_E0030) and [BBa\_E0420](http://parts.igem.org/Part%3ABBa_E0420) are got from 2019 Spring Kit.

![](https://static.igem.org/mediawiki/2020/1/1c/T--XMU-China--07131.png)

![](https://static.igem.org/mediawiki/2020/9/91/T--XMU-China--07132.png)

# 7.16

-[BBa\_K333045](http://parts.igem.org/Part%3ABBa_K333045)is constructed.

![](https://static.igem.org/mediawiki/2020/e/ea/T--XMU-China--07161.png)

# 7.17

-[BBa\_K3332046](http://parts.igem.org/Part%3ABBa_K3332046) and [BBa\_K3332051](http://parts.igem.org/Part%3ABBa_K3332051) are constructed.

![](https://static.igem.org/mediawiki/2020/6/60/T--XMU-China--07171.png)

![](https://static.igem.org/mediawiki/2020/e/e2/T--XMU-China--07172.png)

# 7.18

-[BBa\_K3332056](http://parts.igem.org/Part%3ABBa_K3332056) is constructed

![](https://static.igem.org/mediawiki/2020/e/e8/T--XMU-China--07181.png)

# 7.21

-[BBa\_K3332057](http://parts.igem.org/Part%3ABBa_K3332057)is constructed and identified by PCR .

![](https://static.igem.org/mediawiki/2020/b/b2/T--XMU-China--07211.png)

# 7.24

-[BBa\_K3332061](http://parts.igem.org/Part%3ABBa_K3332061) is constructed successfully to verify INPNC's function and identified by PCR

![](https://static.igem.org/mediawiki/2020/9/91/T--XMU-China--07241.png)

# 7.26

-[BBa\_K3332065](http://parts.igem.org/Part%3ABBa_K3332065) and [BBa\_K3332066](http://parts.igem.org/Part%3ABBa_K3332066) are constructed to get J23100-RBS-phnE1 and J23100-RBS-phnE2

![](https://static.igem.org/mediawiki/2020/1/14/T--XMU-China--07261.png)

![](https://static.igem.org/mediawiki/2020/6/66/T--XMU-China--07262.png)

# 7.27

-[BBa\_K3332068](http://parts.igem.org/Part%3ABBa_K3332068) is constructed successfully.

![](https://static.igem.org/mediawiki/2020/9/92/T--XMU-China--07271.png)

# 7.30

-[BBa\_K3332065](http://parts.igem.org/Part%3ABBa_K3332065) and [BBa\_K3332066](http://parts.igem.org/Part%3ABBa_K3332066) are successfully ligated together to construct [BBa\_K3332067](http://parts.igem.org/Part%3ABBa_K3332067)

![](https://static.igem.org/mediawiki/2020/d/de/T--XMU-China--07301.png)

# 8.1

-[BBa\_K3332069](http://parts.igem.org/Part%3ABBa_K3332069) is successfully constructed by point mutation

# 8.6

-[BBa\_K3332072](http://parts.igem.org/Part%3ABBa_K3332072) is constructed to get J23100-RBS-phnO-His-Tag

# 8.9

-[BBa\_K3332052](http://parts.igem.org/Part%3ABBa_K3332052) is constructed

# 8.10

-[BBa\_K3332047](http://parts.igem.org/Part%3ABBa_K3332047) is constructed successfully.

![](https://static.igem.org/mediawiki/2020/a/a5/T--XMU-China--08101.png)

# 8.12

-[BBa\_K3332049](http://parts.igem.org/Part%3ABBa_K3332049) is constructed.

![](https://static.igem.org/mediawiki/2020/1/1d/T--XMU-China--08121.png)

# 8.14

-[BBa\_K3332073](http://parts.igem.org/Part%3ABBa_K3332073),[BBa\_K3332075](http://parts.igem.org/Part%3ABBa_K3332075),[BBa\_K3332076](http://parts.igem.org/Part%3ABBa_K3332076) and[BBa\_K3332077](http://parts.igem.org/Part%3ABBa_K3332077) are constructed for RNAi

![](https://static.igem.org/mediawiki/2020/c/c2/T--XMU-China--08141.png)

# 8.16

-[BBa\_K3332038](http://parts.igem.org/Part%3ABBa_K3332038) is constructed successfully by PCR

![](https://static.igem.org/mediawiki/2020/0/0f/T--XMU-China--08161.png)

# 8.20

-[BBa\_K3332063](http://parts.igem.org/Part%3ABBa_K3332063) is constructed to verify AIDA's function and identified by PCR.

![](https://static.igem.org/mediawiki/2020/d/d3/T--XMU-China--08201.png)

# 8.25

-[BBa\_K3332040](http://parts.igem.org/Part%3ABBa_K3332040) is constructed.

![](https://static.igem.org/mediawiki/2020/4/4e/T--XMU-China--08251.png)

# 9.3

[BBa\_K3332059](http://parts.igem.org/Part%3ABBa_K3332059) and [BBa\_K3332060](http://parts.igem.org/Part%3ABBa_K3332060)are constructed successfully to anchor GRHPR to the membrane of E. Coli.

![](https://static.igem.org/mediawiki/2020/2/20/T--XMU-China--09031.png)

-[BBa\_K2922039](http://parts.igem.org/Part%3ABBa_K2922039) (3195 bp) successfully constructed.

![](https://static.igem.org/mediawiki/2020/9/95/T--XMU-China--09032.png)

# 9.13

[BBa\_I0500](http://parts.igem.org/Part%3ABBa_K3332059) is constructed

# 9.15

[BBa\_K3332080](http://parts.igem.org/Part%3ABBa_K3332080) and [BBa\_K3332081](http://parts.igem.org/Part%3ABBa_K3332081)is constructed by ligating [BBa\_I0500](http://parts.igem.org/Part%3ABBa_K3332080), [BBa\_Q04510](http://parts.igem.org/Part%3ABBa_Q04510) [BBa\_K1096002](http://parts.igem.org/Part%3ABBa_K1096002).

![](https://static.igem.org/mediawiki/2020/8/83/T--XMU-China--09151.png)

![](https://static.igem.org/mediawiki/2020/8/88/T--XMU-China--09152.png)

# 9.18

[BBa\_K3332091](http://parts.igem.org/Part%3ABBa_K3332091) and [BBa\_K3332092](http://parts.igem.org/Part%3ABBa_K3332092)are constructed as an improvement of [BBa\_K1334002](http://parts.igem.org/Part%3ABBa_1334002) .

![](https://static.igem.org/mediawiki/2020/b/be/T--XMU-China--09181.png)

![](https://static.igem.org/mediawiki/2020/d/d0/T--XMU-China--09182.png)

# 9.19

[BBa\_K3332093](http://parts.igem.org/Part%3ABBa_K3332093) is constructed as an improvement of BBa\_K1334002.

![](https://static.igem.org/mediawiki/2020/3/32/T--XMU-China--09191.png)

# 9.20

[BBa\_K3332084](http://parts.igem.org/Part%3ABBa_K3332084) is constructed by ligating [BBa\_K3332034](http://parts.igem.org/Part%3ABBa_K3332034)and[BBa\_E0420](http://parts.igem.org/Part%3ABBa_E0420).

![](https://static.igem.org/mediawiki/2020/8/8d/T--XMU-China--09201.png)

# 9.23

[BBa\_K3332086](http://parts.igem.org/Part%3ABBa_K3332086) is constructed.

![](https://static.igem.org/mediawiki/2020/4/48/T--XMU-China--XMU-China_2020_-BBa_K3332086-_pTrc-2_E0420_pSB1C3_digested_by_Xba_I_and_Pst_I.png)

# 9.25

[BBa\_K3332087](http://parts.igem.org/Part%3ABBa_K3332087) is constructed by ligating [BBa\_K3332040](http://parts.igem.org/Part%3ABBa_K3332040) and [BBa\_E0420](http://parts.igem.org/Part%3ABBa_E0420).

![](https://static.igem.org/mediawiki/2020/4/43/T--XMU-China--09251.png)

# 9.28

[BBa\_K3332082](http://parts.igem.org/Part%3ABBa_K3332082) is constructed by ligating [BBa\_E0430.](http://parts.igem.org/Part%3ABBa_I10500%3EBBa_I0500%3C/a%3E%20and%20%3Ca%20href%3D)

![](https://static.igem.org/mediawiki/2020/5/50/T--XMU-China--09281.png)

![](https://static.igem.org/mediawiki/2020/1/17/T--XMU-China--09282.png)

# 10.1

[BBa\_K3332064](http://parts.igem.org/Part%3ABBa_K3332064) is constructed to verify BrkA 's function, which is identified by PCR

![](https://static.igem.org/mediawiki/2020/e/eb/T--XMU-China--10011.png)

# 10.7

[BBa\_K3332054](http://parts.igem.org/Part%3ABBa_K3332054) and [BBa\_K3332055](http://parts.igem.org/Part%3ABBa_K3332055)are successfully constructed

![](https://static.igem.org/mediawiki/2020/d/d3/T--XMU-China--10071.png)
![](https://static.igem.org/mediawiki/2020/f/f9/T--XMU-China--10072.png)

# 10.8

[BBa\_K3332090](http://parts.igem.org/Part%3ABBa_K3332090)is constructed to characterize the function of the improved promoter.

![](https://static.igem.org/mediawiki/2020/c/cf/T--XMU-China--10081.png)

# 10.10

[BBa\_K3332097](http://parts.igem.org/Part%3ABBa_K3332097) is constructed by ligating[BBa\_K3332068](http://parts.igem.org/Part%3ABBa_K3332068)are successfully constructed

![](https://static.igem.org/mediawiki/2020/8/8d/T--XMU-China--10101.png)

# 10.11

[BBa\_K3332099](http://parts.igem.org/Part%3ABBa_K3332099) is constructed

![](https://static.igem.org/mediawiki/2020/6/6e/T--XMU-China--10111.png)

# 10.15

[BBa\_K3332097](http://parts.igem.org/Part%3ABBa_K3332097) and [BBa\_K3332068](http://parts.igem.org/Part%3ABBa_K3332098)are constructed

![](https://static.igem.org/mediawiki/2020/a/a1/T--XMU-China--10151.png)

Xiamen University, Fujian, China

No.422, Siming South Road, Fujian, P.R.China 361005

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

# Team:XMU-China/Engineering

[![](https://static.igem.org/mediawiki/2020/c/cb/T--XMU-China--logo.png)](https://2020.igem.org/Team%3AXMU-China)

* [Home](https://2020.igem.org/Team%3AXMU-China)
* Team
  [+ Members](https://2020.igem.org/Team%3AXMU-China/Team)
  [+ Collaborations](https://2020.igem.org/Team%3AXMU-China/Collaborations)
  [+ Partnership](https://2020.igem.org/Team%3AXMU-China/Partnership)
  [+ Attributions](https://2020.igem.org/Team%3AXMU-China/Attributions)
  [+ Notebook](https://2020.igem.org/Team%3AXMU-China/Notebook)
  [+ Experiments](https://2020.igem.org/Team%3AXMU-China/Experiments)
  [+ Safety](https://2020.igem.org/Team%3AXMU-China/Safety)
* Project
  [+ Description](https://2020.igem.org/Team%3AXMU-China/Description)
  [+ Design](https://2020.igem.org/Team%3AXMU-China/Design)
  [+ Models](https://2020.igem.org/Team%3AXMU-China/Model)
  [+ Result](https://2020.igem.org/Team%3AXMU-China/Results)
  [+ Proof of Concept](https://2020.igem.org/Team%3AXMU-China/Proof_Of_Concept)
  [+ Hardware](https://2020.igem.org/Team%3AXMU-China/Hardware)
  [+ Proposed Implementation](https://2020.igem.org/Team%3AXMU-China/Implementation)
* Parts
  [+ Parts](https://2020.igem.org/Team%3AXMU-China/Parts)
  [+ Contributions](https://2020.igem.org/Team%3AXMU-China/Contribution)
  [+ Engineering Success](https://2020.igem.org/Team%3AXMU-China/Engineering)
  [+ Improve](https://2020.igem.org/Team%3AXMU-China/Improve)
* Human Practices
  [+ Overview](https://2020.igem.org/Team%3AXMU-China/Human_Practices)
  [+ Silver](https://2020.igem.org/Team%3AXMU-China/Human_Practices#Silver)
  [+ Gold](https://2020.igem.org/Team%3AXMU-China/Human_Practices#Gold)
  [+ Science Communication](https://2020.igem.org/Team%3AXMU-China/Education)
  [+ Supporting Entrepreneurship](https://2020.igem.org/Team%3AXMU-China/Entrepreneurship)
* Awards
  [+ Awards](https://2020.igem.org/Team%3AXMU-China/Awards)
  [+ Judging](https://2020.igem.org/Team%3AXMU-China/Judging)
  [+ Best Integrated Human Practices](https://2020.igem.org/Team%3AXMU-China/Human_Practices)
  [+ Best Education](https://2020.igem.org/Team%3AXMU-China/Education)
  [+ Best Supporting Entrepreneurship](https://2020.igem.org/Team%3AXMU-China/Entrepreneurship)
  [+ Best Model](https://2020.igem.org/Team%3AXMU-China/Model)
  [+ Best Hardware](https://2020.igem.org/Team%3AXMU-China/Hardware)
  [+ Best Basic Parts](https://2020.igem.org/Team%3AXMU-China/Parts#Basic)
  [+ Best Composite Parts](https://2020.igem.org/Team%3AXMU-China/Parts#Composite)
  [+ Best Part Collection](https://2020.igem.org/Team%3AXMU-China/Parts#Collection)

![](https://static.igem.org/mediawiki/2020/d/dd/T--XMU-China--right2-2.png)
[![](https://static.igem.org/mediawiki/2020/6/62/T--XMU-China--right1-2.png)
[Overview](#Overview)](#Overview)
[![](https://static.igem.org/mediawiki/2020/d/dd/T--XMU-China--right2-2.png)
[Engineering Success](#ES)](#ES)
[![](https://static.igem.org/mediawiki/2020/d/dd/T--XMU-China--right2-2.png)](#Safe_Shipment)

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](https://2020.igem.org/Team%3AXMU-China/Human_Practices)), we found several enzyme candidates. Totally, 101 new BioBricks were designed for our project (See the Parts and Design details in
[Parts](https://2020.igem.org/Team%3AXMU-China/Parts) and
[Design](https://2020.igem.org/Team%3AXMU-China/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](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3332006), [BBa\_K3332011](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3332011) and [BBa\_K3332028](http://parts.igem.org/wiki/index.php?title=Part: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.

