iGEM 2022 — Team zju-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

[Brainstorm](#page-id-1)

[Experiment](#page-id-2)

![](https://static.igem.wiki/teams/4202/wiki/leftlion.png)

## Brainstorm

### wk1 3.1-3.6

### March 2th

Time: 21:15-23:00

Location: Room 234, College of Life Sciences, Zhejiang University, China

Recorder:Li Jiachen

Highlights:meet-and-greet,the first meeting for ZJU-China IGEMers!

#1 Instructor's speech

#2 Team member self-introduction including wet lab group and dry lab group.

#3 Speech by the last captain: introduce and emphasize the attention.

#4 Specifying team rules

Futher plan:

Reading articles and journals.Everyone should do 1-2 self-designed projects and presnt in the next meeting.

### March 6th

Time: 21:15-23:45

Location:

Room 413, National Demonstration Center for Experiment Biology Dducation (ZJU), Zijin'gang Campus, Zhejiang University, China

Recorder: Gao Liaoliao

Highlights: Raise of the programs

#1 Detection of thyroid cancer by specific biological probe -By Pan Zicai

#2 Using biofilm to build a bioengineering platform of nano materials -By Han yibing

#3 Using biofilm to improve the enrichment and recovery efficiency of rare earth elements in e-waste -By Lin Hongying

#4 Using yeast and soybean protein to produce plant meat and improve ordinary vegetarian meat -By Li Jiachen

#5 Turing pattern -By Yan Zhijian

#6 Using algae to solve the treatment of tumor hypoxic area; Drug targeted delivery -By Su Yuyan

Future plan:

Narrow the scope of alternative projects, think about improvement of the programs and put forward new ideas.

### March wk2 3.7-3.13

### Mar 9th

Time: 21:15-23:30

Location:Room 413, National Demonstration Center for Experiment Biology Dducation (ZJU), Zijin'gang Campus, Zhejinag
University, China

Recorder: Pan Zicai

Key summary: Brainstorming and sharing

#1: mold biocomputing --pass -By Yan Zhijian

#2 Make leather from fungi -By Ye Zihang

#3 Thyroid FNA testing -- what are the advantages of mab testing to be solved -By Pan Zicai

#4Artificial meat -By Li Jiachen

#5 Biofilm recovery of e-waste -By Lin Hongying

#6Drug delivery system for intestinal probiotics in the treatment of intestinal bleeding and targeting specific cells -By Su Yuyan:

#7 Ship protective coating made by biofilms, microbial fuel cell -By Han Yibing

#8 Cell factory synthesis of breast milk oligos accharides -By Liu Junyi

#9 Plant vaccines -By Ren Zhuoyun

#10 Bacterial imaging system -By Gao Liaoliao

Future plans

brainstorming

### Mar 13th

Time: 21:15-23:30

Location:Room413,National Demonstration Center for Experiment Biology Dducation(ZJU),Zijin'

Gang Campus, Zhejiang University, China

Recorder: Zihang Ye

Key summary: 3.13 Plan report and brainstorming sharing

#1 In vitro synthesis of FDCA, production of protein plastics -By SuYuyan,YeZihang

# 2Mercury-responsive promoter Pmer,amercaptogen-rich biofilms of Pseudo monassp -By LiJiachen,PanZicai

#3 Modification of nitrogen fixation with the gramineus symbiotic bacterium Klebsiellak.

variicola137 -By LiuJunyi,YanZhijian:

#4 Modified *Bacillus subtilis* for innovative application of pethair and keratin -By HanYibing,Lin

Hongying

#5 Accurate diagnosis of thyroid cancer -By PanZicai

#6 Antibiotic detection--CRISPR-12a, micro integral series -By LinHongying

#7 RPA technology combined with fluorescence quenching is relieved. Antibiotics interfere with RPA amplification and slow down the rate of fluorescence quenching -By Yan ZhiJian

Future plans

#1 Application and Innovation of keratin -By HanYibing

#2 Production of bio-plastics -By Su Yuyan, Ye Zihang

#3 Heavy Metal -By LiJiachen, PanZicai

### March wk3 3.14-3.20

### Mar 16th

Time: 21:15-23:30

Location:
Room 413, National Demonstration Center for Experiment Biology Dducation (ZJU), Zijin’gang Campus, Zhejinag University, China

Recorder: Liu junyi

Highlights: Brain storm

#1 Secreting high-strength biological membrane -By Su Yuyan

#2 Multiple examination of soil pollution -By Li Jiachen

#3 Cell factory producing bio-plastics -By Ye Zihang

#4Modification of nitrogen-fixing bacterial for graminaceous crop; Engineered bactiria resolving hairball in pet’s stomach -By Liu Junyi

Future plan:

#1 bio-plastics -By Su Yuyan, Ye Zihang

#2 biological membrane -By Gao Liaoliao, Ren Zhuoyun

#3 soil pollution examination -By Li Jiachen, Pan Zicai

#4 nitrogen-fixing bacteria -By Liu Junyi, Yan Zhijian

#5 Resolving hairball -By Lin Hongying, Han Yibing

### March wk4 3.21-3.27

### March 21st

Time:21:15-23:00,March 21

Location:Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus,Zhejiang University, China

Recorder:Han Yibing

Highlights:

