iGEM 2019 — Team XMU-China

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:XMU-China/Notebook

* PROJECT
  + [Description](https://2019.igem.org/Team%3AXMU-China/Description)
  + [Design](https://2019.igem.org/Team%3AXMU-China/Design)
  + [Results](https://2019.igem.org/Team%3AXMU-China/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AXMU-China/Demonstrate)
  + [Parts](https://2019.igem.org/Team%3AXMU-China/Parts)
* [MODEL](https://2019.igem.org/Team%3AXMU-China/Model)
* HUMAN PRACTICES
  + [Overview](https://2019.igem.org/Team%3AXMU-China/Human_Practices)
  + [Silver](https://2019.igem.org/Team%3AXMU-China/Human_Practices#Silver)
  + [Gold](https://2019.igem.org/Team%3AXMU-China/Human_Practices#Gold)
  + [Public Engagement](https://2019.igem.org/Team%3AXMU-China/Public_Engagement)
  + [Collaborations](https://2019.igem.org/Team%3AXMU-China/Collaborations)
* OTHER WORK
  + [Contribution](https://2019.igem.org/Team%3AXMU-China/Contribution)
  + [Improve](https://2019.igem.org/Team%3AXMU-China/Improve)
  + [Experiment](https://2019.igem.org/Team%3AXMU-China/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AXMU-China/Notebook)
  + [Safety](https://2019.igem.org/Team%3AXMU-China/Safety)
* Team
  + [Members](https://2019.igem.org/Team%3AXMU-China/Team)
  + [Attributions](https://2019.igem.org/Team%3AXMU-China/Attributions)
  + [Judging](https://2019.igem.org/Team%3AXMU-China/Judging)

![](https://static.igem.org/mediawiki/2019/0/08/T--XMU-China--igemXMU.png)
![](https://static.igem.org/mediawiki/2019/d/d5/T--XMU-China--igem_icon.svg)

* TEAM
  + [Members](https://2019.igem.org/Team%3AXMU-China/Team)
  + [Attributions](https://2019.igem.org/Team%3AXMU-China/Attributions)
  + [Judging](https://2019.igem.org/Team%3AXMU-China/Judging)
* OTHER WORK
  + [Contribution](https://2019.igem.org/Team%3AXMU-China/Contribution)
  + [Improve](https://2019.igem.org/Team%3AXMU-China/Improve)
  + [Experiments](https://2019.igem.org/Team%3AXMU-China/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AXMU-China/Notebook)
  + [Safety](https://2019.igem.org/Team%3AXMU-China/Safety)
* HUMAN PRACTICES
  + [Overview](https://2019.igem.org/Team%3AXMU-China/Human_Practices)
  + [Silver](https://2019.igem.org/Team%3AXMU-China/Human_Practices#Silver)
  + [Gold](https://2019.igem.org/Team%3AXMU-China/Human_Practices#Gold)
  + [Public Engagement](https://2019.igem.org/Team%3AXMU-China/Public_Engagement)
  + [Collaborations](https://2019.igem.org/Team%3AXMU-China/Collaborations)
* [MODEL](https://2019.igem.org/Team%3AXMU-China/Model)
* PROJECT
  + [Description](https://2019.igem.org/Team%3AXMU-China/Description)
  + [Design](https://2019.igem.org/Team%3AXMU-China/Design)
  + [Results](https://2019.igem.org/Team%3AXMU-China/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AXMU-China/Demonstrate)
  + [Parts](https://2019.igem.org/Team%3AXMU-China/Parts)
* [HOME](https://2019.igem.org/Team%3AXMU-China)

![](https://static.igem.org/mediawiki/2019/0/04/T--XMU-China--notebook.png)

* March
* April - May
* June
* Week 1
  7.15 - 7.21
* Week 2
  7.22 - 7.28
* Week 3
  7.29 - 8.4
* Week 4
  8.5 - 8.11
* Week 5
  8.12 - 8.18
* Week 6
  8.19 - 8.25
* Week 7
  8.26 - 9.1
* Week 8
  9.2 - 9.8
* Week 9
  9.9 - 9.15
* Week 10
  9.16 - 9.22
* Week 11-12
  9.23 - 10.6
* Week 13
  10.7 - 10.13
* Week 14
  10.14 - 10.20

![](https://static.igem.org/mediawiki/2019/5/59/T--XMU-China--timeline_left.png)
![](https://static.igem.org/mediawiki/2019/0/01/T--XMU-China--timeline_right.png)

# March

- 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.

# April - May

- We were divided into four groups and started to brainstorm separately (and we reported each’s progress every weekend).
- Our team was formally accepted by iGEM.

# June

-All student members learned laboratory fundamental qualities and received molecular cloning operation training.

# Week 1

-[BBa\_K525998](http://parts.igem.org/Part%3ABBa_K525998) constructed to get the T7 promoter

![](https://static.igem.org/mediawiki/2019/1/19/T--XMU-China--0716.png)

# Week 2

-[BBa\_K2922026](http://parts.igem.org/Part%3ABBa_K2922026) and [BBa\_K2922029](http://parts.igem.org/Part%3ABBa_K2922029) are constructed.

![](https://static.igem.org/mediawiki/2019/c/c6/T--XMU-China--0725.png)

-[BBa\_K2922025](http://parts.igem.org/Part%3ABBa_K2922025) and [Bba\_K2922028](http://parts.igem.org/Part%3ABba_K2922028) are constructed

![](https://static.igem.org/mediawiki/2019/3/3d/T--XMU-China--0727.png)

# Week 3

-[BBa\_K1659003](http://parts.igem.org/Part%3ABBa_K1659003) with linker、[BBa\_K256400](http://parts.igem.org/Part%3ABBa_K256400) with linker、[BBa\_K118022](http://parts.igem.org/Part%3ABBa_K118022) with linker are constructed

![](https://static.igem.org/mediawiki/2019/8/8b/T--XMU-China--0729-1.png)

![](https://static.igem.org/mediawiki/2019/f/f1/T--XMU-China--0729-2.png)

-[BBa\_K1659003](http://parts.igem.org/Part%3ABBa_K1659003) with linker、[BBa\_K118023](http://parts.igem.org/Part%3ABBa_K118023) with linker are constructed

![](https://static.igem.org/mediawiki/2019/3/38/T--XMU-China--0820.png)

-[BBa\_K2922032](http://parts.igem.org/Part%3ABBa_K2922032) is constructed

![](https://static.igem.org/mediawiki/2019/7/72/T--XMU-China--0802-2.png)

-[BBa\_K2922002](http://parts.igem.org/Part%3ABBa_K2922002) is constructed

![](https://static.igem.org/mediawiki/2019/4/48/T--XMU-China--0803.png)

# Week 4

-[BBa\_K2922042](http://parts.igem.org/Part%3ABBa_K2922042) is constructed.

![](https://static.igem.org/mediawiki/2019/7/74/T--XMU-China--0807.png)

-[BBa\_K2922030](http://parts.igem.org/Part%3ABBa_K2922030) and [BBa\_K2922031](http://parts.igem.org/Part%3ABBa_K2922031) are constructed

![](https://static.igem.org/mediawiki/2019/f/f7/T--XMU-China--0810.png)

-[BBa\_K2922008](http://parts.igem.org/Part%3ABBa_K2922008) is constructed by colony PCR

![](https://static.igem.org/mediawiki/2019/0/0b/T--XMU-China--0810-2.png)

# Week 5

-[BBa\_K2922001](http://parts.igem.org/Part%3ABBa_K2922001) is constructed

![](https://static.igem.org/mediawiki/2019/1/1b/T--XMU-China--0814.png)

-[BBa\_K2922000](http://parts.igem.org/Part%3ABBa_K2922000) is constructed

![](https://static.igem.org/mediawiki/2019/c/c5/T--XMU-China--0815.png)

-[BBa\_K2922027](http://parts.igem.org/Part%3ABBa_K2922027) and [BBa\_K2922024](http://parts.igem.org/Part%3ABBa_K2922024) are constructed

![](https://static.igem.org/mediawiki/2019/9/96/T--XMU-China--0815-2.png)

# Week 6

-[BBa\_K2922025](http://parts.igem.org/Part%3ABBa_K2922025) and [BBa\_K2922026](http://parts.igem.org/Part%3ABBa_K2922026) are successfully ligated together to construct further biobricks.

![](https://static.igem.org/mediawiki/2019/9/9e/T--XMU-China--0819.png)

-[BBa\_K2922028](http://parts.igem.org/Part%3ABBa_K2922028) and [BBa\_K2922029](http://parts.igem.org/Part%3ABBa_K2922029) are successfully ligated together to construct further biobricks.

![](https://static.igem.org/mediawiki/2019/3/32/T--XMU-China--0819-2.png)

-[BBa\_K2922028](http://parts.igem.org/Part%3ABBa_K2922028), [BBa\_K2922029](http://parts.igem.org/Part%3ABBa_K2922029) and [BBa\_K2922027](http://parts.igem.org/Part%3ABBa_K2922027) successfully ligated together to construct further biobricks

![](https://static.igem.org/mediawiki/2019/3/36/T--XMU-China--0824.png)

# Week 7

-[BBa\_K2922007](http://parts.igem.org/Part%3ABBa_K2922007) is constructed by colony PCR

![](https://static.igem.org/mediawiki/2019/f/f5/T--XMU-China--0829.png)

-[BBa\_K2922033](http://parts.igem.org/Part%3ABBa_K2922033) is successfully constructed

![](https://static.igem.org/mediawiki/2019/1/18/T--XMU-China--0829-2.png)

-[BBa\_K2922025](http://parts.igem.org/Part%3ABBa_K2922025), [BBa\_K2922026](http://parts.igem.org/Part%3ABBa_K2922026) and [BBa\_K2922024](http://parts.igem.org/Part%3ABBa_K2922024) are successfully ligated together to construct further biobricks.