![](https://static.igem.org/mediawiki/2020/e/e8/T--XMU-China--07181.png)

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.

![](https://static.igem.org/mediawiki/2020/b/b1/T--XMU-China--XMU-China_2020-GRHPR_and_GOX.png)

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.

![](https://static.igem.org/mediawiki/2020/d/d4/T--XMU-China--XMU-China_2020-GRHPR%E9%85%B6%E6%B4%BB.png)

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 Km and Vmax.

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

![](https://static.igem.org/mediawiki/2020/4/4c/T--XMU-China--XMU-China_2020-GRHPR_%E5%8A%A8%E5%8A%9B%E5%AD%A6%E5%8F%82%E6%95%B01-NADPH%E5%BA%95%E7%89%A9.png)

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

![](https://static.igem.org/mediawiki/2020/3/3d/T--XMU-China--XMU-China_2020-GRHPR%E5%8A%A8%E5%8A%9B%E5%AD%A6%E5%8F%82%E6%95%B0-2-glyoxycolic_%E5%BA%95%E7%89%A9.png)

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

| Compound | Km (mM) | kcat (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.

![](https://static.igem.org/mediawiki/2020/e/e2/T--XMU-China--07172.png)

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.

![](https://static.igem.org/mediawiki/2020/b/b1/T--XMU-China--XMU-China_2020-GRHPR_and_GOX.png)

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.

![](https://static.igem.org/mediawiki/2020/c/cf/T--XMU-China--XMU-China_2020-GOX_OD340.png)

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.

![](https://static.igem.org/mediawiki/2020/7/73/T--XMU-China--08061.png)

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

1-2. SDS-PAGE

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

![](https://static.igem.org/mediawiki/2020/7/77/T--XMU-China--XMU-China_2020-%E3%80%90%E8%9B%8B%E7%99%BD%E8%83%B6%E3%80%91PhnO.png)

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. Km and kcat 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.

![](https://static.igem.org/mediawiki/2020/b/be/T--XMU-China--XMU-China_2020-AcCoA_%E5%8F%8C%E5%80%92%E6%95%B0%E6%9B%B2%E7%BA%BF.png)

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

![](https://static.igem.org/mediawiki/2020/8/82/T--XMU-China--XMU-China_2020-AMPA_%E5%8F%8C%E5%80%92%E6%95%B0%E6%9B%B2%E7%BA%BF.png)

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

| Compound | Km (mM) | kcat (s-1) |
| --- | --- | --- |
| acetyl-CoA | 0.1986 | 17.1394 |
| aminomethylphosphonic acid | 0.5637 | 3.2248 |

Table 2. Enzyme kinetic constants with two substrates.

Xiamen University, Fujian, China

No.422, Siming South Road, Fujian, P.R.China 361005

[![](https://static.igem.org/mediawiki/2020/1/1a/T--XMU-China--returntop.png)](#top)

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

# Team:XMU-China/Experiments

[![](https://static.igem.org/mediawiki/2020/c/cb/T--XMU-China--logo.png)](https://2020.igem.org/Team%3AXMU-China)

* [Home](https://2020.igem.org/Team%3AXMU-China)
* Team
  [+ Members](https://2020.igem.org/Team%3AXMU-China/Team)
  [+ Collaborations](https://2020.igem.org/Team%3AXMU-China/Collaborations)
  [+ Partnership](https://2020.igem.org/Team%3AXMU-China/Partnership)
  [+ Attributions](https://2020.igem.org/Team%3AXMU-China/Attributions)
  [+ Notebook](https://2020.igem.org/Team%3AXMU-China/Notebook)
  [+ Experiments](https://2020.igem.org/Team%3AXMU-China/Experiments)
  [+ Safety](https://2020.igem.org/Team%3AXMU-China/Safety)
* Project
  [+ Description](https://2020.igem.org/Team%3AXMU-China/Description)
  [+ Design](https://2020.igem.org/Team%3AXMU-China/Design)
  [+ Models](https://2020.igem.org/Team%3AXMU-China/Model)
  [+ Result](https://2020.igem.org/Team%3AXMU-China/Results)
  [+ Proof of Concept](https://2020.igem.org/Team%3AXMU-China/Proof_Of_Concept)
  [+ Hardware](https://2020.igem.org/Team%3AXMU-China/Hardware)
  [+ Proposed Implementation](https://2020.igem.org/Team%3AXMU-China/Implementation)
* Parts
  [+ Parts](https://2020.igem.org/Team%3AXMU-China/Parts)
  [+ Contributions](https://2020.igem.org/Team%3AXMU-China/Contribution)
  [+ Engineering Success](https://2020.igem.org/Team%3AXMU-China/Engineering)
  [+ Improve](https://2020.igem.org/Team%3AXMU-China/Improve)
* Human Practices
  [+ Overview](https://2020.igem.org/Team%3AXMU-China/Human_Practices)
  [+ Silver](https://2020.igem.org/Team%3AXMU-China/Human_Practices#Silver)
  [+ Gold](https://2020.igem.org/Team%3AXMU-China/Human_Practices#Gold)
  [+ Science Communication](https://2020.igem.org/Team%3AXMU-China/Education)
  [+ Supporting Entrepreneurship](https://2020.igem.org/Team%3AXMU-China/Entrepreneurship)
* Awards
  [+ Awards](https://2020.igem.org/Team%3AXMU-China/Awards)
  [+ Judging](https://2020.igem.org/Team%3AXMU-China/Judging)
  [+ Best Integrated Human Practices](https://2020.igem.org/Team%3AXMU-China/Human_Practices)
  [+ Best Education](https://2020.igem.org/Team%3AXMU-China/Education)
  [+ Best Supporting Entrepreneurship](https://2020.igem.org/Team%3AXMU-China/Entrepreneurship)
  [+ Best Model](https://2020.igem.org/Team%3AXMU-China/Model)
  [+ Best Hardware](https://2020.igem.org/Team%3AXMU-China/Hardware)
  [+ Best Basic Parts](https://2020.igem.org/Team%3AXMU-China/Parts#Basic)
  [+ Best Composite Parts](https://2020.igem.org/Team%3AXMU-China/Parts#Composite)
  [+ Best Part Collection](https://2020.igem.org/Team%3AXMU-China/Parts#Collection)

Molecular Cloning Handbook

#### [Molecular Cloning Handbook 6.0 BETA of XMU-China](#collapse0)

[Molecular\_Cloning\_Handbook.pdf](https://static.igem.org/mediawiki/2020/0/09/T--XMU-China--XMU-China_2020_Molecular_Cloning_Handbook-6.pdf)

Detection System

Confocal Microscope

#### [I. Materials & Apparatus](#collapse11)

● LB liquid medium with 0.1mg/mL chloramphenicol
● Pbs buffer
● Nail polish
● Slide glass and cover glass
● centrifuge
● Leica TCS SP8 X microscope (Leica Microsystems)
● SmartGain: 800 V, 1% Laser Intensity, resolution: 2048 bp, excitation wavelength: 488 nm.

#### [II. Before Experiment](#collapse114_1_1)

1. 200μL bacteria solution and 10μL chloramphenicol were inoculated into 10mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.
2. Pellet 1mL bacteria solution from step 1 in a clean 1.5-mL microcentrifuge tube by centrifugation at 5,000rpm for 1 minute at room temperature. Decant or aspirate medium and discard.
3. Add 1mL pbs buffer, pipet up and down to mix thoroughly. And centrifuge at 5,000rpm for 1 minute at room temperature. Decant or aspirate medium and discard.
4. Repeat step 3 once.
5. Add 500μL pbs buffer, pipet up and down to mix thoroughly.
6. 5μL bacteria solution from step 5 was inoculated in the center of cover glass. And place the slide glass on the cover glass gently.
7. Turn over the slide glass gently and paste nail polish around the edges of cover glass.
8. Turn over the slide glass gently again until nail polish is solidified and last 30min.
9. Turn over the slide glass and make a marker.

#### [III. protocols](#collapse115_1_1)

1. Use Leica TCS SP8 X microscope at appropriate vision and take a picture.
Related parameters: SmartGain: 800 V,
1% Laser Intensity
resolution: 2048 bp
excitation wavelength: 488 nm.

Membrane Protein Characterization-Fluorescence Measurement

#### [I. Materials & Apparatus](#collapse113_1)

● LB liquid medium with 0.1 mg/mL chloramphenicol

#### [II. Before Experiment](#collapse12)

1. 200 μL bacteria solution and 10μL chloramphenicol were inoculated into 10mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.

#### [III. Protocol](#collapse13_13)

1. inoculate 200 μL bacteria solution into 96-well microtiter plates.
2. measure the value of OD600.
3. adjust OD600 of remaining bacterial fluid to 1 with pbs buffer.
4. centrifuge the bacterial fluid at 6,500 rpm for 5 min at 4 °C, decant or aspirate supernatant and discard and add equivalent pbs buffer and pipet up and down to mix thoroughly. And take a picture.
5. inoculate 200 μL bacteria solution into 96-well microtiter plates and measure fluorescence with excitation wavelength 475 nm and emission wavelength 545 nm.
6. use ultrasonic waves to lysis cells at 25 times.
7. centrifuge the bacterial fluid at 20,000 rpm for 20 min at 4 °C. and take a picture.
8. inoculate 200 μL supernatant into 96-well microtiter plates and measure fluorescence with excitation wavelength 475 nm and emission wavelength 545 nm.
9. Decant or aspirate supernatant and discard, add equivalent pbs buffer and pipet up and down to mix thoroughly.
10. inoculate 200 μL supernatant into 96-well microtiter plates and measure fluorescence with excitation wavelength 475 nm and emission wavelength 545 nm.

The Enzyme Activity Determination of Purified GOX & GRHPR

#### [I. Materials & Apparatus](#collapse15_1)

● TECAN® Infinite M200 Pro Microplate Reader
● 96 well microtiter plates
● Glyphosate, NADPH·Na4, Tris-HCl buffer
● Purified GOX enzyme
● Purified GRHPR enzyme

#### [II. Before Experiment](#collapse16)

1. The Enzyme activity determination of Purified GOX
Prepare aqueous solution below.
2.0 mg/mL Glyphosate
2 mM NADPH·Na4
pH=6.8, 50 mM Tris-HCl buffer
Prepare these reaction systems according to the table below. (Unit: μL)

|  | Tris-HCl buffer | NADPH·Na4 | Glyphosate | Purified GRHPR enzyme | Purified GOX enzyme |
| --- | --- | --- | --- | --- | --- |
| A | 80 | 30 | 30 | 30 | 30 |
| B | 110 | 30 | 30 | 30 | 0 |

2. The Enzyme activity determination of Purified GRHPR
Prepare aqueous solution below.
80mM Glyoxylic acid
2mM NADPH·Na4
pH=7.5, 50mM Tris-HCl buffer
Prepare these reaction systems according to the table below. (Unit: μL)

|  | Tris-HCl buffer | NADPH·Na4 | Glyoxylic acid | Purified GRHPR enzyme |
| --- | --- | --- | --- | --- |
| A | 50 | 50 | 50 | 50 |
| B | 100 | 50 | 50 | 0 |

3. Program setting of Microplate Reader：

| Shaking Duration | 5 sec |
| --- | --- |
| Mode | Linear |
| Amplitude | 1 mm |
| Frequency | 886.9 rpm |
| Number of Cycles | 45 |
| Time interval | 10s |
| Wavelength | 340 nm |
| Temperature | 30℃ |
| Number of flash | 25 |

#### [III. Protocol](#collapse16_2)

1. Start testing immediately after preparing the reaction system. Record the value of fluorescence in real time.
2. Analyze the slope.