Summary: Wet Team members report on new ideas gathered this week

#1 Use gene knockout and agrobacterium infestation to solve the problem of crop incompatibility -By Yan Zhijian

#2 Temporarily hiding therapeutic bacteria outside the immune system allows them to treat rheumatoid arthritis more effectively -By Su Yuyan

#3 Producing self-healing cement from *Bacillus* -By Li JiaChen

#4 Hyphal material for the production of bio-plastics or insulating materials -By Li Jia-Chen

#5 Fibrosome of fusion catalysis, assembly, substrate binding, and cell binding modules -By Liu Jun-Yi

#6 Using gut microbes to treat depression -By Gao Liao-Liao

#7 Transform exophytic mycorhize -By Ye Zi Hang

#8 Magnetic protein -By Ren Zhuo Yun

### March 23rd

Time: 21:15-23:15

Location:

Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus,Zhejiang
University, China

Recorder: Zhuoyun Ren

Highlights: deep research on designed project

#1 The advantages and disadvantages of *Bacillus subtilis* as a chassis organism are presented -By Zicai Pan

#2 The application of synthetic organisms in restorative dentistry mentioned -By Yibing Han

#3 Complementary component designed -By Junyi Liu

Summary: Summarizes all the progress of the experiment from the initial design to today

Next steps: get feedback from different stakeholders and supplement our design according to the practical use

### March 27th

Time: 21:15-23:50

Location:

Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus,Zhejiang University, China

Recorder: Zhijian Yan

Highlights:

#1 Cement is more inclined to restoration of cultural relics, and its application in the field of architecture may not be very meaningful proposed -By Liaoliao GAO

#2 An innovative idea: Spirulina transformation system, using Spirulina to produce medical vaccines (can be made into oral vaccines) proposed By -Zihang Ye

#3 Utilize Bioconjugate to degrade corn stover, a new approach raised -By Zihang Ye

#4 Phage MS2 system detects the presence of specific sequence RNAs -By Zhijian Yan

#5 Brainstorm: 1) kraft paper to produce xylitol; 2) astaxanthin; 3) biological stapling; 4)pectin to jam; 5) plant terpane biosynthesis; 6) population sensing -By Zhuoyun Ren

#6 For Cd project: two possible pathways -By Zicai Pan, Hongying Lin

- Enrichment of cadmium by extracellular substrate-bound Pseudomonas aeruginosa

- Surface-based enrichment of cadmium by displaying MT protein

#7 For MICP project: research findings on specific synthetic biology design -By Junyi Liu, Yibing Han, Jiachen Li

- Related items/products are already available

- Use *Bacillus alkalophilus* as a chassis to sense pressure as a repair stop signal

- Expression of filamentous fibronectin to bind calcium carbonate, increase extracellular polysaccharide to improve crystal structure

- Culture material and calcium source can be used waste (calcium source: oyster shell, culture material: sesame seed residue (spore production), soybean meal (bacterium production))

### March wk5 3.28-3.31

### Mar 28th

Time: 21:15-23:00

Location:Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China

Recorder: Su Yuyan

Key summary: Brainstorming and sharing

#1: Develop own ACCBP components/ Promoter of stress perception -by Yan Zhijian

#2 Using microcapsule material packaging/Add alkali resistance system -by Li jiachen

#3 Riboswitch/find Nitrogen responsive promoter BBa\_K216005 -by Ye zihang

#4 Summarize -by Su Yuyan

Using MICP principle to restore stone relics:

1. Chassis organism: Choose *Bacillus subtilis*

2. Metabolic pathways: ① Formation of calcium carbonate: the formation of calcium carbonate precipitation using amorphous calcium carbonate binding protein; Carbonic anhydrase was introduced to enhance the secretion of carbonate.
 ②The l-sucrose gum gene was introduced to synthesize the glue used for gluing calcium carbonate.
③ Secretedilamentous protein, as the supporting combined material.

3. Motor pathway: swrA and SFP genes of flagella were introduced to restore the motor ability of *Bacillus subtilis* and enable it to expand and grow on solids.

4. Other parts: alkali resistance, high salt resistance; Kill switch; Quorum sensing system.

5. Material preparation: soybean meal and other agricultural fertilizers are used as nutrient sources; Oyster shells were used as the source of calcium ions.

### March 31st

Time:21:30-23:00

Location:Room 413, National Demonstration Center for Experiment Biology

Dducation (ZJU), Zijin'gang Campus, Zhejinag University, China

Recorder:Lin Hongying

Highlights:The design of relic restoration project

#1 The introduction of the project from Newcastle in 2010, including germination part, swarming part, CaCO3 precipitation part, filamentous cell formation part and so on. ——By Li Jiachen

#2 The introduction of colloid parts and how to develop our own ACCBP parts and pressure-sensing parts. ——By Yan Zhijian

#3 The design of the safety part and quorum sensing part. ——By Han Yibing

#4 The introduction of the project to detect the concentration of N, P elements in polluted water. ——By Ye Zihang

### April wk2 4.4-4.10

### April 6th

Time: 21:15-23:00

Location: 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China.

Recorder:Li Jiachen

Highlights:we invite the team from the International Union College of Zhejiang University to talk about projects and change the suggestions of each other.