![](https://static.igem.org/mediawiki/2019/2/29/T--XMU-China--0829-3.png)

-[BBa\_K2922034](http://parts.igem.org/Part%3ABBa_K2922034) is constructed

![](https://static.igem.org/mediawiki/2019/0/0f/T--XMU-China--0830.png)

-[BBa\_K2922044](http://parts.igem.org/Part%3ABBa_K2922044) is constructed.

![](https://static.igem.org/mediawiki/2019/0/01/T--XMU-China--0901.png)

# Week 8

-[BBa\_K206000](http://parts.igem.org/Part%3ABBa_K206000) is constructed to get the pBAD (Arabinose promoter)

![](https://static.igem.org/mediawiki/2019/7/73/T--XMU-China--0902.png)

-[BBa\_K1033917](http://parts.igem.org/Part%3ABBa_K1033917) constructed to get the gfasPurple chromoprotein reporter.

![](https://static.igem.org/mediawiki/2019/0/0f/T--XMU-China--0905.png)

-[BBa\_K2922006](http://parts.igem.org/Part%3ABBa_K2922006) is constructed by colony PCR

![](https://static.igem.org/mediawiki/2019/9/97/T--XMU-China--0906.png)

-[BBa\_K2922015](http://parts.igem.org/Part%3ABBa_K2922015), [BBa\_K2922016](http://parts.igem.org/Part%3ABBa_K2922016), [BBa\_K2922017](http://parts.igem.org/Part%3ABBa_K2922017) are constructed by colony PCR.

![](https://static.igem.org/mediawiki/2019/4/4b/T--XMU-China--0906-2.png)

-[BBa\_K2922036](http://parts.igem.org/Part%3ABBa_K2922036) is constructed

![](https://static.igem.org/mediawiki/2019/2/20/T--XMU-China--0907.png)

-[BBa\_K2922038](http://parts.igem.org/Part%3ABBa_K2922038) successfully constructed.

![](https://static.igem.org/mediawiki/2019/9/9d/T--XMU-China--0908.png)

-[BBa\_K2922037](http://parts.igem.org/Part%3ABBa_K2922037) is constructed

![](https://static.igem.org/mediawiki/2019/6/64/T--XMU-China--0908-2.png)

# Week 9

-[BBa\_K2922018](http://parts.igem.org/Part%3ABBa_K2922018)、[BBa\_K2922019](http://parts.igem.org/Part%3ABBa_K2922019)、[BBa\_K2922020](http://parts.igem.org/Part%3ABBa_K2922020) are constructed.

![](https://static.igem.org/mediawiki/2019/9/90/T--XMU-China--0910-2.png)

-[BBa\_K2922012](http://parts.igem.org/Part%3ABBa_K2922012) is constructed by colony PCR.

![](https://static.igem.org/mediawiki/2019/1/1b/T--XMU-China--0915.png)

# Week 10

-[BBa\_K2922013](http://parts.igem.org/Part%3ABBa_K2922013) and [BBa\_K2922014](http://parts.igem.org/Part%3ABBa_K2922014) is constructed by colony PCR

![](https://static.igem.org/mediawiki/2019/0/02/T--XMU-China--0920.png)

-[BBa\_K2922035](http://parts.igem.org/Part%3ABBa_K2922035) is constructed.

![](https://static.igem.org/mediawiki/2019/c/c3/T--XMU-China--0920-2.png)

# Week 11-12

-[BBa\_K1033914](http://parts.igem.org/Part%3ABBa_K1033914) is constructed to get the J23110-RBS-amajlime

![](https://static.igem.org/mediawiki/2019/9/9b/T--XMU-China--1001.png)

-[BBa\_K2922021](http://parts.igem.org/Part%3ABBa_K2922021), [BBa\_K2922022](http://parts.igem.org/Part%3ABBa_K2922022) are constructed by colony PCR.

![](https://static.igem.org/mediawiki/2019/3/32/T--XMU-China--1013.png)

# Week 13

-[BBa\_K516132](http://parts.igem.org/Part%3ABBa_K516132) is constructed.

![](https://static.igem.org/mediawiki/2019/7/7b/T--XMU-China--1009.png)

# Week 14

-[BBa\_K2922023](http://parts.igem.org/Part%3ABBa_K2922023) is constructed.

![](https://static.igem.org/mediawiki/2019/7/71/T--XMU-China--1015.png)

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

![](https://static.igem.org/mediawiki/2019/b/bf/T--XMU-China--1016.png)

Xiamen University, Fujian, China

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


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:XMU-China/Experiments

* PROJECT
  + [Description](https://2019.igem.org/Team%3AXMU-China/Description)
  + [Design](https://2019.igem.org/Team%3AXMU-China/Design)
  + [Results](https://2019.igem.org/Team%3AXMU-China/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AXMU-China/Demonstrate)
  + [Parts](https://2019.igem.org/Team%3AXMU-China/Parts)
* [MODEL](https://2019.igem.org/Team%3AXMU-China/Model)
* HUMAN PRACTICES
  + [Overview](https://2019.igem.org/Team%3AXMU-China/Human_Practices)
  + [Silver](https://2019.igem.org/Team%3AXMU-China/Human_Practices#Silver)
  + [Gold](https://2019.igem.org/Team%3AXMU-China/Human_Practices#Gold)
  + [Public Engagement](https://2019.igem.org/Team%3AXMU-China/Public_Engagement)
  + [Collaborations](https://2019.igem.org/Team%3AXMU-China/Collaborations)
* OTHER WORK
  + [Contribution](https://2019.igem.org/Team%3AXMU-China/Contribution)
  + [Improve](https://2019.igem.org/Team%3AXMU-China/Improve)
  + [Experiment](https://2019.igem.org/Team%3AXMU-China/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AXMU-China/Notebook)
  + [Safety](https://2019.igem.org/Team%3AXMU-China/Safety)
* Team
  + [Members](https://2019.igem.org/Team%3AXMU-China/Team)
  + [Attributions](https://2019.igem.org/Team%3AXMU-China/Attributions)
  + [Judging](https://2019.igem.org/Team%3AXMU-China/Judging)

![](https://static.igem.org/mediawiki/2019/0/08/T--XMU-China--igemXMU.png)
![](https://static.igem.org/mediawiki/2019/d/d5/T--XMU-China--igem_icon.svg)

* TEAM
  + [Members](https://2019.igem.org/Team%3AXMU-China/Team)
  + [Attributions](https://2019.igem.org/Team%3AXMU-China/Attributions)
  + [Judging](https://2019.igem.org/Team%3AXMU-China/Judging)
* OTHER WORK
  + [Contribution](https://2019.igem.org/Team%3AXMU-China/Contribution)
  + [Improve](https://2019.igem.org/Team%3AXMU-China/Improve)
  + [Experiments](https://2019.igem.org/Team%3AXMU-China/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AXMU-China/Notebook)
  + [Safety](https://2019.igem.org/Team%3AXMU-China/Safety)
* HUMAN PRACTICES
  + [Overview](https://2019.igem.org/Team%3AXMU-China/Human_Practices)
  + [Silver](https://2019.igem.org/Team%3AXMU-China/Human_Practices#Silver)
  + [Gold](https://2019.igem.org/Team%3AXMU-China/Human_Practices#Gold)
  + [Public Engagement](https://2019.igem.org/Team%3AXMU-China/Public_Engagement)
  + [Collaborations](https://2019.igem.org/Team%3AXMU-China/Collaborations)
* [MODEL](https://2019.igem.org/Team%3AXMU-China/Model)
* PROJECT
  + [Description](https://2019.igem.org/Team%3AXMU-China/Description)
  + [Design](https://2019.igem.org/Team%3AXMU-China/Design)
  + [Results](https://2019.igem.org/Team%3AXMU-China/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AXMU-China/Demonstrate)
  + [Parts](https://2019.igem.org/Team%3AXMU-China/Parts)
* [HOME](https://2019.igem.org/Team%3AXMU-China)

![](https://static.igem.org/mediawiki/2019/8/85/T--XMU-China--experiments.png)

![](https://static.igem.org/mediawiki/2019/e/e6/T--XMU-China--right50.png)
[![](https://static.igem.org/mediawiki/2019/5/50/T--XMU-China--right51.png)
[Molecular Cloning Handbook](#Molecular_Cloning_Handbook)](#Molecular_Cloning_Handbook)
[![](https://static.igem.org/mediawiki/2019/0/04/T--XMU-China--right52.png)
[Cooperative experiment](#Cooperative_experiment)](#Cooperative_experiment)
[![](https://static.igem.org/mediawiki/2019/d/d0/T--XMU-China--right53.png)
[Aggressive experiment](#Aggressive_experiment)](#Aggressive_experiment)
[![](https://static.igem.org/mediawiki/2019/1/11/T--XMU-China--right54.png)
[Improve experiment](#Improve_experiment)](#Improve_experiment)
[![](https://static.igem.org/mediawiki/2019/b/ba/T--XMU-China--right55.png)
[Common experiment](#Common_experiment)](#Common_experiment)

Molecular Cloning Handbook

#### [Molecular Cloning Handbook 5.0 BETA of XMU-China](#collapse0)

[Molecular\_Cloning\_Handbook.pdf](https://static.igem.org/mediawiki/2019/b/b5/T--XMU-China--Molecular_Cloning_Handbook.pdf)

Cooperative experiment

Congo Red Assay

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

● LB broth agar plates with 0.2% CMC
● 1 mg/mL Congo Red solution
● 1 M NaCl

#### [II. Protocol](#collapse114_1_1)

1. LB broth agar plates with 0.2% CMC are inoculated with the crude enzyme.
2. Then the plates are flooded with 1 mg/mL Congo Red solution for 1 h.
3. Congo Red solution is poured off into a toxic waste bottle and 1 M NaCl is added and leave the plates for another 1 h.
4. Then pour off NaCl solution.
5. Observe the phenomenon on the plates.