Surface display properties of GOX & GRHPR

#### [I. Materials & Apparatus](#collapse17)

● TECAN® Infinite M200 Pro Microplate Reader
● 96 well microtiter plates
● Glyoxylic acid, NADPH·Na4,

#### [II. Before Experiment](#collapse17_1)

1. Surface display properties of GOX
●Prepare aqueous solution below.
●2.0 mg/mL Glyphosate
●2mM NADPH·Na4
●pH=6.8, 50mM Tris-HCl buffer
● Prepare these reaction systems according to the table below. (Unit: μL)

|  | Tris-HCl buffer | NADPH·Na4 | Glyoxylic acid | *E.Coli* Anchored with GOX | Purified GRHPR enzyme |
| --- | --- | --- | --- | --- | --- |
| A(Blank) | 50 | 50 | 50 | 0 | 50 |
| B (INPNC-GOX) | 50 | 50 | 50 | 50 | 0 |
| C (GOX-AIDA) | 50 | 50 | 50 | 50 | 0 |
| D (GOX-Brka) | 50 | 50 | 50 | 50 | 0 |

\*The value of OD600 of all the *E.Coli* need to be the same, 2.2 being a suitable choice.
2. Surface display properties of GRHPR
●Prepare aqueous solution below.
●80mM Glyoxylic acid
●2mM NADPH·Na4
●pH=7.5, 50mM Tris-HCl buffer
● Prepare these reaction systems according to the table below. (Unit: μL)

|  | Tris-HCl buffer | NADPH·Na4 | Glyoxylic acid | *E.Coli* Anchored with GOX | *E.Coli* (J23100-RBS-pSB1C3) |
| --- | --- | --- | --- | --- | --- |
| A(Blank) | 50 | 50 | 50 | 0 | 50 |
| B (INPNC-GRHPR) | 50 | 50 | 50 | 50 | 0 |
| C (GRHPR-AIDA) | 50 | 50 | 50 | 50 | 0 |
| D (GRHPR-Brka) | 50 | 50 | 50 | 50 | 0 |

\*The value of OD600 of all the *E.Coli* need to be the same, 2.2 being a suitable choice.
Program setting of Microplate Reader：

| Shaking Duration | 5 sec |
| --- | --- |
| Mode | Linear |
| Amplitude | 1 mm |
| Frequency | 886.9 rpm |
| Number of Cycles | 45 |
| Time interval | 10s |
| Wavelength | 340 nm |
| Temperature | 30℃ |
| Number of flash | 25 |

#### [III. Protocol](#collapse17_17)

1. Start testing immediately after preparing the reaction system. Record the value of fluorescence in real time.
2. Analyze the slope.

The Enzyme activity determination of Purified iNAP

#### [I. Materials & Apparatus](#collapse15_2)

●Purified iNAP Protein
●Macklin® NADPH
●Corning® Costar 96-Well Black flat-Bottom Plates
●TECAN® Infinite M200 Pro ELIASA

#### [II. Protocol](#collapse15_3)

**A. Preparation of NADPH Solution**
1.9 mg NAPDH powder was dissolved in 6 mL purified water and prepared into 2000 μM solution.
2.Dilute in turn and prepare solutions of concentration of 0.01, 0.05, 0.1, 0.25, 0.5, 1,2.5, 5,10,25,50,100,200 μM
**B．Determination**
1.100 μL of solutions of 0.01, 0.05, 0.1, 0.25, 0.5, 0.5, 5, 2.5, 50, 50, 100, 200 μM are added to a group of wells in the 96-well plate, and 100 μL of purified iNAP protein fluid are added to the group of Wells for detection. Put the 96-well plate into ELIASA.
2.Program setting of ELIASA：
a)Shaking

| Duration | 5 sec |
| --- | --- |
| Mode | Linear |
| Amplitude | 1 mm |
| Frequency | 886.9 rpm |

b)Fluorescence Intensity

| Excitation wavelength | 420 nm |
| --- | --- |
| Emission wavelength | 528 nm |
| Mode | Top |
| Z-Position | Manual(20000 μm) |
| Number of flash | 25 |
| Gain | Manual / Optimal / Calculated from well |

Degradation System

HPLC

#### [I. Materials & Apparatus](#collapse31)

● 1×PBS buffer
● *E.coli* BL21 (DE3) culture (J23100-B0034-phnE1E2\_pSB1C3 and J23100-B0034\_pSB1C3 plasmid, OD600=2.0)
● Glyphosate (Solarbio®)
● Isopropanol (Shanghai Institute of Chemical Reagents)
● Copper sulfate pentahydrate (Aldrich®)
● Double distilled water (ddH2O)
● Agilent Technologies 1200 Series (Degasser, Quat Pump, ALS, TCC)
● Agilent Technologies 1260 Inifinity (VWD)
● Column (Chirex 3126 (D)-penicillamin Column, 250×4.60 mm)

#### [II. Protocol](#collapse32)

**A. Sampling**
1．Take 20 mg of glyphosate in a 15-mL centrifuge tube, and 10 mL of ddH2O is added.
2．Each group was taken 1 mL of glyphosate solution as blank.
3．Add 19 mL *E.coli* BL21 (DE3) culture (experimental group 100-RBS-phnE1E2\_pSB1C3 and control group 100-RBS\_pSB1C3) and 1 ml of glyphosate solution to the 50-ml centrifuge tube.
4．Add 2 mL mixture that mentioned above to each of the experimental group, and mark the number.
5. Take the first group of mixture that the glyphosate solution is just added as blank. The reaction is then carried out at 37 °C, 200 rpm.
6. Centrifugal the 15-mL centrifuge tube with the reaction mixture at 6500 rpm for 5min, take the upper layer, mark the name, number and time period, resuspend the bacterium with PBS buffer, and place the samples in a boiling water bath as soon as possible to quench the reaction.
Gradient : 0, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300 min
**B. Instrument pretreatment**
1. Preparation of mobile phase
a) Add 950ml of water and 50ml Isopropanol to the beaker, and add 0.500g CuSO4·5H2O and transfer them to the beaker.
b) Filter by vacuum pump and ultrasonic defoam for 30 min.
2. Preparation of glucose and cellobiose standard working curve samples (0.01, 0.02, 0.05, 0.1, 0.2, 0.5 g/L)
a) Prepare 2.0 g/L glucose and cellobiose solution.
b) Dilute to 0.1, 0.2, 0.5, 1.0, 2.0 g/L with other volumetric flasks based on a 2.0g/L.
3. Instrument pre-cleaning
a) Loosen the pump valve and clean the outer passage to no air bubbles.
b) Tighten the pump valve. The flow rate was adjusted to 0.8 mL/min. After the baseline was substantially leveled, the flow rate was set to 0.5 mL/min and the pump was turned off.
c) Replace the mobile phase (pre-made i-PrOH-CuSO4-H2O mobile phase) and start the pump.
d) Adjust the solution fill and open the pump valve. (counterclockwise)
f) VMD—Turn on
g) After the baseline is basically leveled, adjust the rectification speed to 0.2 mL/min and the upper pressure limit to 85 bar.
h) Install the column (Chirex 3126 (D)-penicillamin Column, 250×4.60 mm).
i) Open the column oven at 35 °C, adjust the flow rate to 0.8 mL/min, and clean the reference cell for 20 min until the baseline is stable.
**C. Detection**
1. Put in the wash bottle, sequence the parameters, set the path, name and operator, then fill in the sequence table.
2. Take a 1 mL of sample from the syringe and inject it into the sample tube through a 0.22-μm filter.
3. Place all samples in the sample tray according to the sequence table and feed them into the machine.
**D. Post-processing**
1. After running the last sample, manually integrate and save the relevant data file.
2. Rinse for 1 hour and adjust the flow rate to 0.2 mL/min.
3. Close the column oven and turn off the pump and unload the column after the column temperature drops.
4. VMD—Turn off.
5. The flow rate is adjusted to 0.2 mL/min.
6. Turn off the pump, software, liquid meter, and computer in sequence.

The Enzyme Activity Determination of Purified PhnO

#### [I. Materials & Apparatus](#collapse33)

●Purified PhnO enzyme
●TECAN® Infinite M200 Pro Microplate Reader
●96-well microtiter plates
●Reaction system (1 mL) :

| Components | Concentration |
| --- | --- |
| HEPES | 100 mM |
| (NH4)2SO4 | 100 mM |
| 4,4′-dithiodipyridine (DTDP) | 200 μM |
| Ni2+ (NiCl2) | 100 μM |
| AcCoA | 0.1 mM |
| aminomethylphosphonic acid (AMPA) | 2 mM |

#### [II. Protocol](#collapse34)

1. Add 180 μL reaction system into one well in 96-well microtiter plates, and then add 20 μL PhnO enzyme solution into the same well. Put the plate into Microplate Reader.
2.Program setting of Microplate Reader:

| Shaking Duration | 5 sec |
| --- | --- |
| Mode | Linear |
| Amplitude | 1 mm |
| Frequency | 886.9 rpm |
| Number of Cycles | 45 |
| Time interval | 10s |
| Wavelength | 340 nm |
| Temperature | 30℃ |
| Number of flash | 25 |

3.Analyze the relationship between OD324 and time.

Kill Switch Systems

CFU（colony forming units）

#### [I. Materials & Apparatus](#collapse21)

●Culture dish
●LB solid medium
●LB liquid medium
●Chloramphenicol solution (50 mg/mL)
●phosphate buffer saline (PBS)

#### [II. Protocol](#collapse22)

1. Add 200 μL glycerin bacteria into 10 mL LB liquid medium containing 50 mg/mL chloramphenicol. Incubate the culture in a shaker overnight at 37 °C, 200 rpm.
2. Add 1 mL of the culture above into 50 mL LB liquid medium containing 50 mg/mL chloramphenicol, and incubate it in a shaker of 37 °C and 200 rpm until OD600 reaches 1.0.
3. Samples are serially diluted in PBS over a 6-log range and spotted 50μL bacteria solution onto LB solid plate, incubate it at 37°C for 12h.
4. Count the number of colonies, and then calculate the CFU. (CFU/mL=(numbers of colonies)×(dilution factor)/0.05mL)(1)
Reference. C. T. Chan, J. W. Lee, D. E. Cameron, C. J. Bashor, J. J. Collins, 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol 12, 82-86 (2016).

Common experiment

Fluorescence/OD600 Curve

#### [I. Materials & Apparatus](#collapse41)

●1×PBS solution
●Buffer A
●Buffer B
●GE AKTA Prime Plus FPLC System

#### [II. Protocol](#collapse42)

**A. Buffer preparation**
1. Buffer A

| Na3PO4·12H2O | 7.6g |
| --- | --- |
| NaCl | 29.2g |
| Imidazole | 1.36g |
| H2O | 1L |

Adjust pH to 7.4 and be Filtered through 0.22-μm filter membrane. Ultrasonic defoam for 30 min.
2.Buffer B

| Na3PO4·12H2O | 7.6g |
| --- | --- |
| NaCl | 29.2g |
| Imidazole | 34g |
| H2O | 1L |

Adjust pH to 7.4 and be filtered through 0.22-μm filter membrane. Ultrasonic defoam for 30 min.
3.30 % ethanol
4.ddH2O
Be filtered through 0.22-μm filter membrane and ultrasonic defoam for 30 min.
**B. Sampling**
1.40 mL bacterial solution was taken from each group in a 50-mL centrifuge tube, and centrifuged at 6000 rpm for 10 min.
2.Discard the supernatant, then add 40 mL PBS buffer solution to each tube. Solution is resuspended by shock, and centrifuge it at 6000 rpm for 10 min.
3.Add 40 mL Buffer A to shake and resuspended, and place on the ice.
4.Ultrasonic crush.
5.After crushing, centrifuge at 10000 rpm for 20 min to collect supernatant and filter.
**C. AKTA purification**
1.Clean the pipes
Flush the pipe with water at a flow rate of 50 mL/min , and set the pressure Limit as 1.0 MPa.
2.Packing column
a)Set the flow rate at 0.5ml /min and pressure Limit at 1.0mpa.
b)Load the Histrap TM purified column into the pipeline.
3.Balance column
a)Replace the mobile phase of pipeline A with Buffer A and that of pipeline B with Buffer B.
b)Wash 25 mL of pipeline B, and then 50 mL of pipeline A to the baseline horizontally.
4.Load sample
Put the pipeline A into the sample and set the flow rate at 3 mL/min. Pay attention not to let the air enter the pipeline.
5.Wash the miscellaneous
Put pipeline A back into Buffer A and set the flow rate at 5.0 mL/min until the baseline become horizontal.
6.Elute
a) Set Gradient, Length: 100 mL, Target: 100 mL.
b) Set flow rate at 5 mL/min.
c) Set Fraction size at 5 mL/min.
d) Collect protein fluid where the peak is located, then desalt protein fluid with Tris-HCl ( pH7.5)
7.Rebalance the column
8.Dismantle the column
a)Pipeline A and B were rinsed and placed in water. Set the flow rate at 5 mL/min. 25 mL was rinsed with water and 25 mL was rinsed with ethanol.
b)Set the flow rate at 0.5 mL /min and dismantle the HistrapTM column
Set the flow rate at 50 mL/min and rinse 75 mL

Membrane protein extraction

#### [I. Materials & Apparatus](#collapse44)

●LB liquid medium with 0.1mg/mL chloramphenicol
●Membrane and Cytosol Protein Extraction Kit from BestBio company.
●Water bath

#### [II. Before Experiment](#collapse45)

1. 200μL bacteria solution and 10μL chloramphenicol were inoculated into 10mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.

#### [III. Protocol](#collapse46)

1. centrifuge all bacteria solution at 6,500rpm for 15 minutes at room temperature. Decant or aspirate medium and discard.
2. Add 20mL pbs buffer, pipet up and down to mix thoroughly. And centrifuge at 6,500rpm for 15 minutes at 4 °C. Decant or aspirate medium and discard.
3. Blot the supernatant.
4. add 1mL Membrane protein extract A, 10μL Membrane protein extract B and 4μL Protease inhibitor, and pipet up and down to mix thoroughly. Then transform the solution into new 1.5mL tubes.
5. place the tubes in refrigerator at 4 °C and resuspend every 10 minutes lasting about 2~2.5h.
6. centrifuge the solution at 12,000xg for 5 minutes at 4 °C. and inoculate supernatant into new 1.5 mL tubes.
7. place the tubes at 37 °C water bath for 30 minutes. Decant or aspirate supernatant and discard.
8. add 100μL Membrane Protein Solubilizer and leave overnight at 4°C.