#1 Our project introduction

#2 International Union College of Zhejiang University team's project introduction

#3 question and answer

#4 discussion the problems and the solution

Future plan:

Further improve the design of the project, taking into account the water permeability of the stone, contamination by other bacteria, the resistance of bacteria, cost, hardware and so on.

### April 10

Time: 21:15-23:00

Location:

Room 413, National Demonstration Center for Experiment Biology Dducation (ZJU), Zijin’gang Campus, Zhejinag University, China

Recorder: Liu junyi

Highlights: improvement of our current project

#1 Using silica gel as our padding -By Gao Liaoliao

#2 Using hypha to provide support frame -By Yan Zhijian

#3 Adding metal ion to prevent harmful bacterium -By Su Yuyan

#4 Bonding padding material by modified biological membrane -By Pan Zicai

Future plan:

Asking professors for the feasibility of the new designs; exploring new aspects for project improvement

### April wk3 4.11-4.17

### April 11th

Time:20:00-21:00

Location:Online Meeting through Dingtalk

Recorder:Lin Hongying

Highlights:Review of current project about relics restoration

Guest:Associate Professor Dr. Yulan Hu at Zhejiang University, whose main research interest is biotechnology and cultural relics protection.

#1 Evaluation:The project is theoretically feasible in the lab, but most stone relics are unmovable in the environment and other environmental factors like temperature and humidity should be considered.

#2 Detailed problems

1. The lack of oxygen in the deep

When the bacterium swimming into the deep cracks, their proliferation may be influenced due to the lack of oxygen affecting the repair effect.

2. The strength of the adhesive protein

The strength of the adhesive protein should be tested. And the protein strength will gradually reduce as time goes by.

3. The corrosion of cultural relics caused by microorganism.

The organic acids metabolized by microorganisms may cause corrosion to the stone relics, and we can introduce some microorganisms secreting alkaline substances.

4. The water content of the filler

Some stone relics can absorb the water in the medium so that our filler will become dry

5. How to modify our bacterium to gain a more competitive edge than other microorganisms in the cracks?

6. How do they evaluate the effect of the repair work?

We can evaluate the effect from some perspectives like safety, strength, et.al.

### April wk4 4.18-4.24

### April 20th

Time: 21:15-23:50

Location:

Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus,Zhejiang University, China

Recorder: Zhijian Yan

Highlights:

#1 Found parts in iGEM Website to design pathways from Sucrose to Fructans

#2 Analogue research in ACCBP

- How to design our own ACCBP module: Ca2+; Mg2+ aqueous solution

- Cloning of CARP protein from coral for precipitation of calcium carbonate under wide pH conditions

Summary: Some details in project design with different approaches

### April wk5 4.25-4.30

### April 27th

Time:21:15-23:00,April 27

Location:Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus,Zhejiang University, China

Recorder:Pan Zi-Cai

Highlights: Summarize the progress of HP and future planning, explain experiment arrangement and report on this week's progress.

#1 Hardware, construction for artifact crack detection (ultrasound & X-ray) -By Xie Yu-Shu

#2 Model, Ratio optimization of binders, strain growth relationships, screening of optimal quorum sensing systems using machine leaning-By Wang Yu-Kun

#3 Breeding strains gel to protect cultural relics-By Su Yu-Yan

#4 Using sulfate reducing bacteria to clean dirt on cultural relics-By Li Jia-Chen

#5 Improve immobilization efficiency by immobilizing carbonic anhydrase-By Lin Hong-Yin

#6 Produces bacterial cellulose, thus forming crosslink with other substances to form cleaners for rough surfaces-By Han Yi-Bin

#7 Rapid generation of materials with certain structural mechanics using *Bacillus subtilis*, organophosphate and barium alt systems-By Pan Zi-Cai

Future plan:

Carry out preliminary experiment.

Explore the surface cleaning scheme of cultural relics

Design quorum sensing systems

Construct ACCBP related modules

Explore new topics

### May wk1 5.1

### May 1st

Time: 21:15-23:33

Location:

Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China

Recorder: Gao Liaoliao

Highlights: Raise of the programs

#1 Bacterial targeted thrombolysis -By Gao Liaoliao

#2 Using RNAi to Kill nematodes -By Liu Junyi & Lin Hongying

#3 Measuring the expression level of endogenous mRNA by CRISPR system -By Yan Zhijian

#4 Treatment of triple negative breast cancer by simcell & Minisimcell -By Li Jiachen

#5 Fresh keeping of flowers - By Ren Zhuoyun

#6 Cleaning with stable bacteria strains - By Pan Zicai

#7 Biological cleaning, using paper gel bacteria to remove broad-spectrum stains - By Han Yibing

#8 Design of bacterial combined CaCO 3 scaffolds - By Ye Zihang

Future plan:

Cultural relics restoration, drug delivery system in cancer treatment, sewage detection capsule.