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

1. S. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).
2. K. O. Duedu, C. E. French, Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. *Enzyme Microb Technol* 93, 113-121 (2016).

MUC Assay

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

● 4-Methylumbelliferyl *β*-D-cellobioside (MUC)
● Overnight culture grown in suitable medium
● Double distilled water (ddH2O)
● LB broth
● PBS buffer
● 1.5-mL centrifuge tube
● 50-mL centrifuge tube
● High speed refrigerated centrifuge
● Ultrasonic cell crusher
● Shaking incubator of 37 °C
● Black opaque 96-well plate (Costar®)
● Microplate absorbance readers (TECAN® infinite M200 PRO)

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

1. Dissolve MUC in ddH2O to 4 mM as MUC stock (100×), Dilute with ddH2O into working solution (5×).
2. Culture bacteria with target enzyme in suitable medium overnight.

#### [III. Protocol](#collapse13)

1. Measure OD600 of overnight culture grown in suitable medium.
2. Centrifuge at 5000 rpm in 4 °C for 10 min, collect supernatant and resuspend cells in PBS buffer.
3. Crush the cell suspension with ultrasonic cell crusher at power of 200 W for 66 times (3 seconds per time after gap of 6 seconds).
4. Centrifuge at 10000 rpm in 4 °C for 5 min, collect supernatant.
5. Add 200 μL MUC working solution (5×) into 800 μL culture supernatant or crushed cell supernatant as reaction system.
6. Add 200 μL MUC working solution (5×) into 800 μL LB broth or PBS buffer as background group.
7. Incubate under the condition of 37 °C, 200 rpm using a shaking incubator for reaction.
8. Take out one tube of reaction system into boiling water bath for 8 min to stop the reaction at the time of 15, 30, 60, 90, 120 minutes since reaction started.
9. Dilute reaction sample for 100 times and pipet 200 μL diluent into black opaque 96-well plate, measure fluorescence intensity (λex = 364 nm, λem = 640 nm) with TECAN® infinite M200 PRO.
10. Using the ratio of fluorescence intensity and OD600 to determine the activity of exoglucanase in samples.

#### [IV. Reference](#collapse14)

1. S. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).
2. K. O. Duedu, C. E. French, Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. *Enzyme Microb Technol* 93, 113-121 (2016).

OE-PCR (Overlap Extension-PCR)

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

● Accurate® Exp *Taq* DNA Polymerase
● DNA template
● Primers (synthesized by Biosune®)
● Nuclease-free 200-μL PCR tubes
● Biometra TAdvanced Thermal Cycler (Analyticjena®)

#### [II. Protocol](#collapse16)

**1. Prepare reaction system:**
![](https://static.igem.org/mediawiki/2019/7/72/T--XMU-China--11111.png)
**2. Set PCR program:**
![](https://static.igem.org/mediawiki/2019/a/ae/T--XMU-China--Set-PCR-program.png)

Activity of Beta-glucosidase:
 HPLC (High Performance Liquid Chromatography)

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

● 1×PBS buffer
● Crude enzyme solution (store at 4 ℃)
● Glucose (Sangon Biotech®)
● Cellobiose (Sangon Biotech®)
● 5 mM sulfuric acid
● Double distilled water (ddH2O)
● Agilent Technologies 1200 Series (Degasser, Quat Pump, ALS, TCC and RID)
● Agilent Technologies 1260 Inifinity (VWD)
● Column (Aminex HPX-87H Column, 300×7.8 mm)

#### [II. Protocol](#collapse17_1)

● **Sampling**
1. Take 0.25 g of cellobiose in a 250-mL shake flask, and 40 mL of PBS buffer is added to each.
2. Each group was taken 0.8 mL of cellobiose solution as blank.
3. Add 0.8 mL PBS buffer to the shake flask and add 200 μL of PBS to the 1.5-mL centrifuge tube.
4. Add 10 mL crude enzyme solution to each of the experimental group and the control group, and mark the number.
5. Take 1 mL cellobiose solution that the crude enzyme solution is just added as blank. The shaker reaction is then carried out at 37 °C, 200 rpm.
6. Take 1 mL of the reaction mixture at specified intervals, mark the name, number and time period, and place the samples in a boiling water bath as soon as possible to quench the reaction.
Gradient I: 10, 20, 30, 40, 50, 60, 80, 100, 120, 150, 180, 240, 300, 360 min
Gradient II: 10, 20, 30, 40, 50, 60, 80, 100, 120, 150, 180, 240 min
Gradient III: 15, 30, 45, 60, 90, 120, 180 min
● **Instrument pretreatment**
1. Preparation of 5 mM sulfuric acid mobile phase
a) Add a certain amount of water to the beaker, add 535 μL 98% H2SO4 and transfer them to a volumetric flask. After cooling to room temperature, make up to 2 L.
b) Filter by vacuum pump and ultrasonic defoam for 30 min.
2. Preparation of glucose and cellobiose standard working curve samples (0.1, 0.2, 0.5, 1.0, 2.0, 5.0 g/L)
a) Prepare 5.0 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 5.0 g/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 5 mM H2SO4) and start the pump.
d) Adjust the solution fill and open the pump valve. (counterclockwise)
e) RID—open heating valve—45 °C
f) RID—Control—Cleaning Reference Pool—Open—60 min
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 (Aminex HPX-87H Column, 300×7.8 mm).
i) Open the column oven at 65 °C, adjust the flow rate to 0.5 mL/min, and clean the reference cell for 1 h until the baseline is stable.
● **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.
● **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. Replace the mobile phase (pure water), set the filling amount, and adjust the flow rate to 1 mL/mL.
5. RID—Control a cleaning reference pool for 30 min.
6. The flow rate is adjusted to 0.2 mL/min.
7. Turn off the pump (RID control - turn off the heater), software, liquid meter, and computer in sequence.

Growth Curve for verifying that
 Beta-glucosidase can Function Intracellularly

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

● LB Liquid Medium (Sangon Biotech®)
● Complete M9 Medium & restricted M9 Medium
● 0.2 M IPTG
![](https://static.igem.org/mediawiki/2019/9/9a/T--XMU-China--IPTG2.png)
● Chloramphenicol
● Shaking incubator of 37 °C & 20 °C
● Spectrophotometer

#### [II. Before Experiment](#collapse15_3)

1. LB Liquid Medium: Sterilization, at 115 °C, 30 min.
2. IPTG: removal of bacteria by 0.22-μm filter.
3. Chloramphenicol: removal of bacteria by 0.22-μm filter.

#### [III. Steps](#collapse15_4)

● **Preparation of Complete M9 Medium & restricted M9 Medium**
Please sea ***Preparation of Restricted Medium***
● **Resuscitate glycerol bacteria**
1. The single colony, stored in 40% glycerol at -80 °C, is inoculated into LB liquid medium (10 mL) and grown overnight at shaking incubator of 37 °C.
2. 2 M IPTG is added (final concentration: 0.1 mM) to induce the expression at shaking incubator of 37 °C for another 4 hours.
● **Inoculation**
The culture after induced is taken out from the original LB liquid medium and added into the complete M9 medium/restricted M9 medium in accordance with the volumetric ratio of 3% to the new medium. The chloramphenicol and IPTG is added as well (final concentration: 50 μg/mL and 0.1 mM, respectively).
● **Measure the OD600 value**
The growth of bacteria is monitored by the OD600 value measured through spectrophotometer.

Aggressive experiment

Growth curve

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

● UV-1200 Spectrophotometer, MAPADA
● Quartz cuvette（MAPADA）
● Thermostatic oscillator（THZ-320, Shanghai Jinghong laboratory equipment Co.,Ltd.）
● LB liquid medium with 0.1 mg/mL chloramphenicol.

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

1．150 μL bacteria solution was inoculated into LB liquid mediums at 37 °C, 200 rpm until the OD600 value of each reach to 1.
2．25 sterile tubes are needed for the following experiment. 250 μL bacteria solution from step 1 is inoculated into each tube with 3.75 mL LB liquid medium, and then cultured at 37 °C and 200 rpm.
3．Take out one tube every 2 hours, 3 mL bacterial solution is needed for optical density measurement at wavelengths of 600 nm, 577 nm and 458 nm.
4．Take out all of tubes when the OD600 value exceeds 0.6. Then they are cultured at 37 °C and 200 rpm after addition of inducers (working concentration: lactose: 1mg/mL, arabinose: 1mg/mL IPTG: 1μg/mL).

Inhibition zone

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

● Culture dish
● Oxford cup with inner diameter of 6mm, outer diameter of 8mm and height of 10mm
● Chloramphenicol solution（1 mg/mL）
● LB liquid medium
● LB solid medium

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

1．Transfer plasmid pSB1C3 into *E.coli* DH5α and then inoculated it in LB liquid medium at 37 °C, 200 rpm until the OD600 value of each culture reach to 1.
2．150 mL LB solid medium is heated for melt, and then cool it until the temperature decreases to 50 °C. Mix the medium with chloramphenicol solution (180 μL) and indicator bacteria solution (30 mL) and then pour them into the plate (about 12 mL medium per plate). Divide the plate into four areas on average after solidification.
3．First, place the Oxford cup vertically on the surface of the culture medium in the clean bench; then, gently pressurized to make it contact with the culture medium tightly; at last, add the corresponding samples(100 μL) into the Oxford cup.
4．Culture the samples at 37 °C for 16-18 hours, observe phenomena during the experiments and measure the diameter of inhibition zone.