SDS-PAGE with Coomassie Blue Stain

#### [I. Materials & Apparatus](#collapse47)

●Solarbio® SDS-PAGE Gel preparation kit
●Bio-Rad® Mini-PROTEAN System
●Double distilled water (ddH2O)
●Glycine (produced by Solarbio®)
●Tris Powder (produced by Solarbio®)
●SDS (produced by Solarbio®)
●Coomassie Blue R-250
●Isopropanol
●Glacial acetic acid
●Absolute ethyl alcohol
●Protein samples
●Prestained protein marker (produced by TransGen®)
●6 × Protein loaddng buffer (produced by TransGen®)
●50-mL centrifuge tube
●Electrophoresis power supply
●Microwave oven

#### [II. Before Starting](#collapse29)

1. Wash the Bio-Rad® Mini-PROTEAN System with ddH2O.
2. Prepare samples for electrophoresis.

#### [III. Protocol](#collapse28)

**A.Hand casting Polyacrylamide Gels**
1.Wash the glass plate and dry with a mirror paper, assemble the glass cassette sandwich.
2.Prepare the resolving gel solutions without APS and TEMED, mix gently .

|  | Resolving Gel | Stacking Gel |
| --- | --- | --- |
|  | 12% | 5% |
| Range | 12~60 kDa | — |
| ddH2O | 3.3mL | 3.42mL |
| 30% Acrylamide/Bis | 4 mL | 0.83 mL |
| 1 M Tris-HCl, pH 6.8 | — | 0.625 mL |
| 1.5 M Tris-HCl, pH 8.8 | 2.5 mL | — |
| 10% SDS | 100 μL | 50 μL |
| 10% APS | 100 μL | 75 μL |
| TEMED | 10 μL | 7.5 μL |
| Total Volume | 10 mL | 5 mL |

3.Add the APS and TEMED to resolving gel solution, pour 4.3 mL solution into the glass cassette sandwich using a pipet.
4.Using a pipet immediately overlay the monomer solution with ddH2O.
5.Allow the gel to polymerize 45-60 min, the gel is polymerized once you see a line from between the deionized water and the resolving gel. Pour off the overlay water.
6.Dry the area above the resolving gel with filter paper before pouring the stacking gel.
7.Prepare the stacking gel solutions without APS and TEMED, mix gently.
8.Add the APS and TEMED to stacking gel solution, pour the solution above the resolving gel using a pipet, pour until the liquid level reaches the upper edge.
9.Place the comb into the stacking gel and allow the gel to polymerize 30-45 min.
10.Remove the comb by pulling it straight up slowly and gently, rinse the wells completely with ddH2O.
\* Polymerized gel can be storage at 4 °C no more than 1 week when soaking in ddH2O without removing the comb if the electrophoresis not be applied immediately after preparation of the gel.
**B.Performing Electrophoresis**
1.Prepare 2000 mL running buffer:

| Total Volume | 1000 mL | 2000 mL |
| --- | --- | --- |
| Glycine | 18.8 g | 37.6 g |
| Tris Powder | 3.0 g | 6.0 g |
| SDS | 1.0 g | 2.0 g |
| ddH2O | 1000 mL | 2000 mL |

2.Prepare gels and assemble the electrophoresis cell:
a.Remove the comb form the gels and assemble the electrophoresis cell
b.Fill the inner and outer buffer chambers with running buffer. Fill the upper buffer chamber of each core with 200 mL of running buffer; fill the lower buffer chamber to the indicator mark for 2 gels (550 mL) or 4 gels (800 mL) with running buffer
3.Prepare samples
a.Add 6 × Protein loading buffer into 5 times the volume of protein sample, mix gently
b.Heat samples with boiled water bath for 8 minutes
c.Load the appropriate volume of the sample on the gel (~30 μL)
4.Connect the electrophoresis cell to the power supply and perform electrophoresis according to the following conditions:
a.Stage 1: 100V, 80 mA for 30 min
b.Stage 2: 150V, 120 mA for 40 min
5.After electrophoresis is complete, turn the power supply off and disconnect the electrical leads. Pop open the gel cassettes and remove the gel by floating it off the plate into water
6.Stain and image the gel
**C.Coomassie Blue Stain**
1.Prepare Coomassie Blue dyeing liquid and bleaching liquid:

|  | Dyeing liquid | Bleaching liquid |
| --- | --- | --- |
| Coomassie Blue R-250 | 1.0 g | –– |
| Absolute ethyl alcohol | –– | 100 mL |
| Isopropanol | 250 mL | –– |
| Glacial acetic acid | 100 mL | 200 mL |
| ddH2O | 650 mL | 700 mL |
| Glacial acetic acid | 1000 mL | 1000 mL |

2.Staining the gel:
a.Wash gels three times in ddH2O
b.Remove all water from staining container and add Coomassie blue dyeing liquid over the gel
c.Heat with microwave oven in medium-high heat in 1 minute
d.Agitate in 40~60 rpm on a shaking table for 8 min
3.Bleaching the staining
a.Remove all water from staining container and add dyeing liquid back into container
b.Wash gels three times in ddH2O
c.Add bleaching liquid over the gel
d.Heat with microwave oven in medium-high heat in 1 minute
e.Agitate in 40~60 rpm on a shaking table for 20 min
f.Pour the bleaching liquid back to container with activated carbon filtration
g.Repeat step 3d-3f for 2 times
4.Wash the gel with ddH2O and image the gel

#### [IV. Reference](#collapse26)

- Guide to Polyacrylamide Gel Electrophoresis
[1] Bio-Rad. A Guide to Polyacrylamide Gel Electrophoresis and Detection. [M]. Bio-Rad Laboratories, Inc., 2019.
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin\_6040.pdf
[2] Green M R, Sambrook J. Molecular cloning. A Laboratory Manual 4th, 2012.
- Usage of 6 × Loading Buffer
[1] Manual of 6 × Protein loading buffer
http://www.transgen.com.cn/data/upload/pdf/DL101\_2019-10-07.pdf
[2] Chen X, Lu X, Shu N, et al. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system. Scientific Reports, 2017, 7:44304.
[3] Yang S, Meng J, Yang Y, et al. A HSP60-targeting peptide for cell apoptosis imaging. Oncogenesis, 2016, 5(2):e201.
[4] Cheng Y, Liu Y, Wang Y, et al. Chicken DNA virus sensor DDX41 activates IFN-β signaling pathway dependent on chSTING. Developmental & Comparative Immunology, 2017, 76:334-342.

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

# Team:XMU-China/Results

![](https://static.igem.org/mediawiki/2020/c/c3/T--XMU-China--right30.png)
[![](https://static.igem.org/mediawiki/2020/d/d9/T--XMU-China--right31.png)
[Detection](#K1)](#K1)
[![](https://static.igem.org/mediawiki/2020/f/f6/T--XMU-China--right32.png)
[Degradation](#K2)](#K2)
[![](https://static.igem.org/mediawiki/2020/f/fa/T--XMU-China--right33.png)
[Kill switch](#K3)](#K3)

[![](https://static.igem.org/mediawiki/2020/c/cb/T--XMU-China--logo.png)](https://2020.igem.org/Team%3AXMU-China)

* [Home](https://2020.igem.org/Team%3AXMU-China)
* Team
  [+ Members](https://2020.igem.org/Team%3AXMU-China/Team)
  [+ Collaborations](https://2020.igem.org/Team%3AXMU-China/Collaborations)
  [+ Partnership](https://2020.igem.org/Team%3AXMU-China/Partnership)
  [+ Attributions](https://2020.igem.org/Team%3AXMU-China/Attributions)
  [+ Notebook](https://2020.igem.org/Team%3AXMU-China/Notebook)
  [+ Experiments](https://2020.igem.org/Team%3AXMU-China/Experiments)
  [+ Safety](https://2020.igem.org/Team%3AXMU-China/Safety)
* Project
  [+ Description](https://2020.igem.org/Team%3AXMU-China/Description)
  [+ Design](https://2020.igem.org/Team%3AXMU-China/Design)
  [+ Models](https://2020.igem.org/Team%3AXMU-China/Model)
  [+ Result](https://2020.igem.org/Team%3AXMU-China/Results)
  [+ Proof of Concept](https://2020.igem.org/Team%3AXMU-China/Proof_Of_Concept)
  [+ Hardware](https://2020.igem.org/Team%3AXMU-China/Hardware)
  [+ Proposed Implementation](https://2020.igem.org/Team%3AXMU-China/Implementation)
* Parts
  [+ Parts](https://2020.igem.org/Team%3AXMU-China/Parts)
  [+ Contributions](https://2020.igem.org/Team%3AXMU-China/Contribution)
  [+ Engineering Success](https://2020.igem.org/Team%3AXMU-China/Engineering)
  [+ Improve](https://2020.igem.org/Team%3AXMU-China/Improve)
* Human Practices
  [+ Overview](https://2020.igem.org/Team%3AXMU-China/Human_Practices)
  [+ Silver](https://2020.igem.org/Team%3AXMU-China/Human_Practices#Silver)
  [+ Gold](https://2020.igem.org/Team%3AXMU-China/Human_Practices#Gold)
  [+ Science Communication](https://2020.igem.org/Team%3AXMU-China/Education)
  [+ Supporting Entrepreneurship](https://2020.igem.org/Team%3AXMU-China/Entrepreneurship)
* Awards
  [+ Awards](https://2020.igem.org/Team%3AXMU-China/Awards)
  [+ Judging](https://2020.igem.org/Team%3AXMU-China/Judging)
  [+ Best Integrated Human Practices](https://2020.igem.org/Team%3AXMU-China/Human_Practices)
  [+ Best Education](https://2020.igem.org/Team%3AXMU-China/Education)
  [+ Best Supporting Entrepreneurship](https://2020.igem.org/Team%3AXMU-China/Entrepreneurship)
  [+ Best Model](https://2020.igem.org/Team%3AXMU-China/Model)
  [+ Best Hardware](https://2020.igem.org/Team%3AXMU-China/Hardware)
  [+ Best Basic Parts](https://2020.igem.org/Team%3AXMU-China/Parts#Basic)
  [+ Best Composite Parts](https://2020.igem.org/Team%3AXMU-China/Parts#Composite)
  [+ Best Part Collection](https://2020.igem.org/Team%3AXMU-China/Parts#Collection)

Detection of Glyphosate

The Cell-Surface Display System

What work we have done:

Verified that EGFP could be displayed on the surface of the cell by the function of BrkA.

Verified that EGFP could be displayed on the surface of the cell by the function of AIDA.

Verified that EGFP may be displayed on the surface of the cell by the function of INPNC.

Work has not finished yet:

Further certification of the expression and function of INPNC-EGFP.

Proof of expression

Our detection system focuses on NADPH, which is widely found and involved in many biochemical reactions. In order to eliminate the interference of **endogenous** NADPH, we wanted to display our enzymes employed in the detection system on the surface of *E. coli*, more details are shown on the [Design page](https://2020.igem.org/Team%3AXMU-China/Design). A comprehensive analysis of the characteristics of different kinds of anchor proteins was applied and we ultimately determine INPNC, BrkA, and AIDA for experimental implementation.

For the necessity of determining the anchoring ability of these three proteins, we fused anchor protein with EGFP and constructed three genes: *inpnc-egfp* ([BBa\_K3332061](http://parts.igem.org/Part%3ABBa_K3332061)), *egfp-aidA* ([BBa\_K3332063](http://parts.igem.org/Part%3ABBa_K3332063) ) and *egfp-brkA* ([BBa\_K3332064](http://parts.igem.org/Part%3ABBa_K3332064) ). The gene circuits were subsequently constructed to express these three genes of fusion proteins (Fig. 1a, Fig. 2a and Fig. 3a), carried by the pSB1C3 vector.

![](https://static.igem.org/mediawiki/2020/d/d6/T--XMU-China--XMU-China_2020-%E5%9B%9E%E8%B7%AF%2BINPNC-EGFP.png)

**Fig. 1 Gene circuit involved in protein expression and SDS-PAGE analysis of anchored EGFP by silver staining. a**, Gene circuit was constructed by [BBa\_K3332061](http://parts.igem.org/Part%3ABBa_K3332061) to express INPNC-EGFP. **b**, Target bands of INPNC-EGFP (white arrow, 70.4 kDa).

![](https://static.igem.org/mediawiki/2020/d/d9/T--XMU-China--XMU-China_2020-%E5%9B%9E%E8%B7%AF%2BEGFP-BrkA.png)

**Fig. 2 Gene circuit involved in protein expression and SDS-PAGE analysis of anchored EGFP by silver staining. a**, Gene circuit was constructed by [BBa\_K3332064](http://parts.igem.org/Part%3ABBa_K3332064) to express EGFP-BrkA. **b**, Target bands of EGFP-BrkA (black arrow, 76.6 kDa).

![](https://static.igem.org/mediawiki/2020/8/83/T--XMU-China--XMU-China_2020-EGFP-AIDA.png )

**Fig. 3 Gene circuit involved in protein expression and SDS-PAGE analysis of anchored EGFP by silver staining. a**, Gene circuit was constructed by [BBa\_K3332063](http://parts.igem.org/Part%3ABBa_K3332063) to express EGFP-AIDA. **b**, Target bands of EGFP-AIDA (white arrow, 80 kDa).

Target protein from the extracted membrane protein was observed on the SDS-PAGE gel, showing that EGFP-BrkA, INPNC-EGFP and EGFP-AIDA were successfully expressed and anchored into the membrane (Fig. 1b, Fig. 2b and Fig. 3b).

Quantitative analysis of fluorescence intensity

Positive colonies were cultivated to express the target protein. Then we harvested bacteria by centrifugation. The harvested bacteria, the lysate underwent centrifugation and its supernatant, were photographed, and then the fluorescence changes were quantitatively detected *via* microplate reader (Fig. 4 and Fig. 5).