### May wk2 5.2-5.8

### May 8th

Time: 21:15-23:00

Location:Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China

Recorder: Han Yibing

Key summary: content introduction of iGEM opening ceremony and experimental schedule

# 1 Introduction to relevant contents of the opening ceremony (providing a slack to communicate with foreign iGEM teams and follow-up in-depth exchanges with China Agricultural University) -By Liu Junyi

Future plans:

#1 Experimental design of carbonic anhydrase -By Li Jiachen, Liu Junyi

#2 Experimental design of accbp mineralized peptide -By Ye Zihang, Lin Hongying, Su Yuyan

#3 Experimental design of high sugar suicide module -By Han Yibing, Ren zhuoyun, Yan Zhijian

#4 Experimental design of quorum sensing -By Gao Liaoliao, Pan Zicai

#5 Experimental design of measurement improvement -By Gao Liaoliao, Pan Zicai, Liu Junyi

#6 Write the experimental application -By Pan Zicai

#7 Docking ppt with Guo Donglai -By Han Yibing

### May wk3 5.9-5.15

### May 14th

Time:21:15-23:00

Location:Room413,National Demonstration Center for Experiment Biology Dducation(ZJU),Zijin'gang Campus,Zhejinag University,China

Recorder:YeZiHang

Key summary:rule explanation,experiment division

#1 Parts related matters interpretation -By Yan Zhijian

#2 Introduction to iGEM rules -By Su Yuyan

#3 A sensor literature explanation -By Yan Zhijian

#4 Grouting method; Modeling based on fracture conditions and media fluidity -By Lin Hongying

Future plans:

#1 Lin Hongying:Responsible for safety certification

#2 Su Yuyan:Contact Teacher Wang Baojun

#3 Su Yuyan,Yan Zhijian:Experimental design of cultural relic removal

#4 Jiachen Li,Junyi Liu: Carbonian hydrase partial experimental design

#5 ZihangYe,HongyingLin:ACCBP part experimental design

#6 HanYibing,YanZhijian: Experimental design of part of suicide module

#7 Gao Liaoliao,PanZicai: Experimental design of quorum sensing

#8 WangYu kun: Modeling based on fracture conditions and media fluidity

## Experiment

### 5.9-5.15

**- Public**

Purify a strain of WB600 by plate streaking from commercialize B.subtilis strain.

Prepare culture media containing Tetracycline and test Shuttle plasmid pHY300PLK

Get the GFP and mRFP sequence from distribution plate.

### 5.15-5.22

**- Public**

Separately construct pHY300PLK plasmids with GFP and RFP, in order to test transforming conditions of B.subtilis.

### 5.15-5.22

**- Public**

Separately construct pHY300PLK plasmids with GFP and RFP, in order to test transforming conditions of B.subtilis.

**- Part Measurement**

Amplifate luciferae, luciferin-regenerating enzyme, and separately construct three pet28(a)+ plasmids:

(a) pet28a(+) with luciferase,(b) pet28a(+) with luciferase regenerating enzyme,(c) pet28a(+) with both luciferase and luciferin-regenerating enzyme.
Transform three plasmids to E.coli BL21

### 5.22-5.29

**- Public**

Try electric transform of B.subtilis but failed

### June 6.20-6.26

**- Biological scafforld**

Synthesis plasmids PHY300PLK-HagT209C-SpyT , PHY300PLK-EutM-SpyC , PET-28(a)- spyT-mRFP , PET-28(a)-spyC-mRFP by Genscript .

### July wk2 7.4-7.10

**- Quorum Sensing Part**

construction of plasmids: Oxylimited-GFP-terminator

**- Biological scafforld**

Transform PET-28(a)- spyT-mRFP , PET-28(a)- spyT-mRFP in BL21

Explore the protein inducing condition

Complete the induction of spyT-mRFP and spyT-mRFP

**- Biosafety**

Improve the 2012 LMU's *Bacillus subtilis* genome extraction protocol: only chloroform isoamyl alcohol (24:1) is used to extract protein, and the extraction time of *Bacillus subtilis* genome DNA is shortened to within 2h.

spaBCTS and PsacB sequences have been successfully cloned from genomic DNA.

Improvement of protein SDS-PAGE process: a new protein glue dyeing solution was developed.

**- Calcium Carbonate Production part**

Do codon optimization for Carbonic Anhydrase(CA) and construct the plasmid PHY300PLK-P43-CA1

Plasmids PHY300PLK-P43-CA1 and PHY300PLK-ACCBP synthesis (by company)

### July wk3 7.11-7.17

**- Quorum Sensing Part**

Construction of plasmids: Oxylimited-GFP-terminator

**- Biological scafforld**

Explore the proprior condition for the HA-tag protein IP purification

**- Biosafety**

PHY-PSacB-GFP-Terminator plasmid and PHY-Pveg-PsacB-GFP-Terminator plasmid were constructed successfully.

**- Calcium Carbonate Production part**

Transform PHY300PLK-P43-CA1 in BL21

PHY300PLK-P43-CA1 plasmid extraction

**- Public**

Several attempts to electrotransform *Bacillus subtilis* failed.

We have done a successful attempt at chemical transformation of B.subtilis

### July wk4 7.18-7.24

**- Quorum Sensing Part**

construction of plasmids signal-GFP, and Characterization in BL2; Characterization of immunity part in BL21;explore the effect of *Bacillus subtilis* inhibitory concentration

**- Biological scafforld**

Explore the chemical confromation method of *Bacillus subtilis* .

Explore the protein expression condition of *Bacillus subtilis* .

**- Biosafety**

Try to refer to the protocol of LMU to chemically transform *Bacillus subtilis*, and succeed.