Improve experiment

Fluorescence Microscopy

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

● IPTG
● 1M Tris-HCl buffer (pH 7.5)
● NaCl
● LB liquid medium
● Chloramphenicol
● BL21 (DE3) glycerin bacteria to be tested
● 5(6)-carboxyfluorescein (Sigma-Aldrich®)
● Cryogenic refrigerated centrifuge
● Coverslip
● Glass slide
● Clear nail polish
● FiveEasy Benchtop pH Meter FE20
● Olympus IX73 fluorescence microscope

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

● **Culture and induction of bacteria**
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 0.6.
3. Add IPTG to a final concentration of 500 μM to induce the expression of target genes.
4. Add the fluorescent dye 5(6)-carboxyfluorescein to a concentration of 10 mM.
5. Incubate the culture at 20 °C for 24 h.
● **Preparation of wash buffer**
1. Take 2.5 mL of 1 M Tris-HCl solution (pH 7.5) into a new 50-mL centrifuge tube.
2. Weigh 438 mg of analytically pure NaCl and pour it into the centrifuge tube above.
3. Add 50 mL of cold purified water. Adjust the pH to 7.5 with a pH meter. The wash buffer of 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 is obtained.
● **Preparation of samples and shooting**
1. Pour the bacterial solution into a 50-mL centrifuge tube after induction. Centrifuge at 5000 rpm for 10 min to collect bacteria bodies.
2. Discard the supernatant, add 5 mL of wash buffer. Resuspend the pellets, and centrifuge at 5000 rpm for 10 min.
3. Repeat step 2. Wash the pellets until the color (orange) of the fluorescent dye 5(6)- carboxyfluorescein can not be seen. Discard the supernatant.
4. Add 25 mL of wash buffer and resuspend the cell pellets.
5. Take 20 μL of the resuspended droplets onto the coverslip. Gently attach the slide and coverslip together.
6. Apply a layer of clear nail polish around the coverslip to secure the coverslip and slide.
7. After the nail polish has dried, place it under the fluorescence microscope.
8. Select the oil microscope of the fluorescence microscope. The magnification is 100 times, shoot with bright field and BWA channel.
9. Save the bright field and fluorescent field photos with obvious effects.

#### [III. Reference](#collapse27)

1. J. Shin, *et al.*, Display of membrane proteins on the heterologous caveolae carved by caveolin-1 in the Escherichia coli cytoplasm. *Enzyme Microb Technol* **79-80**, 55–62 (2015).

Quantitative Analysis for the Capacity of Lead Absorption

#### [I. Materials & Apparatus](#collapse23)

● IPTG
● Pb(NO3)2
● Nitric acid
● LB liquid medium
● Chloramphenicol
● BL21 (DE3) glycerin bacteria to be tested
● Cryogenic refrigerated centrifuge
● ELAN ICP-DRC-*q*MS (PerkinElmer, SCIEX, Canada)
● 0.22-μm filter

#### [II. Protocol](#collapse24)

● **Preparation of samples to be tested**
1. Add 200 μL glycerin bacteria into 10 mL LB liquid medium containing 50 μg/mL chloramphenicol. Incubate the culture in a shaker overnight at 37 °C, 200 rpm.
2. Add 1 mL the culture above into 50 mL LB liquid medium containing 50 μg/mL chloramphenicol, and incubate it in a shaker of 37 °C and 200 rpm until OD600 reaches 0.5.
3. Add IPTG to a final concentration of 500 μM to induce the expression of target genes (DsbA-MBP, DsbA-PbBD and DsbA-MBP-CAV1).
4. Incubate the culture at 30 °C for 0.5 h.
5. Add Pb(NO3)2 to a final concentration of 50 μM. Shake the bacteria solution and divide them into four 50-mL centrifuge tubes evenly. Induce in a shaker at 37 °C, 200 rpm.
6. According to the set incubation time, periodically take out the centrifuge tubes from the shaker to stop the incubation.
7. Centrifuge at 5000 rpm for 10 min to collect bacterial bodies.
8. Discard the supernatant and wash the pellets with 1 mL of water. Repeat twice.
9. Resuspend the pellets in 1 mL of water and transfer the solution to a 1.5-mL centrifuge tube that has been weighed in advance.
10. Centrifuge at 5000 rpm for 5 min to collect bacterial bodies.
11. Discard the supernatant, dehydrate the bacteria at 60 °C for 12 h.
12. Weigh the tube and calculate the dry weight of the bacteria.
13. Add 1 mL of 2% dilute nitric acid to each 1.5-mL centrifuge tube. Digest bacteria in the water bath at 60 °C for 8 h avoiding light, and leave them at room temperature for 48 h to ensure digestion completely.
14. Filter the samples through a 0.22-μm filter after the digestion. The filtrates was diluted with purified water to obtain samples which can be tested.
● **Determination of lead concentration in samples**
1. The 208Pb element detection instrument is ELAN ICP-DRC-*q*MS (PerkinElmer, SCIEX, Canada).
2. Set mass spectrometry conditions: RF power, 1200 W; plasma gas flow, 15 L/min; auxiliary gas flow, 1.1 L/min; nebulizer gas flow, 0.88 L/min; lens voltage, 7.2 V; dwell time, 100 ms.
3. Set the injection mode as direct injection mode. Measure the signal intensity of 208Pb element in the samples, and substitute it into the corresponding range standard curve to calculate the lead concentration in the original samples. Obtain the Pb(Ⅱ) absorption amount per unit mass of bacteria.

#### [III. Reference](#collapse43)

1. <https://static.igem.org/mediawiki/2010/0/08/Protocol_for_chemical_inducible_expression_of_MBP_%26_sample_preparation.pdf>
2. C. Hui *et al.*, Surface display of metal binding domain derived from PbrR on Escherichia coli specifically increases lead(II) adsorption. *Biotechnol Lett* **40**, 837-845 (2018).

Common experiment

Preparation of Restricted Medium

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

● 1 M MgSO4
![](https://static.igem.org/mediawiki/2019/1/11/T--XMU-China--MgSO4-2.png)
● 1 M CaCl2
![](https://static.igem.org/mediawiki/2019/3/3a/T--XMU-China--CaCl2.png)
● 5×M9 Salt Solution
![](https://static.igem.org/mediawiki/2019/3/38/T--XMU-China--Salt-Solution.png)
● 20% Glucose
![](https://static.igem.org/mediawiki/2019/4/49/T--XMU-China--glu.png)
● 2% Cellobiose
![](https://static.igem.org/mediawiki/2019/8/85/T--XMU-China--cel.png)

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

1. Sterilize the 5×M9 Salt Solution, 1 M CaCl2 and 1 M MgSO4 at 115 °C for 30 min.
2. Pass the 20% Glucose and 2% Cellobiose through a 0.22-μm syringe filter.
3. Mix the medium according to the following formula:
![](https://static.igem.org/mediawiki/2019/4/40/T--XMU-China--medium.png)

Protein Expression and the Subsequent Treatment of Samples

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

● LB Liquid Medium (Sangon Biotech®)
● 1×PBS Buffer
![](https://static.igem.org/mediawiki/2019/2/2a/T--XMU-China--PBS.png)
● 0.2 M IPTG
![](https://static.igem.org/mediawiki/2019/c/c2/T--XMU-China--IPTG.png)
● Shaking incubator of 37 °C & 20 °C
● Large centrifuge
● Spectrophotometer

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

1. LB liquid medium: Sterilization, at 115 °C, 30 min.
2. IPTG: removal of bacteria by 0.22-μm filter membrane.

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

● **Cultivation**
1. A single colony from LB broth agar plates stored in 40% glycerol at -80 °C is inoculated into 10 mL of LB liquid medium and grown overnight at shaking incubator of 37 °C.
2. Then inoculated at 1% by volume into fresh LB liquid medium.
3. Cell’s growth is monitored with a spectrophotometer equipped.
● **Induced**
1. When the value of OD600 is between 0.6 and 0.8, added 0.1 mM IPTG to induce protein expression.
2. After induced under the condition of “20 °C, 200 rpm” for 24 h, the cells are harvested.
● **Get the protein samples from the culture medium and the cells**
1. Then the protein samples from the culture medium is obtained by centrifugation for 5 min, at 6500 rpm, 4 ℃.
2. The cell precipitation is washed twice with PBS buffer, and the final precipitation is resuspended in PBS buffer, which is equal to the volume of the original medium, and then the cells are broken on the ice. Then complete lysis on ice.
3. The supernatant is obtained by centrifugation for 5 min at 10000 rpm.

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 loading buffer (produced by TransGen®)
● 50-mL centrifuge tube
● Electrophoresis power supply
● Microwave oven

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

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

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

● **Hand casting Polyacrylamide Gels**
Please see ***SDS-PAGE with Silver Stain***.
● **Electrophoresis**
Please see ***SDS-PAGE with Silver Stain***.
● **Coomassie Blue Stain**
1. Prepare Coomassie Blue dyeing liquid and bleaching liquid:
![](https://static.igem.org/mediawiki/2019/2/2a/T--XMU-China--Coomassie-Blue.png)
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 min.
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 min.
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**
Please see ***SDS-PAGE with Silver Stain***.
● **Usage of 6×Loading Buffer**
Please see ***SDS-PAGE with Silver Stain***.