![](https://static.igem.org/mediawiki/2020/2/2d/T--XMU-China--XMU-China_2020-AIDA_BrkA_%E8%8D%A7%E5%85%89.png)

**Fig. 4 Quantitative analysis of fluorescence intensity of EGFP-AIDA and EGFP-BrkA. a**, bacteria of *E. coli* with EGFP-AIDA#3 (left), EGFP (middle) and Blank (right) (J23100-B0034). **b**, bacteria of *E. coli* with EGFP-AIDA#4 (left), EGFP (middle) and Blank (right) (J23100-B0034). **c**, bacteria of *E. coli* with EGFP-BrkA, EGFP, and Blank (right) (J23100-B0034). **d**, the lysate underwent centrifugation and its supernatant of *E. coli* with Blank (J23100-B0034) (left), EGFP-AIDA#3 (middle) and EGFP (right), **e**, the lysate underwent centrifugation and its supernatant of *E. coli* with Blank (J23100-B0034) (left), EGFP-AIDA#4 (middle) and EGFP (right). **f**, the lysate underwent centrifugation and its supernatant of *E. coli* with Blank (J23100-B0034) (left), EGFP-BrkA (middle) and EGFP (right). **g**, Quantitative detection of fluorescence intensity of the resuspension and supernatant of broken samples.

![](https://static.igem.org/mediawiki/2020/e/ee/T--XMU-China--XMU-China_2020-INPNC_%E8%8D%A7%E5%85%89.png)

**Fig. 5 Quantitative analysis of fluorescence intensity of INPNC-EGFP. a**, bacteria of *E. coli* with Blank (J23100-B0034) (left), INPNC-EGFP (middle) and EGFP (right). **b**, the lysate underwent centrifugation and its supernatant of *E. coli* with Blank(J23100-B0034) (left), INPNC-EGFP (middle) and EGFP (right). **c**, Quantitative detection of fluorescence intensity of the resuspension and supernatant of broken samples.

By comparing the fluorescence intensity of samples shown on the photos, we could find that both the bacteria, the lysate underwent centrifugation and its supernatant of *E. coli* with J23100 had no fluorescence signal. While both the bacteria, the lysate underwent centrifugation and its supernatant of *E. coli* with EGFP had a **strong** fluorescence signal. Notably, after ultrasonication, the fluorescence signal was dispersed to both the supernatant and the precipitation, which meant the EGFP protein was normally expressed on the intracellular. However, samples of EGFP-fusion proteins had fluorescence signals most on the precipitation, which suggests that our anchor proteins successfully display EGFP on the surface of *E. coli*.

We further quantitatively detected fluorescence of the resuspension and supernatant of broken samples. And we found ratios of fluorescence intensity of the resuspension and supernatant had an obvious difference between experimental groups.

Confocal laser scanning microscope imaging

We used a confocal laser scanning microscope (CLSM) to further confirm the cell-surface display system successfully worked and the results are shown in Fig. 6.

![](https://static.igem.org/mediawiki/2020/c/c3/T--XMU-China--XMU-China_2020-confocal_laser_scanning_microscope_%28clsm%29_imaging.png )

**Fig. 6 Confocal laser scanning microscope (CLSM) imaging.**  J23100-B0034 was a negative control and EGFP was a positive control. Scale bars, 5 μm.

*E. coli* BL21 (DE3) carrying BBa\_E0040 (EGFP) was rod-shaped and the fluorescence was equably distributed in the bacteria. The fluorescence of *E. coli* BL21 (DE3) carrying [BBa\_K3332063](http://parts.igem.org/Part%3ABBa_K3332063) (EGFP-AIDA) or [BBa\_K3332064](http://parts.igem.org/Part%3ABBa_K3332064) (EGFP-BrkA) was observed to be **dotted** and dispersed on the surface of bacteria. The results proved that EGFP has been anchored to the surface of the *E. coli* by the function of AIDA or BrkA, which is very exciting.

However, the fluorescence of *E. coli* BL21 (DE3) carrying [BBa\_K3332061](http://parts.igem.org/Part%3ABBa_K3332061) (INPNC-EGFP) was observed to be equally distributed in the bacteria and rarely dispersed on the surface of *E. coli*. The results proved that EGFP might not be anchored to the surface of the *E. coli* by the function of INPNC. More certifications are needed for this part.

Glyphosate Oxidase (GOX)

What work we have done:

Assayed the enzymatic activities of the glyphosate oxidase GOX.

Verified that GOX could be displayed on the surface of the cell by the function of INPNC.

Verified that GOX could be displayed on the surface of the cell by the function of BrkA.

Verified that GOX could be displayed on the surface of the cell by the function of AIDA.

Assayed the enzymatic activities of the three fusion protein of GOX and anchor protein and found that INPNC-GOX has the best activity.

Work has not finished yet:

The kinetics of GOX.

Proof of expression

In order to detect glyphosate, we selected three genes from different sources: *gox* ([BBa\_K3332006](http://parts.igem.org/Part%3ABBa_K3332006) ), *grhpr* ([BBa\_K3332011](http://parts.igem.org/Part%3ABBa_K3332011) ), and *inap* ([BBa\_K3332001](http://parts.igem.org/Part%3ABBa_K3332001) ). GOX and GRHPR can degrade glyphosate into glycolic acid while consuming NADPH as a cofactor, and iNap can bind NADPH and show apparent fluorescence changes.

The constructed plasmid was transformed into *E. coli* BL21 (DE3). Positive colonies were selected by chloramphenicol preliminarily and then verified by colony PCR. The colonies, confirmed by sequencing finally, were cultivated to express target protein. And the total protein was gained from the supernatant after ultrasonication and centrifugation. GE AKTA Prime Plus FPLC System was used to harvest purified protein from the broken supernatant. An apparent protein peak in the AKTA FPLC System demonstrated the correct purified protein. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis, and Coomassie blue staining (Fig. 7).

![](https://static.igem.org/mediawiki/2020/4/45/T--XMU-China--XMU-China_2020-GOX%E7%BA%AF%E5%8C%96.png)

**Fig 7. Gene circuit involved in protein expression and SDS-PAGE analysis of GOX by Coomassie blue staining. a**, Gene circuit was constructed by [BBa\_K880005](http://parts.igem.org/Part%3ABBa_K880005) and [BBa\_K3332006](http://parts.igem.org/Part%3ABBa_K3332006) to express GOX. **b**, Target bands of GOX (pink arrow, 34.6 kDa).

As shown in SDS-PAGE of GOX, the target protein could be observed at 34.6 kDa on the purified protein lanes and broken supernatant lanes, but not in the control groups. These results demonstrated that the GOX protein with His-Tag was successfully expressed in *E. coli* BL21 (DE3) and purified using GE AKTA Prime Plus FPLC System.

*in vitro* characterization of GOX

After transforming glyphosate into glyoxylic acid through GOX, we employed GRHPR, a glyoxylate reductase from the human liver, to reduce glyoxylic acid with NADPH consumption. NADPH was easy to be detected **by the signal of OD340**.

GE AKTA Prime Plus FPLC System was used to purify GOX and GRHPR protein. Then, glyphosate solution, NADPH solution, and GOX & GRHPR purified protein dissolved in Tris-HCl (pH 7.5) were mixed to detect the changes of OD340 immediately using TECAN® Infinite M200 Pro to investigated the performance of this system. (Fig. 8)

![](https://static.igem.org/mediawiki/2020/c/cf/T--XMU-China--XMU-China_2020-GOX_OD340.png )

**Fig. 8 OD340-Time curve of GOX & GRHPR and negative control.**

As shown in Fig. 8, OD340 decreased as time went on in the group of GOX & GRHPR, while no decrease was observed in the negative control group (using GRHPR only). These results proved that GOX catalyzed the **degradation** reaction of glyphosate.

Expression of GOX fused with anchor protein

In order to display our protein on the surface of *E. coli*, GOX, GRHPR, and iNap with three types of anchor protein were fused (Figure 9a). They were inserted into the expression vectors with J23100 and B0034 ([BBa\_K880005](http://parts.igem.org/Part%3ABBa_K880005) ) to obtain composite parts. Then, these recombinant plasmids were transformed into *E. coli* DH5α, and the correct recombinant one was confirmed by chloramphenicol, PCR or restriction enzyme digestion, and sequencing (Fig. 9b-i).

PCR and enzyme-digested products were purified by agarose gel electrophoresis (Fig. 9b-d). Due to the limited time, we just successfully constructed eight fusion protein designed.

![](https://static.igem.org/mediawiki/2020/b/bf/T--XMU-China--XMU-China_2020-surface_display_system.png)

**Fig. 9 Gene circuit involved in protein expression and agarose gel electrophoresis of PCR or enzyme-digested product.**  Lane M: Marker. **a**, Gene circuits of enzymes used in our detection system fused with anchor proteins. **b**, the target band of *inpnc-gox*. **c**, the target band of *gox-aidA*. **d**, the target band of *gox-brkA*. **e**, the target band of *inpnc-grhpr*. **f**, the target band of *grhpr-aidA*. **g**, the target band of *grhpr-brkA*. **h**, the target band of *inpnc-inap*. **i**, the target band of *inap-aidA*.

After centrifugation, the harvested cells were resuspended and treated by membrane protein extraction kit to extract membrane protein. Membrane protein was verified by SDS-PAGE and silver staining (Fig. 10).

![](https://static.igem.org/mediawiki/2020/0/00/T--XMU-China--XMU-China_2020-INPNC-GOX_%E7%BA%AF%E5%8C%96.png)

**Fig. 10 Gene circuit involved in protein expression and SDS-PAGE analysis of INPNC-GOX by silver staining. a**, Gene circuit was constructed ([BBa\_K2922052](http://parts.igem.org/Part%3ABBa_K2922052)) to express INPNC-GOX. **b**, Target bands of INPNC-GOX (black arrow, 68.8 kDa).

Target protein from the membrane protein was observed through SDS-PAGE, which indicated that INPNC-GOX was successfully expressed and **anchored** into the membrane. Due to the limited time, the proof of expression of GOX-AIDA and GOX-BrkA was not finished finally, which was a great regret for us indeed.

Characterization of GOX fused with anchor protein

After mixed the glyphosate solution, NADPH solution, purified GRHPR protein dissolved in Tris-HCl (pH 7.5), and bacteria solution carrying GOX fused with anchor protein, OD340 was immediately detected using TECAN® Infinite M200 Pro to investigate the performance of GOX fused with anchor protein (Fig. 11).

![](https://static.igem.org/mediawiki/2020/5/5b/T--XMU-China--XMU-China_2020-GOX锚定酶活_.png )

**Fig. 11 OD340-Time curve of three fusion proteins of GOX and negative control.**

As shown in Fig. 11, OD340 decreased as time went on, when using bacteria solution carrying three types of anchored GOX. The OD340 decreased rate of J23100-B0034 and GOX-His samples was less than that of the INPNC-GOX samples. The results proved that INPNC-GOX, GOX-AIDA, and GOX-BrkA have been **displayed** on the surface and convert glyphosate as expected.

Glyoxylate reductase (GRHPR)

What work we have done:

Assayed the enzymatic activities of the glyoxylate reductase GRHPR.

Assayed the kinetics of GOX.

Verified that GRHPR could be displayed on the surface of the cell by the function of INPNC.

Verified that GRHPR could be displayed on the surface of the cell by the function of BrkA.

Verified that GRHPR could be displayed on the surface of the cell by the function of AIDA.

Assayed the enzymatic activities of the three fusion protein of GRHPR and anchor protein and found that INPNC-GRHPR has the best activity.

Proof of expression

GRHPR can convert glyoxylic acid into glycolic acid while NADPH is **consumed** as a cofactor. So, we constructed [BBa\_K3332056](http://parts.igem.org/Part%3ABBa_K3332056) which consists of [BBa\_K880005](http://parts.igem.org/Part%3ABBa_K880005) and [BBa\_K3332011](http://parts.igem.org/Part%3ABBa_K3332011) .

The constructed plasmid was transformed into *E. coli* BL21 (DE3). Positive colonies were selected by chloramphenicol preliminarily and verified by colony PCR. The colonies, confirmed by sequencing finally, were cultivated to express target protein. And the total protein was gained from the supernatant after ultrasonication. GE AKTA Prime Plus FPLC System was used to harvest purified protein from the broken supernatant. An apparent protein peak in the AKTA FPLC System demonstrated the correct purified protein. A purified protein was verified by the sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis and Coomassie blue staining (Fig. 12).

![](https://static.igem.org/mediawiki/2020/e/e8/T--XMU-China--XMU-China_2020-GRHPR_%E7%BA%AF%E5%8C%96.png)

**Fig 12. Gene circuit involved in protein expression and SDS-PAGE analysis of GRHPR by Coomassie blue staining. a**, Gene circuit was constructed by [BBa\_K880005](http://parts.igem.org/Part%3ABBa_K880005) and [BBa\_K3332011](http://parts.igem.org/Part%3ABBa_K3332011) to express GRHPR. **b**, Target bands of GRHPR (blue arrow, 36.7 kDa).

As showed by SDS-PAGE of GRHPR, the target protein was observed at 36.7 kDa on the purified protein lanes and broken supernatant lanes, but not in the control groups. The results showed that the GRHPR protein with His-Tag was successfully expressed in *E. coli* BL21 (DE3) and was successfully purified using GE AKTA Prime Plus FPLC System.

*in vitro* characterization of GRHPR

GRHPR was a glyoxylate reductase from the human liver, which reduce glyoxylic acid with NADPH consuming. NADPH was easy to be detected by the signal of OD340.

GE AKTA Prime Plus FPLC System was used to get purified GRHPR protein. After mixed Glyoxylic acid solution, NADPH solution, Tris-HCl, and GRHPR purified protein dissolved in Tris-HCl (pH 7.5), OD340 was immediately detected using TECAN® Infinite M200 Pro to investigate the performance of GRHPR (Fig. 13).