PHY-PsacB-Pveg-GFP-Terminator plasmid was characterized in *Bacillus subtilis*. It had no induction activity, but had expression activity

PHY-PsacB-GFP-Terminator plasmid was characterized in *Bacillus subtilis*, with no induction activity and suspected weak expression activity

**- Calcium Carbonate Production part**

PHY300PLK-P43-CA1 electrotransformation to WB600 (failed)

We have done a successful attempt at chemical transformation of B.subtilis

### July wk5 7.25-7.31

**- Quorum Sensing Part**

Confirm the function of immunity part in BL21.

**- Biological scafforld**

Purify the spyC-mRFP (with 6\*His tag) by His-tag Purification Resin

Purify the spyT-mRFP (with HA tag) by Anti-HA Magnetic Beads

**- Biosafety**

The concentration of sucrose (1% - 10% sucrose) was changed to characterize the induction effect of PsacB plasmid. It was found that it had only weak expression activity and no induction activity. It was considered to optimize the promoter to reduce leakage expression.

Transforming PHY psacb MazE / F into E. coli and extracting plasmid

**- Calcium Carbonate Production part**

Restructuring plasmid (add Terminater7 to PHY300PLK-P43-CA1)

### August wk1 8.1-8.7

**- Quorum Sensing Part**

Transform plasmids into WB600, including Oxylimited-GFP-terminator, Oxylimited-SpaBCTS-terminator,signal-GFP. Run SDS-Page to confirm the phenotype. However, failed.

**- Biological scafforld**

Construct the PHY-PsacB-EutM-spyC-terminator (add the promotor and terminator)

**- Biosafety**

The plasmid was reconstructed and the reporter gene was inserted into the HindIII digestion site (shortening the distance between the start codon and RBS) to try to improve the expression efficiency

**- Calcium Carbonate Production part**

Construct the plasmid PHY300PLK-P43-CA2-T1-T7(from Parts: BBa\_K2232014)

### August wk2 8.8-8.14

**- Quorum Sensing Part**

Construction of plasmids: P43-GFP,P43-SpaBCTS, and confirm the phenotype in BL21 using 0.1mg/ml *Bacillus subtilis* to trigger the function of signal-GFP plasmid in WB600

**- Biological scafforld**

Induce the expression of EutM-SpyC in *Bacillus subtilis* .

Explore the experimental protocol of EutM-SpyC

Construct the PET-28(a)-EutM-spyC plasmid

**- Biosafety**

The characterization could not be completed due to the dispersion of SDS-PAGE small molecule proteins.

Try to improve the efficiency of promoter induced expression

replacement of reporter gene BBa\_K1159001(FirelyLuciferase) BBa\_I712019(NanoLuciferase) add RBS (Spovg-RBS, available before Pveg)

**- Calcium Carbonate Production part**

PHY300PLK-P43-CA2-T1-T7 chemical transformation to WB600. Cultivating the engineered WB600 in LB (Tc+) for 24hExpression of carbonic anhydrase in *Bacillus subtilis*

Construct the PET-28(a)-ACCBP plasmid

### August wk3 8.15-8.21

**- Quorum Sensing Part**

Purify the plasmid and transform plasmids into WB600. Confirm the phenotype of P43-GFP plasmid in WB600 by SDS-Page.

**- Biological scafforld**

Construct the PHY-P43-HagT209C-SpyT (add the promotor and terminator)

Complete the western blotting verification of spy system

**- Biosafety**

Optimize promoter recombinant full-length Pveg-sacB (linearized vector with homologous arm and Pveg have been prepared).

Recombinant PVG leader RNA (recombine the constitutive promoter PVG and the leader RNA upstream of sacB gene).

**- Calcium Carbonate Production part**

Transforme the PET-28a(+)-ACCBP into the BL21.

### August wk4 8.22-8.28

**- Quorum Sensing Part**

Confirm the GFP expression triggered by 0.1 mg/ml *Bacillus subtilis* inWB600 and run SDS-Page.

**- Biological scafforld**

Transform PET-28(a)-EutM-SpyC in BL21

Induce the EutM-SpyC and purify the EutM-SpyC(with 6\*His tag ) by His-tag Purification Resin

Verify the function of protein

**- Biosafety**

Construct plasmids, but they have been failing.

**- Calcium Carbonate Production part**

Purification of carbonic anhydrase and SDS-PAGE electrophoresis

Detection of esterase activity of carbonic anhydrase

### August wk5 8.29-8.31

**- Quorum Sensing Part**

Construction of plasmids: Oxylimited-SpaBCTS-terminator-New. Transform plasmids into WB600.

**- Biological scafforld**

Construct the PHY-P43-HagT209C::SpyTag588-DT plasmid by overlapping PCR

**- Biosafety**

- The PHY-PsacB-FierlyLuciferase-Terminator plasmid, PHY-PsacB-NanoLuciferase-Terminatorplasmid, and PHY-Pveg-LR-spoVGRBS plasmid were successfully constructed.

**- Calcium Carbonate Production part**

Cultivating the engineered WB600 in SMM (Tc+) for 48h

### September wk1 9.1-9.7

**- Biological scafforld**

Induce the expression of EutM-SpyC in E.coli .