SDS-PAGE with Silver Stain

#### [I. Materials & Apparatus](#collapse25)

● Solarbio® SDS-PAGE Gel preparation kit
● Bio-Rad® Mini-PROTEAN System
● Double distilled water (ddH2O)
● Milli-Q Water
● Glycine (produced by Solarbio®)
● Tris Powder (produced by Solarbio®)
● SDS (produced by Solarbio®)
● Beyotime® Fast Silver Stain Kit
● Protein samples
● Prestained protein marker (produced by TransGen®)
● 6×Protein loading buffer (produced by TransGen®)
● 50-mL centrifuge tube
● Electrophoresis power supply
● Microwave oven

#### [II. Before Experiment](#collapse37)

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

#### [III. Protocol](#collapse38)

● **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.
![](https://static.igem.org/mediawiki/2019/7/74/T--XMU-China--resolving-gel.png)
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 for 45-60 min, the gel is polymerized once you see a line from between the ddH2O 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 for 30-45 min.
10. Remove the comb by pulling it straight up slowly and gently, rinse the wells completely with ddH2O.
*Tips: 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.*
● **Electrophoresis**
**1.** Prepare 2000 mL running buffer:
![](https://static.igem.org/mediawiki/2019/9/96/T--XMU-China--total_V.png)
**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 min.
c) Load the appropriate volume of the sample on the gel (about 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.
● **Silver Stain**
**1.** Wash the staining container and transform gel into staining container.
**2.** Fixation：
a) Prepare fixative liquid: 50 mL ethanol/10 mL acetic acid/40 mL Milli-Q water.
b) Pour fixative liquid over the gel.
c) Agitate in 60-70 rpm on a shaking table for 40 min.
**3.** Ethanol wash:
a) Discard fixation liquid, add 100 mL 30% ethanol over the gel.
b) Agitate in 60-70 rpm on a shaking table for 10 min.
**4.** Water Wash:
a) Discard 30% ethanol, add 200 mL Milli-Q water over the gel.
b) Agitate in 60-70 rpm on a shaking table for 10 min.
**5.** Sensitize:
a) Prepare sensitizer liquid: 99 mL Milli-Q water/1 mL sensitizer liquid (100×).
b) Discard Milli-Q water, add 100 mL sensitizer liquid over the gel.
c) Agitate in 60-70 rpm on a shaking table for 2 min.
**6.** Water Wash:
a) Discard sensitizer liquid, add 200 mL Milli-Q water over the gel.
b) Agitate in 60-70 rpm on a shaking table for 1 min.
c) Repeat water wash.
**7.** Silver stain:
a) Prepare silver reagent: 99 mL Milli-Q water/1 mL silver reagent (100×).
b) Discard Milli-Q water, add 100 mL silver reagent over the gel.
c) Agitate in 60-70 rpm on a shaking table for 10 min.
**8.** Water Wash:
a) Discard sensitizer liquid, add 100 mL Milli-Q water over the gel.
b) Agitate in 60-70 rpm on a shaking table for 1 min (No more than 1.5 min).
**9.** Developing:
a) Prepare developer reagent: 80 mL Milli-Q water/20 mL silver stain developer (5×)/0.05 mL accelerator.
b) Discard Milli-Q water, add 100 mL developer reagent over the gel.
c) Agitate in 60-70 rpm on a shaking table for about 2.5 min, until brown band appears.
**10.** Stop:
a) Prepare stop reagent: 95 mL Milli-Q water/20 mL silver stain stop reagent (20×).
b) Discard developer reagent, add 100 mL stop reagent over the gel.
c) Agitate in 60-70 rpm on a shaking table for 10 min.
**11.** Water Wash:
a) Discard stop reagent, add 100 mL Milli-Q water over the gel.
b) Agitate in 60-70 rpm on a shaking table for 4 min.
**12.** Wash the gel with ddH2O and image the gel.

#### [IV. Reference](#collapse39)

● **Guide to Polyacrylamide Gel Electrophoresis**
[1. Bio-Rad. A Guide to Polyacrylamide Gel Electrophoresis and Detection. 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](http://www.transgen.com.cn/data/upload/pdf/DL101_2019-10-07.pdf%20)
2. X. Chen, *et al.*, Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system. *Sci Rep* **7**, 44304 (2017).
3. S. Yang, *et al.*, A HSP60-targeting peptide for cell apoptosis imaging. *Oncogenesis* **5**, e201 (2016).
4. Y. Cheng, *et al.*, Chicken DNA virus sensor DDX41 activates IFN-β signaling pathway dependent on chSTING. *Dev Comp Immunol* **76**, 334-342 (2017).
● **Silver Stain**
1. Manual of Beyotime® Fast Silver Stain Kit:
<https://www.beyotime.com/Manual/P0017S%20%E5%BF%AB%E9%80%9F%E9%93%B6%E6%9F%93%E8%AF%95%E5%89%82%E7%9B%92.pdf>
2. D. Xiang, *et al.*, Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli. *Protein Expression Purif* **69**, 153-158 (2010).

Xiamen University, Fujian, China

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

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# Team:XMU-China/Results

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![](https://static.igem.org/mediawiki/2019/9/9e/T--XMU-China--rignt0.png)
[![](https://static.igem.org/mediawiki/2019/d/db/T--XMU-China--right1.png)
[Cooperative](#Cooperative)](#Cooperative)
[![](https://static.igem.org/mediawiki/2019/c/cd/T--XMU-China--right2.png)
[Aggressive](#Aggressive)](#Aggressive)
[![](https://static.igem.org/mediawiki/2019/1/1c/T--XMU-China--right3.png)
[Supplement](#Supplementary_Materials)](#Supplementary_Materials)

* PROJECT
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* TEAM
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* OTHER WORK
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Cooperative

## What work we have done:

Assayed the enzymatic activities of three cellulases separately.

Verified that YebF could be secreted out of the cell.

Verified that YebF-cellulase fusion protein could be secreted out of cell.

Assayed the enzymatic activity of YebF-cellulase fusion protein out of and inside cell.

Verified that cellulases could be secreted out of cell with the help of Kil secretion cassette.

Assayed the enzymatic activity of cellulases out of and inside cell in the presence of Kil secretion cassette.

Verified that cellobiose could be degraded by beta-glucosidase intracellularly.

## Work has not finished yet:

Quantitative analysis for the enzymatic activity of CenA.

## Get more information of our experiments, please [click here](https://2019.igem.org/Team%3AXMU-China/Experiments).

# The Expression of Cellulases

In order to degrade cellulose, we selected three genes from different sources: *bgl1A* ([BBa\_K2564000](http://parts.igem.org/Part%3ABBa_K2564000)), *cex* ([BBa\_K118022](http://parts.igem.org/Part%3ABBa_K118022)) and *cenA* ([BBa\_K118023](http://parts.igem.org/Part%3ABBa_K118023)). Cex and CenA degraded cellulose into cellobiose, and Bgl1A degraded cellobiose into glucose (*1*). We use T7 promoter and RBS ([BBa\_K525998](http://parts.igem.org/Part%3ABBa_K525998)) to express these proteins at a high level.

The constructed plasmid was transformed into *E. coli* BL21 (DE3). Positive colonies that were selected by chloramphenicol preliminarily and then by colony PCR, while finally confirmed by sequencing were cultivated and induced by IPTG to express cellulases. The supernatant of culture, namely *sup*, was obtained by centrifugation. And the total protein was gained by ultrasonication. The lysate underwent centrifugation and its supernatant, namely *broken sup*, was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 1-3).

![](https://static.igem.org/mediawiki/2019/8/8c/T--XMU-China--XMU-China_result_1.png)

**Fig. 1. Gene circuit involved in protein expression and SDS-PAGE analysis of CenA by Coomassie blue staining.** (**A**) Gene circuit was constructed by BBa\_K525998 and BBa\_K118023 to express endoglucanase (CenA). (**B**) Target bands of CenA (red arrow, 47 kDa).

![](https://static.igem.org/mediawiki/2019/1/1e/T--XMU-China--XMU-China_result_2.png)

**Fig. 2. Gene circuit involved in protein expression and SDS-PAGE analysis of Cex by Coomassie blue staining.** (**A**) Gene circuit was constructed by BBa\_K525998 and BBa\_K118022 to express exoglucanase (Cex). (**B**) Target bands of Cex (red arrow, 47 kDa).

![](https://static.igem.org/mediawiki/2019/e/eb/T--XMU-China--XMU-China_result_3.png)

**Fig. 3. Gene circuit involved in protein expression and SDS-PAGE analysis of Bgl1A by Coomassie blue staining.** (**A**) Gene circuit was constructed by BBa\_K525998 and BBa\_K2564000 to express beta-glucosidase (Bgl1A). (**B**) Target bands of Bgl1A (red arrow, 53 kDa).

As showed by SDS-PAGE of CenA/Cex/Bgl1A, the target protein can be observed at 47 kDa/47 kDa/53 kDa on the broken *sup* lanes, but not in the control groups. The results showed that the three cellulases were successfully expressed in *E. coli* BL21 (DE3). And there were no target protein bands in the supernatant of the culture, indicating that these proteins could not be secreted by themselves.

# The Secretion of YebF

YebF ([BBa\_K1659003](http://parts.igem.org/Part%3ABBa_K1659003)) is an *E. coli* motor protein. When the coding sequences of other protein are fused to the *yebF* sequence, YebF can help secrete these proteins out of the *E. coli* membrane (*2*). YebF was verified to be secreted out of *E. coli* BL21 (DE3) through SDS-PAGE (Fig. 4B).