![](https://static.igem.org/mediawiki/2020/d/d4/T--XMU-China--XMU-China_2020-GRHPR%E9%85%B6%E6%B4%BB.png)

**Fig.13 OD340-Time curve of GRHPR and negative control.**

As shown in Fig. 13, OD340 of samples adding GRHPR **decreased** very quickly while the OD340 of control stay almost the same. These results proved that GRHPR works very well.

Expression of GRHPR fused with anchor protein

In order to display our protein on the surface of *E. coli*, GOX, GRHPR, and iNap with three types of anchor protein were fused. Then recombinant plasmids were transformed into *E. coli* DH5α, and the correct recombinant one was confirmed by chloramphenicol, PCR or restriction enzyme digestion, and sequencing.

PCR and enzyme-digested products were purified by agarose gel electrophoresis (Fig. 9e-g). INPNC-GRHPR, GRHPR-AIDA, and GRHPR-BrkA were successfully constructed.

After centrifugation, the harvested cells were resuspended and treated by membrane protein extraction kit to extract membrane protein. Membrane protein was verified by SDS-PAGE and silver staining (Fig. 14).

![](https://static.igem.org/mediawiki/2020/4/45/T--XMU-China--XMU-China_2020-GRHPR-BrkA_%E7%BA%AF%E5%8C%96.png)

**Fig 14. Gene circuit involved in protein expression and SDS-PAGE analysis of GRHPR-BrkA by silver staining. a**, Gene circuit was constructed ([BBa\_K2922060](http://parts.igem.org/Part%3ABBa_K2922060) ) to express GRHPR-BrkA. **b**, Target bands of GRHPR-BrkA (Black arrows, 85.4 kDa).

The target protein from the membrane protein was observed through the SDS-PAGE gel, which indicated that GRHPR-BrkA was successfully expressed and anchored into the membrane. Due to the limited time, the proof of expression of INPNC-GRHPR and GRHPR-AIDA was not finished finally, which was a great regret for us indeed.

Characterization of GRHPR fused with anchor protein

After mixed glyoxylic acid solution, NADPH solution, and bacteria solution carrying GRHPR fused with anchor protein, OD340 changes were immediately measured. When NADPH is consumed, OD340 **declines** (Fig. 15).

![](https://static.igem.org/mediawiki/2020/a/a5/T--XMU-China--XMU-China_2020-GRHPR%E9%94%9A%E5%AE%9A%E9%85%B6%E6%B4%BB.png)

**Fig. 15 OD340-Time curves of GRHPR fused with 3 types of anchor protein.**

We successfully got OD340-Time curves of GRHPR fused with 3 types of anchor protein. When using GRHPR fused with anchor protein, the decrease of OD340 was observed as the reaction went on. Compared with J23100-B0034 and GRHPR-His-Tag, great changes took place in the slope of the experimental group, especially for INPNC-GRHPR, which means that fusion protein is displayed on the surface and works very well.

Notably, the enzyme activity of **INPNC-GRHPR** was the best one of them.

iNap

What work we have done:

Assayed the enzymatic activities of the NADPH sensor iNap and got the working curve in the presence of NADPH.

Verified that iNap could be displayed on the surface of the cell by the function of INPNC.

Verified that iNap could be displayed on the surface of the cell by the function of AIDA.

Assayed the enzymatic activities of the two fusion protein of iNap and anchor protein and found that INPNC-iNap has better activity.

Work has not finished yet:

iNap-BrkA has not been got.

Proof of expression

iNap is a chimera of circularly permuted YFP (cpYFP) and the NADP(H) binding domain of Rex from *Thermus aquaticus* (T-Rex). It has an excitation peak near 420 nm and an emission peak near 528 nm. After **NADPH binding**, iNap showed apparent fluorescence changes. So, we constructed [BBa\_K3332046](http://parts.igem.org/Part%3ABBa_K3332046) which consists of [BBa\_K880005](http://parts.igem.org/Part%3ABBa_K880005) and [BBa\_K3332001](http://parts.igem.org/Part%3ABBa_K3332001).

The constructed plasmid was transformed into *E. coli* BL21 (DE3). Positive colonies were cultivated to express the target protein. And the total protein from the supernatant after ultrasonication and centrifugation. GE AKTA Prime Plus FPLC System was used to harvest purified protein from the broken supernatant. An apparent protein peak in the AKTA FPLC System demonstrated the correct purification of His-Tag-iNap protein. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis and Coomassie blue staining (Fig. 16).

![](https://static.igem.org/mediawiki/2020/b/b0/T--XMU-China--XMU-China_2020-iNAP_%E7%BA%AF%E5%8C%96.png)

**Fig.16 Gene circuit involved in protein expression and SDS-PAGE analysis of GRHPR by Coomassie blue staining. a**, Gene circuit was constructed by [BBa\_K880005](http://parts.igem.org/Part%3ABBa_K880005) and [BBa\_K3332001](http://parts.igem.org/Part%3ABBa_K3332001) to express iNap. **b**, Target bands of iNap (black arrow, 42.2 kDa).

As shown in SDS-PAGE of GRHPR, the target protein can be observed at 42.2 kDa on the purified protein lanes and broken supernatant lanes, but not in the control groups. The results showed that the iNap with His-Tag was successfully expressed in *E. coli* BL21 (DE3) and purified.

*in vitro* characterization of iNap

We mixed different concentrations of NADPH half-in-half with purified iNap protein dissolved in Tris-HCl (pH 7.5) and measure the fluorescence changes immediately. TECAN® Infinite M200 Pro was used to detect fluorescence intensity. The samples were excited at 420 nm, and the emission was measured at 528 nm (Fig. 17).

![](https://static.igem.org/mediawiki/2020/f/f7/T--XMU-China--XMU-China_2020-iNAP%E5%B7%A5%E4%BD%9C%E6%9B%B2%E7%BA%BF.png)

**Fig. 17 iNap fluorescence intensity which was excited at 420 nm in the presence of different concentrations of NADPH. Emission was measured at 528 nm.**

We successfully got a working curve of iNap in the presence of different concentrations of NADPH, which indicated that iNap is a **sensitive** detector of NADPH.

Expression of iNap fused with anchor protein

In order to display our protein on the surface of *E. coli*, GOX, GRHPR, and iNap with three types of anchor protein were fused. Then constructed plasmids were transformed into *E. coli* DH5α, and the correct recombinant one was confirmed by chloramphenicol, restriction enzyme digestion, and sequencing.

Enzyme-digested products were purified by agarose gel electrophoresis (Fig. 9h-i). INPNC-iNap and iNap-AIDA were successfully constructed.

Characterization of iNap fused with anchor protein

After culturing the engineering bacteria to OD600 = 1.8~2.0, *E. coli* with INPNC-iNap or iNap-AIDA were obtained by centrifuging at 4000 rpm. Then, the cell precipitation was washed three times with PBS buffer (pH 8.0), and the final precipitation was resuspended in PBS buffer, which was equal to the volume of the original medium.

NADPH solutions were mixed with washed INPNC-iNap cell precipitation dissolved in PBS buffer half-in-half (pH 8.0), and fluorescence changes were measured in the presence of different concentrations of NADPH. TECAN® Infinite M200 Pro was used to detect fluorescence intensity (Fig. 18).

![](https://static.igem.org/mediawiki/2020/f/fd/T--XMU-China--XMU-China_2020-iNAP%E9%94%9A%E5%AE%9A%E9%85%B6%E6%B4%BB.png)

**Fig. 18 a**, Fluorescence Intensity-Time curve of INPNC-iNap and negative control in the presence of different NADPH concentrations. **b**, Fluorescence Intensity-Time curve of iNap-AIDA, and negative control in the presence of different NADPH concentrations.

When using engineered bacteria expressing INPNC-iNap or iNap AIDA, we successfully got fluorescence intensity gradient in a certain concentration range of NADPH. However, when using *E. coli* carrying BBa\_K880005 (negative control in this case), no significant gradient could be observed through a vast range of NADPH’s concentration. In conclusion, our anchor proteins selected could **work well** and iNap could sense **extracellular** NADPH even displayed on the surface of bacteria.

Brief summary of detection

Taken the experiments’ results above together, GOX, GRHPR, and iNap could work effectively and some of our enzymes in **fusion protein-form** with different anchor proteins were **well characterized** anyway. In view of all of this, we finally completed the design and construction of the ultimate gene circuits and some necessary characterization works in advance of [demonstration](https://2020.igem.org/Team%3AXMU-China/Proof_Of_Concept).

Degradation of Glyphosate

Phosphonate acid transmembrane transport

What work we have done:

Verified that PhnE1E2 can obviously enhance the transport capability of chassis bacteria to glyphosate.

Work has not finished yet:

Verify the function of PhnE1 and PhnE2 separately.

PhnE1E2

We use the *phnE1* and *phnE2* gene from *Sinorhizobium meliloti* 1021 to enhance the ability of our chassis bacteria to transport glyphosate. We expressed the two genes in a **polycistron** way on the pSB1C3 vector (Fig. 19).

![](https://static.igem.org/mediawiki/2020/e/e7/T--XMU-China--XMU-China_2020-phnE1E2.png)

****Fig. 19 Gene circuit of *phnE1* and *phnE2*.****

The constructed plasmid was transformed into *E. coli* BL21 (DE3). Positive colonies that were selected by chloramphenicol preliminarily and then by colony PCR, restriction enzyme digestion (Fig. 20) and sequencing were cultivated to express target protein. And the total protein was gained by ultrasonication. The supernatant of culture and cells was separated by centrifugation. The harvested cells were then resuspended, treated by membrane protein extraction kit and subsequently got membrane protein, followed by SDS-PAGE and silver staining (Fig. 21). Notably, the bands of PhnE1 and PhnE2 were a little bit **obscure**. We attributed this to the difficulty of membrane protein’s expression in the engineered bacteria because the exogenous expression of membrane protein might change the permeability of membrane, to some extent, which seemed to be slightly poisonous.

![](https://static.igem.org/mediawiki/2020/0/01/T--XMU-China--XMU-China_2020-The_agarose_gel_electrophoresis_of_enzyme-digested_product_%28EcoR_I_%26_Pst_I%29.png)

**Fig. 20 The agarose gel electrophoresis of enzyme-digested product (*Eco*R I & *Pst* I).**

![](https://static.igem.org/mediawiki/2020/b/b8/T--XMU-China--XMU-China_2020-SDS-PAGE_analysis_of_PhnE1_%26_PhnE2_by_silver_staining.png)

**Fig. 21 SDS-PAGE analysis of PhnE1 and PhnE2 by silver staining.**

After verifying that the two proteins can be expressed, we used HPLC to verify the effect of PhnE1 and PhnE2 on the transport ability of glyphosate by chassis bacteria. After being cultured in LB medium containing glyphosate for a certain time, the content of glyphosate in the medium was determined by HPLC. The amount of glyphosate was represented by peak area. We found that compared with the blank control, the transformed bacteria could absorb **more** glyphosate (Fig. 22).

![](https://static.igem.org/mediawiki/2020/f/fb/T--XMU-China--XMU-China_2020-Relationship_between_the_peak_area_of_glyphosate_and_culture_time.png)

**Fig. 22 Relationship between the peak area of glyphosate and culture time.**

C-P cleavage of glyphosate

What work we have done:

Verified the relative inhibit effect to target genes of *phnJ* 0.94, *phnJ* 0.69, *phnF* 0.97 and *phnF* 0.75.

Verified the relative inhibit effect to target genes of *phnF* 0.97-*phnJ* 0.94 and *phnF* 0.97-phnJ 0.75.

Work has not finished yet:

Verify the relative inhibit effect to target genes of *phnF* 0.97-*phnJ* 0.94 and *phnF* 0.97-*phnJ* 0.75.

Verify the relative inhibit effect to target genes of each RNAi sequence with different promoters.

Verify the degradation activity of *phnJ* mut45.

RNAi

We designed siRNA sequences through **siRCon** which is developed by [Team Bielefeld-CeBiTec](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Software) and used them to hinder endogenous *phnF* and *phnJ* gene from expression. OmpA 5’ UTR and Hfq binding sequence are added to the flanks of siRNA sequences to enhance the binding its ability with target sequence as well as increase the half-life of siRNA. Promoter BBa\_J23101 and terminator BBa\_J61048 were used to transcribe the siRNA sequences on the pSB1C3 vector (Fig. 23).

![](https://static.igem.org/mediawiki/2020/1/14/T--XMU-China--XMU-China_2020-RNAicircuit.png)

**Fig. 23 Gene circuit of RNAi sequences.**

Due to sequencing errors of some sequences, we finally chose *phnF* 0.97, *phnJ* 0.94 and *phnJ* 0.75 for subsequent experiments. We assembled the RNAi sequence of *phnF* 0.97 with *phnJ* 0.94 and *phnJ* 0.75 separately, each of them was transcribed by promoter BBa\_J23101 and terminator BBa\_J61048 on the pSB1C3 vector (Fig. 24).

![](https://static.igem.org/mediawiki/2020/8/8a/T--XMU-China--XMU-China_2020-final_RNAi_sequences.png)

**Fig. 24 Gene circuit of final RNAi sequences we used.**

The constructed plasmid was transformed into *E. coli* BL21 (DE3). Positive colonies that were selected by chloramphenicol preliminarily and then by colony PCR, restriction enzyme digestion (Fig. 25) and sequencing were cultivated to extract total RNA and then verified the hindering rate of RNAi sequences to target genes by reverse transcription and *q*PCR (Fig. 26).