**- Calcium Carbonate Production part**

Determination of the ability of Carbonic Anhydrase to catalyze the hydration of CO2

Veify the ACCBP expression in the E.coli

### September wk2 9.8-9.14

**- Biological scafforld**

Purify the EutM-SpyC from E.coli and utilize the TEM to detect the morphology of EutM.

Transform the PHY-Hag into the *Bacillus subtilis*.

**- Calcium Carbonate Production part**

Determination the ability of Carbonic Anhydrase to catalyze the precipitation of CaCO3

Explore the expression condition of ACCBP in the E.coli

### September wk3 9.15-9.21

**- Calcium Carbonate Production part**

SEM exploring the crystal structure of precipitated amorphous calcium carbonate

Explore the expression condition of ACCBP in the E.coli

Construct the PET-28a(+)-Δ16 ACCBP plasmid

### After 9.21

**- Biological scafforld**

Transform the plasmid PHY-P43-Hag into the *Bacillus subtilis*

Verify the function of Hag

Verify the feasibility of biological scaffold

**- Biosafety**

Transform pHY300PLK containing [**BBa\_K4202045**](http://parts.igem.org/Part%3ABBa_K4202045)(PsacB-mazE-mazF), [**BBa\_K4202033**](http://parts.igem.org/Part%3ABBa_K4202033)(Pveg-1xLR-GFP), [**BBa\_K4202034**](http://parts.igem.org/Part%3ABBa_K4202034)(Pveg-2xLR-GFP), [**BBa\_K4202035**](http://parts.igem.org/Part%3ABBa_K4202035)(Pveg-3xLR-GFP), and [**BBa\_K4202036**](http://parts.igem.org/Part%3ABBa_K4202036)(Pveg-1xLR-GFP) into Bacillus subtilis WB600 strain.

Characterized the above parts in media containing different sucrose concentrations. The growth curve was measured using Nanodrop (only in the characterization of [**BBa\_K4202045**](http://parts.igem.org/Part%3ABBa_K4202045)) and the fluorescence intensity was measured using flow cytometer.

**- Calcium Carbonate Production part**

ACCBP was expressed and purified according to the conditions obtained in the pre-experiment

Protein structure and function verification of ACCBP.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

[Overview](#page-id-1)

[Part1](#page-id-2)

[Part2](#page-id-3)

[Part3](#page-id-4)

![](https://static.igem.wiki/teams/4202/wiki/leftlion.png)

## Overview

As EngineerGirl puts it,  *“When we say engineering principles, we mean the ideas, rules, or concepts that need
to be kept in mind when solving an engineering problem.”* We knew that engineering principles are very
different from scientific research ideas, and the solution to engineering problems pays more attention to specific
design and practice. Our team's engineering follows the principle of *design-build-test-learn*and is
committed to the combination of dry and wet labs to improve the efficiency of each cycle.

During the design phase, we fully follow the principle of *top-down* and carefully consider the
recommendations of stakeholders in human practice. From determining abstract biological circuits to selecting
actual DNA sequences, from the expected results of the design to the actual tests, we will refine all the relevant
details as much as possible at this stage. At the same time, we will work to promote the integration of wet and
dry laboratories, by using computers to help wet laboratories model and predict, and designing hardware to
optimize wet laboratory workflows and efficiency.

During the construction phase, we will consider the experimental method, the experimental materials, and the
expected results in advance, and pay attention to the correlation between the integrity and individuality of the
module.

During the testing phase, our focus is on the standardization of the measured data, which we will convert using
internationally harmonized units to ensure accurate delivery of information. At the same time, data processing and
analysis in the wet lab will also be carried out after communication with the team members of the dry lab.

During the learning phase, we will reflect on the problems that arise and the conclusions that can be drawn from
the design-build-test process, and integrate what we think into the next design cycle. **We understand that the
process of engineering does not happen overnight, so we take an objective look at the implications of each
failure or success and use it as much as possible to optimize the engineering of our projects.**

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-0.jpg)

## Part1 Commit suicide and minimize the leakage

Our goal for engineering the sucrose inducible promoter was to characterize the native sucrose inducible promoter
and improved it to implement a system that was of practical use when coupled with mazE/F system.

1. The inducible activity should have a wide range to produce a sufficient amount of MazE/F at the beginning of
the culture.

2. The promoter should have a lower leaky expression to enable the degradation of the antitoxin mazE.

### Cycle1 Basic inducible promoter

The first step was to find an inducible promoter and characterized it to base future iteration on.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-1.png)

### Cycle2 Change the length of the spacer

Next, we needed to find out why the promoter didn’t exhibit the inducible activity. The first concern was the
distance between the promoter and the initiation codon (ATG) of the mRFP gene.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-2.png)

### Cycle3 Improve the transcription initiation activity of the promoter

In order to use the promoter in our project, we needed to make structural changes to the promoter.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-3.png)

## Part2 Enable ***Bacillus subtilis*** to precipitate calcium carbonate with fixed structure outside the cell.

Our goal for part2 was to transform the carbonic anhydrase and ACCBP gene into *Bacillus subtilis* to obtain
the ability to precipitate calcium carbonate with fixed structure outside the cell.