![](https://static.igem.org/mediawiki/2019/8/8d/T--XMU-China--XMU-China_result_4.png)

**Fig. 4. Gene circuit involved in protein expression and SDS-PAGE analysis of YebF by Coomassie blue staining.** (**A**) Gene circuit was constructed by BBa\_K525998 and BBa\_K1659003 to express YebF. (**B**) Target bands of YebF (red arrow, 13 kDa).

Target protein from the supernatant of culture was observed at about 13 kDa on the SDS-PAGE gel, showing that YebF was successfully expressed and secreted into the medium.

# The Secretion of YebF-cellulase Fusion Protein

In order to secrete our cellulases out of the *E. coli* membrane, we fused the cellulase gene fragments with *yebF* gene fragment at the N-terminal using a **flexible** GS linker (GGGGS) by overlap extension polymerase chain reaction (OE-PCR). PCR products were purified by agarose gel electrophoresis (Fig. 5).

![](https://static.igem.org/mediawiki/2019/7/7f/T--XMU-China--XMU-China_result_5.png)

**Fig. 5. Agarose gel electrophoresis of OE-PCR product. Lane M: Marker.** (**A**) The target bands of *yebF* (about 363 bp) and *bgl1A* (about 1400 bp) in the first step of OE-PCR to fuse the two sequences. (**B**) The target band of *yebF-bgl1A* (about 1764 bp) in the second step of OE-PCR. (**C**) The target bands of *yebF* (about 363 bp) and *cenA* (about 1353 bp) in the first step of OE-PCR to fuse the two sequences. (**D**) The target band of *yebF-cenA* (about 1731 bp) in the second step of OE-PCR. (**E**) The target bands of *yebF* (about 363 bp) and *cex* (about 1469 bp) in the first step of OE-PCR to fuse the two sequences. (**F**) The target band of *yebF-cex* (about 1839 bp) in the second step of OE-PCR. All target bands are showed in red boxes.

These parts were inserted into the expression vectors with T7 and RBS ([BBa\_K525998](http://parts.igem.org/Part%3ABBa_K525998)) by restriction sites *Eco*RI and *Pst*I. Then the ligation mixture was transformed into *E. coli* DH5α, and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.

The supernatant of culture and cells was separated by centrifugation. The harvested cells was then resuspended, treated by ultrasonication and subsequently centrifuged to gain supernatant, followed by SDS-PAGE and silver staining or Coomassie blue staining (Fig. 6, fig. S1 and fig. S2).

![](https://static.igem.org/mediawiki/2019/6/64/T--XMU-China--XMU-China_result_6.png)

**Fig. 6. Gene circuit involved in protein expression and SDS-PAGE analysis of YebF-Bgl1A by silver staining.** (**A**) Gene circuit was constructed (BBa\_K2922006) to express YebF-Bgl1A. (**B**) Target bands of YebF-Bgl1A (red and blue arrows, 63.4 kDa). The character “R” stands for the flexible linker.

The target bands existing in *sup* (red arrow in Fig. 6B) and broken *sup* (blue arrow in Fig. 6B) of experimental groups only indicated that YebF-Bgl1A was expressed and secreted out of the cell successfully. The same experiments to YebF-CenA and YebF-Cex also showed similar results that the target bands were observed in *sup* of experimental groups while not in the control groups (fig. S1-S2). In contrast to cellulases without YebF fused at the N-terminal, which showed no target bands in the *sup* through SDS-PAGE, and given all of this, we safely concluded that protein YebF contributed to the secretion of these cellulases. In addition, the results of SDS-PAGE confirmed the success of OE-PCR again, while indirectly.

# The Characterization of Kil Secretion Cassette

The second secretory mechanism adopted involved the Kil secretion cassette. The *kil* gene ([BBa\_K1350001](http://parts.igem.org/Part%3ABBa_K1350001)) encodes a kind of lysis protein. Get more information about the secretory mechanism, please [click here](https://2019.igem.org/Team%3AXMU-China/Design). However, the secretion can be achieved without cell lysis and death only if the expression intensity of the promoter is proper. Here we applied various constitutive promoters to regulate the expression of Kil protein to investigate its secretion ability. The constitutive promoters we used were [BBa\_J23114](http://parts.igem.org/Part%3ABBa_J23114), [BBa\_J23109](http://parts.igem.org/Part%3ABBa_J23109) and [BBa\_J23112](http://parts.igem.org/Part%3ABBa_J23112) (in the order of strong to weak), respectively. The secretion of Bgl1A was proved by performing SDS-PAGE (Fig. 7). Similar results of CenA and Cex were also obtained except for Cex (114-kil-) (fig. S3-S8).

![](https://static.igem.org/mediawiki/2019/2/2c/T--XMU-China--XMU-China_result_7.png)

**Fig. 7. Gene circuits involved in protein expression and SDS-PAGE analysis of Bgl1A (114/109/112-kil-) by Coomassie blue staining.** (**A-C**) Gene circuits were constructed (BBa\_K2922012, BBa\_K2922013 and BBa\_K2922014) to express Bgl1A. (**E-F**) The target bands on SDS-PAGE gels (red and blue arrows, 53 kDa).

Just like the results about YebF, the target bands only existed in both *sup* and broken *sup* of experimental groups but not in the control groups. These results confirmed that the Kil secretion cassette successfully helped secrete the target protein out of the cell under the regulation of constitutive promoters. In fact, the light shade of bands in *sup* convinced us that the bacteria were not dead on a large scale, which further indicated the initiation intensity of J23114, J23109 and J23112 was appropriate enough to avoid cell lysis and death during the culturing process.

However, for the secretory section, no matter the YebF or Kil or some others, a vital issue could not be ignored and skipped was the extracellular enzymatic activity of those target proteins after secretion was achieved successfully. How to choose a most appropriate secretory mechanism so that cellulases can be secreted out of the cell with high activity was the next problem we needed to handle. Actually, we did series of assays for the enzymatic activity of three kinds of cellulases we selected and then made decisions.

# Enzymatic Activity of Endoglucanase (CenA)

Congo Red assay was utilized to qualitatively test the enzymatic activity of CenA in the form of crude enzyme, and this method was from iGEM18-UESTC-China, who had a nice [collaboration](https://2019.igem.org/Team%3AUESTC-China/Collaborations) with us this year. As Congo Red only binds to long chain polysaccharides but not short chain, the short chain therefore are washed off during staining procedure resulting in halo formation (*3*). The results are shown in Fig. 8-9.

![](https://static.igem.org/mediawiki/2019/6/6c/T--XMU-China--XMU-China_result_8.png)

**Fig. 8. Congo Red assay for enzymatic activity of CenA.** Left: *sup* of PT7-RBS-*cenA* and blank control (LB liquid medium); Right: broken *sup* of PT7-RBS-*cenA* and blank control (PBS).

Areas with the broken *sup* of CenA added showed due to the hydrolysis of CMC (carboxymethyl cellulose) whereas the blank control didn't show any clearance zones. The obvious difference showed that the broken *sup* of CenA had enzymatic activity, but the *sup* of culture had not. This was to say that the enzyme, Endoglucanase (CenA), which was expressed successfully, had a certain level of enzymatic activity to hydrolyze cellulose. Besides, what the results showed was in accordance with the results of SDS-PAGE (Fig. 6B) as well.

The same methods were applied to test the enzymatic activity of the broken *sup* of YebF-CenA fusion protein and CenA (114/109/112-kil-) and *sup* of those. The results are shown as follows:

![](https://static.igem.org/mediawiki/2019/9/9e/T--XMU-China--XMU-China_result_9.png)

**Fig. 9. Congo Red assay for enzymatic activity of YebF-CenA and CenA (109/112/114-kil-).** (**A**) The *sup* of YebF-CenA and blank control (LB liquid medium). (**B**) The broken *sup* of YebF-CenA and blank control (PBS). (**C-E**) The results of BBa\_K2922021, BBa\_K2922016 and BBa\_K2922017 for CenA (114/109/112-kil-) respectively.

All the areas with CenA crude enzyme, no matter YebF-CenA or CenA assembled with Kil secretion cassette under the regulation of those three promoters added showed clearance zones produced by hydrolysis of CMC (Fig. 9A-9E). While the blank control didn't show any zones of clearance. The results showed that both intracellular and extracellular CenA possessed considerable enzymatic activity.

In order to quantitatively analyze the enzymatic activity of series of CenA, we firstly tried to apply high performance liquid chromatography (HPLC) to separate the products after the substrates were digested by CenA, but the results were not satisfactory enough. Due to the limitation of time, the quantitative analysis for the enzymatic activity of CenA was not finished finally, which was a great regret for us indeed.

# Enzymatic Activity of Exoglucanase (Cex)

In order to test the enzymatic activity of Cex, 4-Methylumbelliferyl *β*-D-cellobioside (MUC) assay was carried out. MUC is broken down into methylumbelliferone (4-MU) and cellobiose in the presence of exoglucanase. The fluorescence intensity of methylumbelliferone can be measured at the emission wavelength of 460 nm (excitation wavelength: 364 nm) (*4*). In other words, the activity of exoglucanase can be reflected by the fluorescence intensity with time. In this part of assay, all BioBricks containing *cex* in our project this year were analyzed carefully where the enzymatic activity of *sup* and broken *sup* were evaluated respectively (Fig. 10).