![](https://static.igem.org/mediawiki/2020/3/32/T--XMU-China--XMU-China_2020-The_agarose_gel_electrophoresis_of_enzyme-digested_product.png)

**Fig. 25 The agarose gel electrophoresis of enzyme-digested product (*Eco*R I & *Pst* I).**

![](https://static.igem.org/mediawiki/2020/b/be/T--XMU-China--XMU-China_2020-The_transcription_rate_of_phnJ_and_phnF_verified_by_reverse_transcription_and_qPCR.png)

**Fig. 26 The transcription rate of *phnJ* and *phnF* verified by reverse transcription and *q*PCR.**

It can be seen that the RNAi sequences are able to **downregulate** the expression of target genes, and the relative hindering rate was consistent with the prediction given by the siRCon.

PhnJ

The crack of C-P bond is a crucial step during the degradation process of glyphosate, where PhnJ protein is an **essential** subunit from C-P lyase that can crack C-P bond. Therefore, the *phnJ* gene from *Enterobacterales* was used to enhance the capability of our chassis bacteria to degrade glyphosate. We use promoter [BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100) , RBS [BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034) and terminator [BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015) to express the exogenous *phnJ* on the pSB1C3 vector (Fig. 27).

![](https://static.igem.org/mediawiki/2020/6/63/T--XMU-China--XMU-China_2020-phnjjj.png)

**Fig. 27 Gene circuit of *phnJ*.**

The constructed plasmid was transformed into *E. coli* BL21 (DE3). Positive colonies that were selected by chloramphenicol preliminarily and then by colony PCR, restriction enzyme digestion (Fig. 28) and sequencing were cultivated to express target protein. However, there are too many intracellular proteins with similar molecular weight to PhnJ that we couldn’t distinguish the target band on the polyacrylamide gel after staining. We also tried to purify the protein PhnJ-His-Tag with GE AKTA Prime Plus FPLC System, but due to the special structure of PhnJ, the His-Tag will be **wrapped** inside the protein so the protein couldn’t be ‘captured’ by the column. Finally, we did not get the SDS-PAGE results of PhnJ.

![](https://static.igem.org/mediawiki/2020/4/4d/T--XMU-China--XMU-China_2020-Figure_10_agarose.png)

**Fig. 28 The agarose gel electrophoresis of enzyme-digested product (*Eco*R I & *Pst* I).**

Based on our [modeling results](https://2020.igem.org/Team%3AXMU-China/Model), we designed three mutated *phnJ* sequences to further improve the glyphosate degradation ability of chassis bacteria (Fig. 29). In the first sequence, the 21st arginine was mutated to methionine; in the second sequence, the 16th threonine was mutated to serine, and the 40th arginine was mutated tyrosine; in the third sequence, the 45th proline was mutated to glutamine.

![](https://static.igem.org/mediawiki/2020/3/3f/T--XMU-China--XMU-China_2020-circuitphnj.png)

**Fig. 29 Gene circuits of three mutated *phnJ* sequences.**

The constructed plasmid was transformed into *E. coli* BL21 (DE3). Positive colonies that were selected by chloramphenicol preliminarily and then by sequencing were cultivated to express target protein. For the same reason, we did not get the SDS-PAGE results of PhnJ mutated sequences. We didn’t perform colony PCR and restriction enzyme digestion since these methods could not verify whether the mutation was successful or not.

AMPA degradation

What work we have done:

Verify that PhnO can obviously enhance the degradation capability of chassis bacteria to AMPA.

Work has not finished yet:

None.

PhnO

We use the *phnO* gene from *Salmonella enterica* LT2 to enhance the ability of our chassis bacteria to degrade aminomethylphosphoric acid (AMPA), which is the main metabolites of glyphosate. We expressed the proteins with promoter [BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100) and RBS [BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034) on the pSB1C3 plasmid backbone, [BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015) was used as a terminator (Fig. 30).

![](https://static.igem.org/mediawiki/2020/1/19/T--XMU-China--XMU-China_2020-circuit_of_phnO.png)

**Fig. 30 Gene circuit of *phnO*.**

The constructed plasmid was transformed into *E. coli* BL21 (DE3). Positive colonies that were selected by chloramphenicol preliminarily and then by colony PCR, restriction enzyme digestion (Fig. 31) and sequencing were cultivated to express target protein. And the total protein was gained by ultrasonication. We used GE AKTA Prime Plus FPLC System to get purified protein from broken sup and successfully corrected purified protein. Purified protein was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 32).

![](https://static.igem.org/mediawiki/2020/f/fb/T--XMU-China--XMU-China_2020-Figure_13._The_agarose_gel.png)

**Fig. 31 The agarose gel electrophoresis of enzyme-digested product (*Eco*R I & *Pst* I).**

![](https://static.igem.org/mediawiki/2020/a/af/T--XMU-China--XMU-China_2020-SDS-PAGE_analysis_of_PhnO.png)

**Fig. 32 SDS-PAGE analysis of PhnO by Coomassie blue staining.**

Velocities for the reaction of PhnO were determined by 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. Enzyme activity was determined through the microplate reader.

*K*m and *k*cat of different substrates were determined with a concentration gradient (Fig. 33, Table 1).

![](https://static.igem.org/mediawiki/2020/5/5e/T--XMU-China--XMU-China_2020-The_relationship_of_1Enzyme.png)

**Fig. 33 The relationship of 1/enzyme activity and 1/concentration of acetyl-CoA (A) and AMPA (B).**

| Compound | Km (mM) | kcat (s-1) |
| --- | --- | --- |
| acetyl-CoA | 0.1986 | 17.1394 |
| AMPA | 0.5637 | 3.2248 |

**Table 1. Enzyme kinetic constants with two substrates.**

Brief Summary of Degradation

In conclusion, the experimental result of *phnE1E2*, RNAi and *phnO* works well respectively. While *phnJ* was just verified in ultimate gene circuits (please see[https://2020.igem.org/Team:XMU-China/Proof\_Of\_Concept](https://2020.igem.org/Team%3AXMU-China/Proof_Of_Concept) ). These works ensured our following experiments of characterization of ultimate gene circuits of degradation system.

Kill switches

Kill Switch in Detection System

![](https://static.igem.org/mediawiki/2020/7/7e/T--XMU-China--XMU-China_2020-pBAD_inverter_mazF_B0015.png)

**Fig.34 Gene circuit of kill switch in detection system ([BBa\_K3332081](http://parts.igem.org/Part%3ABBa_K3332081)).**

This kill switch is mainly composed of three parts: pBAD/araC ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)), inverter ([BBa\_Q04510](http://parts.igem.org/Part%3ABBa_Q04510)) and *mazF* ([BBa\_K1096002](http://parts.igem.org/Part%3ABBa_K1096002)). When the *E. coli* is in our detection device where *L*-arabinose exists, the toxin gene *mazF* won't express and the *E. coli* won’t die. As long as the *E. coli* escape from our device to somewhere without *L*-arabinose, the *mazF* will express and the *E. coli* will be killed. More details could be seen in the [Design page](https://2020.igem.org/Team%3AXMU-China/Design).

What work we have done:

Verified that pBAD/araC can express downstream genes when induced by *L*-arabinose.

Verified that inverter has a reverse induction effect.

Verified that MazF have the function of killing *E. coli*.

Assayed the killing effect of kill switch in detection system ([BBa\_K3332081](http://parts.igem.org/Part%3ABBa_K3332081)).

The characterization of the function of inverter

Given the use of *L*-arabinose inducible promoter involved in the kill switch (Fig. 34) and the purpose to characterize the function of inverter which is also employed in the kill switch in Fig. 34, we use pBAD/araC ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)), inverter ([BBa\_Q04510](http://parts.igem.org/Part%3ABBa_Q04510)) and B0034-*eyfp*-B0015 ([BBa\_E0430](http://parts.igem.org/Part%3ABBa_E0430)) to construct the following circuits:

![](https://static.igem.org/mediawiki/2020/e/ea/T--XMU-China--XMU-China_2020-pBAD-EYFP%E6%8B%BC%E5%9B%BE.png)

**Fig. 35 Construction of gene circuits involved in characterization of inverter. a**, Comparing circuit ([BBa\_K3332082](http://parts.igem.org/Part%3ABBa_K3332082)) of *L*-arabinose inducible promoter, pBAD/araC. **b**, Characterization circuit for inverter ([BBa\_K3332079](http://parts.igem.org/Part%3ABBa_K3332079)). **c**, DNA gel electrophoresis of [BBa\_K3332082](http://parts.igem.org/Part%3ABBa_K3332082) (vector: pSB1C3, digested by *Spe* I and *Pst* I). **d**, DNA gel electrophoresis of [BBa\_K3332079](http://parts.igem.org/Part%3ABBa_K3332079) (vector: pSB1C3, digested by *Eco*R I and *Pst* I).

*eyfp* was designed in the downstream of pBAD/araC (Fig. 35a and 35c) to be set as a comparison of characterization circuit for inverter (Fig. 35b and 35d). By comparing the fluorescence intensity of induction group and non-induction group, the function of inverter can be characterized.

0.2% *L*-arabinose solution was added to induce for 6 hours, then the fluorescence intensity and corresponding OD600 of two groups was measured.

![](https://static.igem.org/mediawiki/2020/3/3d/T--XMU-China--XMU-China_2020-BY_BNY_fluorescence.png)

**Fig. 36 Fluorescence intensity/OD600 for induction group and non-induction group (6 hours).**  Data are collected and analyzed according to iGEM standard data analysis form.

The fluorescence intensity indicates the expression level of *eyfp*. The result shows an extremely high fluorescence intensity of the characterization circuit without inducer added and a much lower fluorescence intensity after inducing. It demonstrated that the inverter did actually **convert** the input inducible signal of *L*-arabinose to the shut-down of the downstream gene’s (*eyfp* in this case) expression (Fig. 36). While in the control group, which has no inverter constructed in it, had an opposite behavior to the characterization circuit. Taken together, the function of inverter was characterized and this part could work well to support subsequent constructions and applications.

Verified that MazF has the function of killing *E. coli*

We use pBAD/araC ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)), RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034) ), Terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015) ), *mazF* ([BBa\_K1096002](http://parts.igem.org/Part%3ABBa_K1096002)) to construct the following circuit (Fig. 37), [BBa\_K3332083](http://parts.igem.org/Part%3ABBa_K3332083)
, to verify the MazF's function to kill the bacteria carrying the *mazF* expression plasmid.

![](https://static.igem.org/mediawiki/2020/0/02/T--XMU-China--XMU-China_2020-pBAD-MazF.png)

**Fig.37. Construction of characterization circuit for MazF. a**, Gene circuit involved in characterize the function of MazF ([BBa\_K3332083](http://parts.igem.org/Part%3ABBa_K3332083)
). **b**, DNA gel electrophoresis of [BBa\_K3332083](http://parts.igem.org/Part%3ABBa_K3332083)
(vector: pSB1C3, colony PCR).

CFU assay was implemented to characterize the effect of toxin MazF (Fig. 38a). We discovered that the number of colonies decreased significantly (about 2-fold) after 5 hours, while there was no significant change in the non-induced group (Fig. 38b). This indicates that the *mazF*’s expression is successfully induced and toxin MazF could be lethal to the bacteria which has just toxin gene expressed but no antitoxin gene expressed/in them.

![](https://static.igem.org/mediawiki/2020/0/0e/T--XMU-China--XMU-China_2020-CFU1%E6%8B%BC%E5%9B%BE.png)

**Fig. 38 CFU assay for characterizing the function of MazF. a**, Solid medium plates of CFU assay, where plates marked “A” belonged to the non-induction group while those marked “B” belonged to the induction group. **b**, CFU (per mL liquid culture) calculated of different groups are plotted against time (h).

Assayed the killing effect of kill switch in detection system

We use pBAD/araC ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)), inverter ([BBa\_Q04510](http://parts.igem.org/Part%3ABBa_Q04510)), RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034) ), Terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015) ), *mazF* ([BBa\_K1096002](http://parts.igem.org/Part%3ABBa_K1096002)) to construct the following circuit:

![](https://static.igem.org/mediawiki/2020/b/b9/T--XMU-China--XMU-China_2020-pBAD-inverter-MazF.png)

**Fig. 39 Construction of gene circuit of kill switch in detection system. a**, Gene circuit of kill switch in detection system. **b**, DNA gel electrophoresis of [BBa\_K3332081](http://parts.igem.org/Part%3ABBa_K3332081) (vector: pSB1C3, colony PCR).

CFU assay was also carried out to characterize the effect of kill switch (Fig. 40a). We discovered that the number of colonies of *E. coli* carrying [BBa\_K3332081](http://parts.igem.org/Part%3ABBa_K3332081) (without induction) and pBAD/araC-*mazF* (induction) decreased significantly after 8 hours, while there was no significant change in pBAD/araC-inverter-*mazF* (induction) and pBAD/araC-*mazF* (without induction) groups (Fig. 40b), which could verify that [BBa\_K3332081](http://parts.igem.org/Part%3ABBa_K3332081) has the killing function without *L*-arabinose. In conclusion, the kill switch ([BBa\_K3332081](http://parts.igem.org/Part%3ABBa_K3332081)) in detection system did work to prevent engineered *E. coli* escaping and could achieve our purpose of bio-containment.

![](https://static.igem.org/mediawiki/2020/c/c4/T--XMU-China--XMU-China_2020-CFU2%E6%8B%BC%E5%9B%BE.png)

**Fig. 40 CFU assay for characterizing the killing effect of kill switch in detection system. a**, Solid medium plates of CFU assay, where plates marked “C” belonged to the non-induction group while those marked “D” belonged to the induction group. **b**, CFU (per mL liquid culture) calculated of different groups are plotted against time (h).