### Cycle1:Validation of the P43 promoter as a strong constitutive promoter of *Bacillus subtilis*

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-4.png)

### Cycle2 Express carbonic anhydrase in *Bacillus subtilis*

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-5.png)

### Cycle3 Add terminators and Change the medium

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-6.png)

### Cycle4 Explore the esterase activity of carbonic anhydrase

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-7.png)

### Cycle5 Verify the esterase activity of purified carbonic anhydrase and Exclude endogenous interference

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-8.png)

### Cycle6 Verify the function of carbonic anhydrase catalyzing biomineralization from the macroscopic level

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-9.png)

## Part3 Multi-channel fluorescence detection system

Multi-channel fluorescence detection system had practical significance in many fields. Our goal was to design a
multi-channel multifunctional fluorescence detection device to help wet lab to complete the detection of
experiments related to luminescent substances.

1.Detection of fluorescent protein concentration

2.Characterization of the oxidation and regeneration process of D-Luciferin

### Cycle1 Preliminary setup of the device

First we needed to complete the preliminary setup of the device to test the oxidation of D-Luciferin.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-10.png)

### Cycle2 Exploration of the optimum temperature for luciferase

After determining the shooting parameters, we further detected the reaction of luciferase and D-Luciferin at
different temperature conditions, which will helped us determine the thermal stability of luciferase.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-11.png)

### Cycle3 Characterization of luciferase regenerating enzyme

Oxyluciferin, which was the product of the luciferase reaction, had a strong inhibitory effect on the firefly
luciferase in a manner competitive with firefly luciferin. In the presence of luciferase regenerating enzyme and
D-cysteine, oxyluciferin was supplied as the substrate luciferin for next light emission, so that the fluorescence
luminescence time of the system could be prolonged.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-12.png)

### Cycle4 Detection of fluorescent protein concentration

After characterization of the oxidation and regeneration process of D-Luciferin, we hoped the device to meet the
needs of detecting fluorescent protein concentrations.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-13.png)

### Cycle5 Linearity and detection limit

Linearity was the basis of fluorescent protein concentration detection. We expected to find ranges where there
was a strong linear relationship between gray values and fluorescent protein concentrations.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-engineering-14.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

[Module 1: Calcium Carbonate Production](#page-id-1)

[Module 2: Biological Scaffold](#page-id-2)

[Module 3: Quorum Sensing](#page-id-3)

[Module 4: Biosafety](#page-id-4)

![](https://static.igem.wiki/teams/4202/wiki/leftlion.png)

Our experiment was also divided into four parts for validation. We started with the most
basic molecular biology
experiments and ended with macroscopic validation, trying to demonstrate the feasibility of the overall design.

## Module 1: Calcium Carbonate Production

### 1.1 The reinforcement of HCO3- production

This part describes our efforts to transfer and express of Carbonic Anhydrase (CA) and
ACCBP genes in the
*Bacillus subtilis*
WB600, followed by protein purification to verify the activity of CA in catalyzing HCO3-
production, and the function of ACCBP to transform ACC.

1.1.1 Plasmids construction

Firstly, we built two plasmids, pHY300PLK-P43-CA1-T7(from iGEM parts library No.
[BBa\_K2232014](http://parts.igem.org/Part%3ABBa_K2232014)) and
pHY300PLK-P43-CA2-T7 (CA2 is the codon optimization product of CA1) using Snapgene and sent the sequence to
GenScript for synthesis. The plasmids were amplified by DH5α, then extracted, purified, and verified by
sequencing.

1.1.2 Expression of CA

After chemical transformation of plasmids, the transformed *Bacillus subtilis* WB600
strain
were cultured in
optimized LB and SMM medium, and obtained crude enzyme solution of carbonic anhydrase by centrifugation and
ultrasonic disruption. We name the carbonic anhydrase expressed by BBa\_K2232000 as **CA1**, while name the
carbonic anhydrase expressed by BBa\_K4202004 as **CA2**. Then
we detected the molecular mass of carbonic anhydrase by SDS-PAGE and coomassie blue staining.

**Result:**  SDS-PAGE displayed bands of 37kDa for CA monomer, which didn' t
exist in the
control group.

![](https://static.igem.wiki/teams/4202/wiki/zju-result-1-1.png)

**Fig 1-1** SDS-PAGE and Coomassie brilliant blue staining results of whole protein
lysates
of strain CA1, strain CA2 and blank WB600. Lane 1: Protein Ladder; Lane2: CA2 strain grown in LB medium, Lane3:
CA2 strain grown in SMM medium, Lane4: CA1 strain grown in LB medium, Lane5: CA1 strain grown in SMM medium,
Lane6: blank WB600 strain.

1.1.3 CA purification

We added a 6X His tag to the N-terminus of CA on the vector, and the CA was purified by
agarose-nickel column
affinity chromatography. SDS-PAGE analysis was performed on the flowthrough, washing fluid and eluent during the
purification of CA1 and CA2 by Ni-column.

**Result:** The eluate of CA1 and CA2 had two obvious bands at 37kDa and 74kDa, which
were
monomer CA and dimer
CA, respectively. However, the Ni-NTA resin had much non-specific protein binding, we can add a small amount of
imidazole to reduce non-specific protein binding.