![](https://static.igem.org/mediawiki/2019/2/25/T--XMU-China--XMU-China_result_10.png)

**Fig. 10. Quantitative analysis for the enzymatic activity of Exoglucanase (Cex) through MUC assay.**  (**A**) The fluorescence intensity at different time points of the *sup* in different experimental groups and the control group. (**B**) The fluorescence intensity at different time points of the broken *sup* in different experimental groups and the control group.

The results of *sup* showed that the supernatant of the culture of control ([BBa\_K525998](http://parts.igem.org/Part%3ABBa_K525998)) had no enzymatic activity, as there was no change in fluorescence intensity, which was caused by the lack of secretory ability. By contrast to the control, the other experimental groups all presented a certain degree of enzymatic activity, which was reflected by the increase of fluorescence intensity due to the produce of fluorescent 4-MU. It was worth noting that the increase of fluorescence intensity of Cex (109-kil-) group encoded by [BBa\_K2922018](http://parts.igem.org/Part%3ABBa_K2922018) was most evident and the intensity produced by this group’s *sup* at 120 min (17037.33) was almost 3 times (even more) higher than that of the other Cex groups (Fig. 10A). Different to *sup*, the broken *sup* of all experimental groups showed not so obvious difference among groups (Fig. 10B). Before 90 minutes, the signal of broken *sup* of Cex (109-kil-) was highest, which convinced us that J23109 was the most proper promoter could be applied in subsequent experiments including demonstration, together with the results of *sup*.

Additionally, the results of Cex (114-kil-) showed that the enzyme in the supernatant of the culture indeed had activity like the Cex (109-kil-) and Cex (112-kil-) whose secretion had been confirmed through SDS-PAGE, which could be evidence of the secretion of Cex (114-kil-) to some extent.

# Quantitative Analysis for the Enzymatic Activity of Beta-glucosidase (Bgl1A)

Given the fact that the beta-glucosidase (Bgl1A) can catalyze the hydrolysis of cellobiose to glucose, we came out that the products (glucose) and substrates (cellobiose) in the reaction system could be separated by high performance liquid chromatography (HPLC) and quantified through the peak areas in the chromatogram, which contributed to analyzing the enzymatic activity of Bgl1A. First of all, we investigated whether the cellobiose and glucose could be separated in our HPLC equipment. Fortunately, these two vital molecules could be distinguished obviously with 5 mM H2SO4 as the mobile phase (retention time of cellobiose: 9.5 min, retention time of glucose: 11.5 min, Fig. 11A). Then, the standard working curves (SWC) of cellobiose and glucose in certain chromatographic conditions were determined and plotted (Fig. 11B), which was the quantitative basis for the latter analysis.

We have noticed that the buffer and medium we used a lot might bring unnecessary affects to subsequent analysis, therefore the LB medium and PBS was tested before we carried out the assay, respectively. The chromatograms of LB liquid medium and PBS presented strong peaks at similar time about 8 min (Fig. 11C & 11D), which would not hinder the analysis of cellobiose and glucose.

![](https://static.igem.org/mediawiki/2019/c/c8/T--XMU-China--XMU-China_result_11.png)

**Fig. 11. Quantitative analysis was supported by HPLC.**  (**A**) The chromatograms of cellobiose and glucose of different concentrations. (**B**) The SWC of cellobiose and glucose. (**C**) The chromatogram of LB liquid medium. (**D**) The chromatogram of PBS.

The quenched reaction systems of different reaction times were analyzed through HPLC (Fig. 12, fig. S9). For the Bgl1A (PT7-RBS-*bgl1A*), the *sup* of the culture showed no enzymatic activity as the concentration (conversed according to SWC) of cellobiose and glucose in the supernatant of the culture hardly changed, which was same to that of control group (Fig. 12A). While the broken *sup* of experimental and control groups showed significant difference. The concentration of cellobiose decreased fiercely with time and the concentration of glucose increased rapidly with time, for the experimental group. While, there was no change of the concentration of the two molecules for the control group. Given all of this, we could summarized that the crude enzyme of Bgl1A (PT7-RBS-*bgl1A*), just as the results of broken *sup* showed, had enzymatic activity of a certain level whereas the supernatant of the culture had not. It is worth mentioning that this conclusion can be derived from the comparison of results between all experimental groups and their correspondent control groups in this assay (fig. S9). Regardless of this, the further calculations of the average consuming rate of cellobiose and producing rate of glucose in 0.5 h were done to compare the enzymatic activity of Bgl1A in different experimental groups (Tab. S1). The results of the three constitutive promoters were at a same level, where the consuming rate of cellobiose and producing rate of glucose of J23114-RBS-*kil*-PT7-RBS-*bgl1A* was the highest (8.724 g·L-1·h-1 and 8.683 g·L-1·h-1 respectively) among all groups. The enzymatic activity of YebF-Bgl1A was at the lowest level, which may be caused by the fusion of two protein molecules with the steric effect. But it was just our guess without further investigation.

![](https://static.igem.org/mediawiki/2019/c/c9/T--XMU-China--XMU-China_result_12.png)

**Fig. 12. Quantitative analysis for the enzymatic activity of Bgl1A (PT7-RBS-*bgl1A*) was supported through HPLC.**  (**A**) The concentration of cellobiose and glucose of *sup* at different times. (**B**) The concentration of cellobiose and glucose of broken *sup* at different times.

All experimental groups except Bgl1A (PT7-RBS-*bgl1A*) showed no enzymatic activity, which confused us much, because the enzyme was actually secreted out of the cell (verified through SDS-PAGE). That is to say that the beta-glucosidase (Bgl1A) cannot function as normal intracellularly when it is secreted out of the cell. In fact, once we had suspected that the conditions caused by our treatment to the samples of *sup* made the loss or decease of the enzymatic activity of Bgl1A, especially the pH condition of the culture. However, the following experiments we carried out to explore the influence of pH for the enzymatic activity of Bgl1A in the supernatant of the culture was failed (data not shown). In that case, we started to consider whether some changes in the structure of Bgl1A had emerged or not. Unluckily, due to the limit of time, we haven’t continue to investigate this issue further.

# Beta-glucosidase (Bgl1A) can Function Intracelluarly

With the help of [Prof. Zeng](https://2019.igem.org/Team%3AXMU-China/Human_Practices#Silver), we knew that the Bgl1A could catalyze the hydrolysis of cellobiose **intracellularly** in *E.coli* BL21 (DE3) due to the function of beta-galactoside permease encoded by *lacY* gene (*5*). Thus, we carried out the following experiment to prove it. The two recombinant bacteria, one carrying PT7-RBS-*bgl1A* and the other carrying [BBa\_K525998](http://parts.igem.org/Part%3ABBa_K525998) as control, were cultured in different M9 restriction medium with the cellobiose and glucose as the sole carbon source respectively. The growth curves of all these experimental and control groups were monitored through the OD600 value. The results proved that the Bgl1A could function successfully intracellularly, since the bacteria carrying PT7-RBS-*bgl1A* had grown normally in the restriction medium with cellobiose as the sole carbon source rather than the bacteria of control group (Fig. 13).

![](https://static.igem.org/mediawiki/2019/c/c8/T--XMU-China--XMU-China_result_13.png)

**Fig. 13. The growth curves for the verification of beta-glucosidase functioning intracellularly.**

# Brief Summary of *Cooperative*

Combining the experiments’ results above, the cellulases working effectively and the YebF and Kil secretion cassette functioning successfully was verified anyway. In view of all of this, we finally completed the [design](https://2019.igem.org/Team%3AXMU-China/Design) and construction of the ultimate circuits which would be applied to the [demonstration](https://2019.igem.org/Team%3AXMU-China/Demonstrate).

Aggressive

## What work we’ve done:

Verified Nimm, Eimm, Nkil and Ekil can be expressed successfully.

Verified the virulence of Colicin-N be able to kill other bacteria nearby successfully.

Verified the virulence of Colicin-E1 be able to kill other bacteria nearby successfully.

Verified the activity of Nkil to promote the secretion of Colicin-N.

Verified the activity of Ekil to promote the secretion of Colicin-E1.

## Work hasn’t finished yet:

Characterization of the virulence of Colicin-N and Colicin-E1.

## Get more information of our experiments, please [click here](https://2019.igem.org/Team%3AXMU-China/Experiments).

# Expression of Nimm、Eimm、Nkil、Ekil

Plasmids pSB1C3 carrying three different BioBricks [BBa\_K2922032](http://parts.igem.org/Part%3ABBa_K2922032) (T7 promoter-RBS-*cni* (*Nimm*)), [BBa\_K2922030](http://parts.igem.org/Part%3ABBa_K2922030) (T7 promoter-RBS-*imm* (*Eimm*)), [BBa\_K2922033](http://parts.igem.org/Part%3ABBa_K2922033) (T7 promoter-RBS-*cnl* (*Nkil*)) and [BBa\_K2922031](http://parts.igem.org/Part%3ABBa_K2922031) (T7 promoter-RBS-*kil* (*Ekil*)) were built and transformed into *E.coli* BL21 (DE3) successfully.

After culture and induction, the proteins (Nimm, Eimm, Nkil and Ekil) expressed were shown in the graphs of SDS-PAGE gels (Fig. 14).

![](https://static.igem.org/mediawiki/2019/7/70/T--XMU-China--XMU-China_result_14.png)

**Fig. 14. Coomassie stain of SDS-PAGE gels.** (**A**) The target bands of Eimm (white box, 13 kDa). (**B**) The target bands of Ekil (red box, 4.8 kDa). (**C**) The target bands of Nkil (black box, 5.6 kDa). (**D**) Nimm (red box, 15 kDa).