In addition to the above design, we also came up with other kill switch schemes during the brainstorming, which were not well characterized due to the limited time.

Alternative Kill Switch in Detection System

“Deadman” kill Switch:

![](https://static.igem.org/mediawiki/2020/5/56/T--XMU-China--XMU-China_2020-deadman-2.png)

**Fig. 41 “Deadman” kill switch.**

In this circuit, LacI can repress pTrc-2 promoter and pTrc-2 derivative promoter while TetR can repress pLtetO-1 promoter. When ATc (anhydrotetracyclin) exits, the pLtetO-1 promoter can’t be repressed due to the combination of ATc and TetR repressor. Then LacI which is expressed by pLtetO-1 can repress pTrc-2 promoter and pTrc-2 derivative promoter. As a result, *mf-lon* and *mazF* can’t be expressed. As a kind of *E. coli* toxin, MazF can cause the *E. coli* death. So there comes the conclusion that as long as the engineered *E. coli* are cultured in the environment with ATc, it won’t be killed by the function of MazF, but when the *E. coli* escape from our detection device, the effect can be reversed, that is to say, the *E. coli* will be killed by the MazF. In the same way, we can conclude that at the presence of IPTG, *mazF* can be expressed to cause *E. coli* death.

What work we have done:

Verified that pLtetO-1 promoter could be repressed by TetR protein, which can be inhibited by ATc.

Verified that pTrc-2 promoter and pTrc-2 derivative promoter could be repressed by LacI protein, which can be inhibited by IPTG.

Work has not finished yet:

Verify that mf-lon hydrolases has the function of recognizing and degrading the genes with a corresponding degron.

Assay the killing effect of Deadman kill switch.

Verified that pLtetO-1 promoter could be repressed by TetR protein, which can be inhibited by ATc

We use pLtetO-1 ([BBa\_K3332034](http://parts.igem.org/Part%3ABBa_K3332034)), B0034-*ecfp*-B0015 ([BBa\_E0420](http://parts.igem.org/Part%3ABBa_E0420)), [BBa\_J23106](http://parts.igem.org/Part%3ABBa_J23106) and [BBa\_P0140](http://parts.igem.org/Part%3ABBa_P0140) to construct the following circuits in Fig. 42.

![](https://static.igem.org/mediawiki/parts/a/ac/T--XMU-China--XMU-China_2020-106-tetR-pLtetO-1-ecfp.png)

**Fig. 42 Construction of gene circuits for verifying the function of pLtetO-1 promoter and TetR repressor. a**, Gene circuit ([BBa\_K3332084](http://parts.igem.org/Part%3ABBa_K3332084)) for characterizing pLtetO-1 promoter. **b**, Gene circuit ([BBa\_K3332085](http://parts.igem.org/Part%3ABBa_K3332085)) for verifying the function of TetR expressed by *tetR* gene. **c**, DNA gel electrophoresis of [BBa\_K3332084](http://parts.igem.org/Part%3ABBa_K3332084) (vector: pSB1C3 or pUC57, digested by *Eco*R I and *Pst* I). **d**, DNA gel electrophoresis of [BBa\_K3332085](http://parts.igem.org/Part%3ABBa_K3332085) (vector: pSB1C3, digested by *Spe* I and *Pst* I).

100 ng/mL ATc solution was added to induce for 6 hours, then measure the fluorescence intensity and corresponding OD600 of two groups of induction and non-induction (Fig. 43). There is no significant difference of fluorescence intensity/OD600 between the induction group and non-induction group of [BBa\_K3332084](http://parts.igem.org/Part%3ABBa_K3332084), indicating the constitutive expression of pLtetO-1 promoter when no TetR exists in the system. And the fluorescence intensity/OD600 of the non-induction group of [BBa\_K3332085](http://parts.igem.org/Part%3ABBa_K3332085) is rather low, compared to that of the induction group of this circuit, revealing that the gene *tetR* and its product TetR repressor could function well under the circumstance of the absence of ATc. Besides, the repressed pLtetO-1 promoter shows a **dramatic** change in expression level after induction, which presents an excellent inducibility for further utilization.

![](https://static.igem.org/mediawiki/2020/d/d5/T--XMU-China--XMU-China_2020-pLtetO.png)

**Fig. 43 Fluorescence intensity/OD600 for induction and non-induction group (6 hours).**  Data are collected and analyzed according to iGEM standard data analysis form.

Verified that pTrc-2 promoter and pTrc-2 derivative promoter could be repressed by LacI protein, which can be inhibited by IPTG

We use pTrc-2 promoter ([BBa\_K3332038](http://parts.igem.org/Part%3ABBa_K3332038)), pTrc-2 derivative promoter ([BBa\_K3332040](http://parts.igem.org/Part%3ABBa_K3332040)), B0034-*ecfp*-B0015 ([BBa\_E0420](http://parts.igem.org/Part%3ABBa_E0420)), pLtetO-1 ([BBa\_K3332034](http://parts.igem.org/Part%3ABBa_K3332034)), RBS1 ([BBa\_K3332036](http://parts.igem.org/Part%3ABBa_K3332036)
) and LacI-ssrAtag (mf-lon) ([BBa\_K3332037](http://parts.igem.org/Part%3ABBa_K3332037) ), Terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015) ) to construct the following circuits (Fig. 44):

![](https://static.igem.org/mediawiki/2020/b/b7/T--XMU-China--XMU-China_2020-pTrc-2%E7%B3%BB%E5%88%97.png)

**Fig. 44 Construction of gene circuits for characterizing pTrc-2 promoter and pTrc-2 derivative promoter.** Gene circuits: a, [BBa\_K3332087](http://parts.igem.org/Part%3ABBa_K3332087); b, [BBa\_K3332088](http://parts.igem.org/Part%3ABBa_K3332088); c, [BBa\_K3332089](http://parts.igem.org/Part%3ABBa_K3332089). DNA gel electrophoresis: d, [BBa\_K3332087](http://parts.igem.org/Part%3ABBa_K3332087) (vector: pUC57, digested by *Eco*R I and *Pst* I); e, [BBa\_K3332088](http://parts.igem.org/Part%3ABBa_K3332088) (vector: pSB1C3, digested by *Pst* I); f, [BBa\_K3332089](http://parts.igem.org/Part%3ABBa_K3332089) (vector: pUC57, digested by and *Pst* I).

We added IPTG solution to induce for 6 hours, then measure the fluorescence intensity and corresponding OD600 of four groups. We can see, after adding IPTG to induce the two promoters, the fluorescence intensity/OD600 of [BBa\_K3332088](http://parts.igem.org/Part%3ABBa_K3332088) and [BBa\_K3332089](http://parts.igem.org/Part%3ABBa_K3332089) both increased. The change of fluorescence intensity/OD600 after induction of pLtetO-1-lacI-pTrc-2-E0420 (ECFP) group ([BBa\_K3332088](http://parts.igem.org/Part%3ABBa_K3332088)) is larger than that of pLtetO-1-lacI-pTrc-2 derivative-E0420 (ECFP) group ([BBa\_K3332089](http://parts.igem.org/Part%3ABBa_K3332089)) (Fig. 45), so it can confirm us that the LacI has a weak inhibitory effect on pTrc-2 promoter and a strong inhibitory effect on pTrc-2 derivative promoter.

![](https://static.igem.org/mediawiki/2020/6/6f/T--XMU-China--XMU-China_2020-pTrc2_2d_%E6%8B%BC%E5%9B%BE.png)

**Fig. 45 Characterization of pTrc-2 and pTrc-2 derivative promoters. a**, photographs of liquid culture of the four groups. **b**, Fluorescence intensity/OD600 for induction and non-induction groups (6 hours). Data are collected and analyzed according to iGEM standard data analysis form.

Kill switch in degradation system

![](https://static.igem.org/mediawiki/2020/9/9b/T--XMU-China--XMU-China_2020-pHCHO_inverter_mazF_B0015.png)

**Fig. 46 Gene circuit of kill switch in degradation system** ([BBa\_K3332078](http://parts.igem.org/Part%3ABBa_K3332078)).

Formaldehyde promoter (pHCHO) provides an inducible scheme for downstream genes. Inverter plays the role of gene inversion which can **reverse** induction effect when existing. *mazF* is designed to follow formaldehyde promoter and inverter, which makes sense of our kill switch system in degradation: Formaldehyde promoter can be activated after formaldehyde induction and expression of *mazF* is blocked by reversion effect of inverter. In contrast, without formaldehyde, *mazF* can express under pR promoter inside inverter and cause cell death by MazF’s protein toxicity.

What work we have done:

Verified that formaldehyde promoter could be induced by formaldehyde.

Verified that inverter has a reverse induction effect.

Verified that MazF could kill *E. coli*.

Work has not finished yet:

Construct the total circuit and then verify its function.

Verified that formaldehyde promoter could be induced by formaldehyde

We use formaldehyde promoter ([BBa\_K1334002](http://parts.igem.org/Part%3ABBa_K1334002)), B0034\_ecfp\_B0015 ([BBa\_E0420](http://parts.igem.org/Part%3ABBa_E0420)) to construct the following circuit, [BBa\_K3332090](http://parts.igem.org/Part%3ABBa_K3332090) (Fig. 47a), which is used to characterize formaldehyde promoter.

![](https://static.igem.org/mediawiki/2020/0/0f/T--XMU-China--XMU-China_2020-pHCHO-ecfp%E6%8B%BC%E5%9B%BE.png)

**Fig. 47 Construction of gene circuit for characterizing formaldehyde promoter. a**,Gene circuit[BBa\_K3332090](http://parts.igem.org/Part%3ABBa_K3332090). **b**, DNA gel electrophoresis of [BBa\_K3332090](http://parts.igem.org/Part%3ABBa_K3332090) (vector: pSB1C3, digested by *Eco*R I and *Pst* I).

Because the formaldehyde (HCHO) is poisonous to *E. coli*, the growth curve of *E. coli* in different concentrations of formaldehyde was tested. Different concentration gradients of formaldehyde were added into the culture environment of *E. coli* and the growth curves were observed and recorded (Fig. 48).

![](https://static.igem.org/mediawiki/2020/9/91/T--XMU-China--XMU-China_2020-ProFormaldehyde_Expression_Model.png)

**Fig.48 [BBa\_K3332090](http://parts.igem.org/Part%3ABBa_K3332090) (ProFormaldehyde-ECFP) groups and [BBa\_K1334002](http://parts.igem.org/Part%3ABBa_K1334002) (ProFormaldehyde-pSB1C3) groups induced by different concentrations of formaldehyde.**

After induction, [BBa\_K3332090](http://parts.igem.org/Part%3ABBa_K3332090) groups (ProFormaldehyde-ECFP) had a generally higher fluorescence intensity/OD600 than that of [BBa\_K1334002](http://parts.igem.org/Part%3ABBa_K1334002) (ProFormaldehyde-pSB1C3) groups (Fig. 48), which indicates that formaldehyde promoter could work well. However, according to the growth curves recorded, the toxicity of formaldehyde is not obvious.

We have designed different improvement versions of formaldehyde promoters, which were characterized together with the original formaldehyde promoter ([BBa\_K1334002](http://parts.igem.org/Part%3ABBa_K1334002)). More details could be seen in the [Improve page](https://2020.igem.org/Team%3AXMU-China/Improve).

Verified that inverter could reverses expression pattern when exists

Refer to the above (kill switch in the detection system).

Verified that MazF have the function of killing *E. coli*

Refer to the above (kill switch in the degradation system).

Alternative Kill Switch in Degradation System

![](https://static.igem.org/mediawiki/2020/4/4b/T--XMU-China--XMU-China_2020-%E5%AE%9A%E6%97%B6%E5%BC%80%E5%85%B3.png )

**Fig. 49 Alternative kill switch in degradation system.**

PO promoter (pPO) and delta protein are used to realize the gradually increasing expression of toxin after ATc induction: when engineered *E. coli* are applied in soil, ATc can induce Tet promoter (pTet) to produce delta protein which can activate PO promoter. As a result, pPO could express delta protein and the toxin protein MazF, among which delta protein could continue activating pPO and generating more and more delta and toxin protein (forming a **positive** feedback). At the same time, the antitoxin MazE could be expressed by placing different constitutive promoters which have different expression levels in front of *mazE*. So the concentration of intracellular MazE is relatively constant, but as long as adding ATc, the kill switch will be **kicked off**, which means the concentration of MazF will increase gradually. When the amount of toxin exceeds the amount of antitoxin, the *E. coli* will be killed. By adding ATc before putting our engineered *E. coli* in the tea garden, the *E. coli* will be dead after a certain time which can be controlled by selecting different constitutive promoters for expressing antitoxin MazE.

Due to the limit of time, although we have these two mature kill switch designs for our degradation system, we choose the former as our main design. But the latter one inspired CSU and helped their [modeling part](https://2020.igem.org/Team%3ACSU_CHINA/Partnership).

Brief Summary of Kill Switch

In the sum of the experimental results above altogether, for detection system, the kill switch can **effectively** prevent engineered bacteria from escaping. Although the alternative has not been constructed completely, the three promoters: pTrc-2, pTrc2-derivative, pLtetO-1 of alternative were successfully characterized. For degradation system, the function of each part in the circuit of kill switch: formaldehyde promoter, MazF and inverter was verified but whether the total circuit could work or not hasn’t been testified yet. Meanwhile, the alternative of kill switch in degradation system still remains in the theory inquisition stage, which also inspired the modeling part of CSU.

Xiamen University, Fujian, China

No.422, Siming South Road, Fujian, P.R.China 361005

[![](https://static.igem.org/mediawiki/2020/1/1a/T--XMU-China--returntop.png)](#top)