# The virulence of Colicin-N and Colicin-E1 to kill other bacteria nearby

Plasmids pSB1C3 carrying three different BioBricks [BBa\_K2922034](http://parts.igem.org/Part%3ABBa_K2922034) (The Colicin-N operon under T7 promoter [BBa\_K525998](http://parts.igem.org/Part%3ABBa_K525998) control), [BBa\_K2922035](http://parts.igem.org/Part%3ABBa_K2922035) (The Colicin-N operon under pBAD [BBa\_K206000](http://parts.igem.org/Part%3ABBa_K206000) control), [BBa\_K2922036](http://parts.igem.org/Part%3ABBa_K2922036) (The Colicin-E1 operon under T7 promoter [BBa\_K525998](http://parts.igem.org/Part%3ABBa_K525998) control) and [BBa\_K2922037](http://parts.igem.org/Part%3ABBa_K2922037) (The Colicin-E1 operon under pBAD [BBa\_K206000](http://parts.igem.org/Part%3ABBa_K206000) control) were built and transformed into *E.coli* BL21 (DE3) successfully, which conformed by colony PCR and sequencing (Fig. 15).

![](https://static.igem.org/mediawiki/2019/4/43/T--XMU-China--XMU-China_result_15.png)

**Fig. 15. Colony PCR in 1% agarose gel.** (**A**) The target bands of BBa\_K2922037 (about 2400 bp), (**B**) The target bands of BBa\_K2922035 (about 2100 bp), BBa\_K2922036 (about 2300 bp) and BBa\_K2922034 (about 2000 bp).

The *E.coli* BL21 (DE3) holding corresponding plasmids were cultured and expressed colicin kit through induction. The culture medium was collected as the sample for inhibition zone experiment, of which the protocol can be viewed in [Notebook-Experiment-Inhibition zone](https://2019.igem.org/Team%3AXMU-China/Experiments). The virulence of Colicin-N and Colicin-E1 were verified by measuring the size of inhibition zone between the experimental and control group.

![](https://static.igem.org/mediawiki/2019/b/b2/T--XMU-China--XMU-China_result_16.png)

**Fig. 16. The inhibition zone experiments through Oxford cup method.** The results of experimental groups (induced) and the control groups (non-induced) were shown in the left and right in every petri dish respectively. (**A**) TN, namely the inhibition zone of strains expressing Colicin-N kit regulated by T7 promoter. (**B**) PN, namely the inhibition zone of strains expressing Colicin-N kit regulated by pBAD (arabinose promoter). (**C**) TE, namely the inhibition zone of strains expressing Colicin-E1 kit regulated by T7 promoter. (**D**) PE, namely the inhibition zone of strains expressing Colicin-E1 kit regulated by pBAD (arabinose promoter).

# The secretion of Colicin-N and Colicin-E1 promoted by Nkil and Ekil

The *E.coli* BL21 (DE3) holding BioBricks [BBa\_K2922034](http://parts.igem.org/Part%3ABBa_K2922034) and [BBa\_K2922036](http://parts.igem.org/Part%3ABBa_K2922036) were cultured and expressed by adding corresponding inducer. The supernatant was obtained after centrifuging at 13000 rpm and used as the sample of inhibition zone experiment, of which the protocol can be viewed in [Notebook-Experiment-Inhibition zone](https://2019.igem.org/Team%3AXMU-China/Experiments). The secretion of Colicin-N and Colicin-E1 was analyzed by measuring the size of the inhibition zone.

Finally, we found that the sizes of inhibition zone in the experimental group were larger than that in the control group (Fig. 17) and the data were recorded (Tab. S2).

![](https://static.igem.org/mediawiki/2019/d/d1/T--XMU-China--XMU-China_result_17.png)

**Fig. 17. The inhibition zone experiments through Oxford cup method.** The results of experimental groups (induced) and the control groups (non-induced) were shown in the left and right in every petri dish respectively. The supernatant was used as sample in each group, which obtained from corresponding culture medium. (**A**) TN sup, namely the inhibition zone of strains expressing Colicin-N kit regulated by T7 promoter. (**B**) TE sup, namely the inhibition zone of strains expressing Colicin-E1 kit regulated by T7 promoter.

# Brief Summary of *Aggressive*

Globally, we can say that our cloning was successful although we met some troubles in growing the resulting transformed bacteria. We successfully demonstrate the virulence of Colicin-E1 and Colicin-N, and expressed proteins needed in our design verified by SDS-PAGE or spectrophotometer. In view of all of this, we finally completed the [design](https://2019.igem.org/Team%3AXMU-China/Design) and demonstrate its designed function primarily.

# Reference

1. G. Xie, *et al.*, Genome sequence of the cellulolytic gliding bacterium Cytophaga hutchinsonii. *Appl Environ Microbiol* **73**, 3536-3546 (2007).

2. [http://parts.igem.org/Part:BBa\_K1610300](http://parts.igem.org/Part%3ABBa_K1610300)

3. S. S. Lakhundi, Synthetic biology approach to cellulose degradation Doctoral Dissertation, The University of Edinburgh, UK, 2012.

4. K. O. Duedu, C. E. French, Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. *Enzyme Microb Technol* **93-94**, 113-121 (2016).

5. M. Crandall, *et al.*, Temperature-Sensitive Mutants of Escherichia coli Affecting β-Galactoside Transport. *J Bacteriol* **105**, 609-619 (1971).

Supplementary Materials

![](https://static.igem.org/mediawiki/2019/1/1c/T--XMU-China--XMU-China_result_s1.png)

**Fig. S1. Gene circuit involved in protein expression and SDS-PAGE analysis of YebF-CenA by silver staining.** (**A**) Gene circuit was constructed (BBa\_K2922007) to express YebF-CenA. (**B**) Target bands of YebF-CenA (red arrow, 60 kDa). The character “R” stands for the flexible linker.

![](https://static.igem.org/mediawiki/2019/2/24/T--XMU-China--XMU-China_result_s2.png)

**Fig. S2. Gene circuit involved in protein expression and SDS-PAGE analysis of YebF-Cex by Coomassie blue staining.** (**A**) Gene circuit was constructed (BBa\_K2922008) to express YebF-Cex. (**B**) Target bands of YebF-Cex (red arrow, 60 kDa). The character “R” stands for the flexible linker.

![](https://static.igem.org/mediawiki/2019/7/77/T--XMU-China--XMU-China_result_s3.png)

**Fig. S3. Gene circuit involved in protein expression and SDS-PAGE analysis of CenA (114-kil-) by silver staining.** (**A**) Gene circuit was constructed (BBa\_K2922017) to express Kil and CenA. (**B**) Target bands of CenA (blue arrow, 47 kDa) in a brighter graph. (**C**) Target bands of CenA (red arrow, 47 kDa) in a darker graph.

![](https://static.igem.org/mediawiki/2019/2/20/T--XMU-China--XMU-China_result_s4.png)

**Fig. S4. Gene circuit involved in protein expression and SDS-PAGE analysis of CenA (109-kil-) by silver staining.** (**A**) Gene circuit was constructed (BBa\_K2922021) to express Kil and CenA. (**B**) Target bands of CenA (red and blue arrows, 47 kDa).

![](https://static.igem.org/mediawiki/2019/5/5b/T--XMU-China--XMU-China_result_s5.png)

**Fig. S5. Gene circuit involved in protein expression and SDS-PAGE analysis of CenA (112-kil-) by silver staining.** (**A**) Gene circuit was constructed (BBa\_K2922016) to express Kil and CenA. (**B**) Target bands of CenA (red and blue arrows, 47 kDa).

![](https://static.igem.org/mediawiki/2019/1/12/T--XMU-China--114-cex-2.png)

**Fig. S6. Gene circuit involved in protein expression and SDS-PAGE analysis of Cex (114-kil-) by silver staining.** (**A**) Gene circuit was constructed (BBa\_K2922020) to express Kil and Cex. (**B**) No target bands on the gel.

![](https://static.igem.org/mediawiki/2019/1/14/T--XMU-China--XMU-China_result_s7.png)

**Fig. S7. Gene circuit involved in protein expression and SDS-PAGE analysis of Cex (109-kil-) by Coomassie blue staining.** (**A**) Gene circuit was constructed (BBa\_K2922018) to express Kil and Cex. (**B**) Target bands of Cex (red and blue arrows, 47 kDa).

![](https://static.igem.org/mediawiki/2019/8/80/T--XMU-China--XMU-China_result_s8.png)

**Fig. S8. Gene circuit involved in protein expression and SDS-PAGE analysis of Cex (112-kil-) by Coomassie blue staining.** (**A**) Gene circuit was constructed (BBa\_K2922019) to express Kil and Cex. (**B**) Target bands of Cex (red and blue arrows, 47 kDa).

![](https://static.igem.org/mediawiki/2019/7/79/T--XMU-China--XMU-China_result_s9.png)

**Fig. S9. Quantitative analysis for the enzymatic activity of YebF-Bgl1A and Bgl1A (109/112/114-kil-) was supported through HPLC. (A, C, E, G)** The concentration of cellobiose and glucose of *sup* at different times, for different experimental and control groups respectively. (**B, D, F, H**) The concentration of cellobiose and glucose of broken *sup* at different times, for different experimental and control groups respectively.

**Tab. S1.** The calculation results of consuming and producing rate

![](https://static.igem.org/mediawiki/2019/f/fa/T--XMU-China--table1.png)

**Tab. S2.** The results of the inhibition zone experiments

![](https://static.igem.org/mediawiki/2019/7/7b/T--XMU-China--table2.png)

Xiamen University, Fujian, China

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