![](https://static.igem.wiki/teams/4202/wiki/zju-result-1-2.png)

**Fig 1-2** A: SDS-PAGE analysis of CA1 protein purified by Ni-NTA resin. Lane1:
flowthrough,
Lane2-4: the 1-4 washing fluid, Lane5-9: the 1-5 eluate, Lane10: protein Maker B: SDS-PAGE analysis of CA2
protein
purified by Ni-NTA resin. Lane1-5: the 1-5 eluent, Lane6-8: the 1, 2 and 4 washing fluid, Lane9: the
flowthrough,
Lane10: protein Maker.

1.1.4 Determinate the ability of CA to catalyze the hydration of
CO2

To measure the activity of CA, we used the modified Wilbur-Anderson's method **[1]**. As
we know, CA can
catalyze CO2 hydration and at the same time release H+ reducing the pH. According to that,
we chose bromothymol blue, an acid-base indicator that appears yellow when pH≤6 and blue when pH>7.6. Therefore,
the color development of bromothymol blue can indirectly reflect the change of pH from 8.0 to 6.0 by CA.

**Result:** After adding icy saturated CO2 solution for 10min, the color of
the
tubes containing
crude enzyme solution CA1 and CA2 began to change, indicating that the pH of the solution began to decrease.
After
10min, the tubes containing the crude enzyme solution showed significant discoloration, and after 5 days, the
tubes containing the CA1 crude enzyme solution turned completely yellow, implying that the pH had decreased from
8.0 to 6.0 due to the formation of H+ during CO2 hydration.

The activity of CA1 and CA2 were verified in this experiment, but the enzyme activities
were
weak, possibly due
to low enzyme expression or insufficient concentration of unpurified enzyme. In addition, the catalytic rate of
CA2 crude enzyme solution was lower than that of CA1（without codon optimization）.

![](https://static.igem.wiki/teams/4202/wiki/zju-result-1-3.png)

**Fig 1-3** The activity of CA detected by the Wilbur-Anderson's method. From left to
right,
the four tubes were pH=6.0 Tris-HCl buffer with bromothymol blue indicator, reaction system with blank WB600
lysate, CA2 crude enzyme solution, CA1 crude enzyme solution. A: Initial reaction solution（0min); B: 10min after
adding ice-saturated CO2 solution; C: 5d after adding ice-saturated CO2 solution.

1.1.5 Detection of esterase activity of CA

The common α-CA is a secreted monomeric protein with esterase activity that can catalyze
the
catalyzes the
formation of p-nitrophenol from p-nitrophenyl acetate and show the color(yellow) **[2]**. Although it was
reported that the CA naturally produced by *Bacillus Halodurans* TSLV1 had no esterase activity, we still
wanted to test whether the recombinant CA expressed in *Bacillus subtilis* WB600 had esterase activity. In
this part, we used the Nanodrop and microplate reader to measure the color reaction rates of the CA crude enzyme
solutions. We also measured the esterase activity of purified CA solution in order to avoid influence of the
other
proteins in the cell lysis solution.

**Result:** The system with crude enzyme solution which quickly changed from colorless
to
yellow had an
absorption peak at about 400nm(Fig.1-4 A). Absorption-time curve at 405nm showed that CA1 and CA2 crude enzyme
solutions had high esterase activity(Fig.1-4B). For purified CA solution,the results showed that purified CA1
and
CA2 both still had strong esterase activity(Fig. 1-4 C), which was inconsistent with the literature we checked.
This may be because the recombinant CA expressed in *Bacillus subtilis* WB600 has both monomer and dimer
forms, and some monomer CA own esterase activity.

![](https://static.igem.wiki/teams/4202/wiki/zju-result-1-4.png)

**Fig 1-4** Detection of esterase activity of CA. A: absorbance of the system with crude
enzyme solution at different wavelengths, B: esterase activity of CA1 and CA2 crude enzyme solution, x: Time
(min), Y: A405nm, Blank: no bacterial split solution, WB600: lysate of blank WB600 was added, CA1:
the lysate of
transformant-CA1, CA2:the lysate of transformant-CA2, C: esterase activity of purified CA1 and CA2 enzyme
eluent,
x: Time (min), Y: A405nm, Blank: no enzyme solution, WB600: lysate of blank WB600, CA1: purified CA1
enzyme
solution, CA2: purified CA2 enzyme solution.

1.1.6 Determinate the ability of CA to catalyze the precipitation of
CaCO3

To test the ability of engineered *Bacillus subtilis* WB600 with CA genes to
precipitate
CaCO3,
we cultured the engineered bacteria in 30ml of LB medium at 25oC for 3 days and added 5 ml of 100mM
CaCl2 solution on the first and second days. We filtered the culture medium through a Whatman
filter paper to separate the bacteria and CaCO3. The bacteria and CaCO3 were dried and
weighed, respectively. We can calculate CaCO3 precipitation capacity of engineered bacteria by the
formula: CaCO3 dry weight (mg)/cell dry weight (g) **[3]**.

After three days of cultivation, we could clearly see that the culture medium of the CA1 and CA2
transformant
became turbid. The precipitate was filtered and dried. According to the the formula: CaCO3 production
capacity = CaCO3 dry weight (mg)/cell dry weight (g), we found that the the precipitation efficiency
of
CA1 was higher than that of CA2.

![](https://static.igem.wiki/teams/4202/wiki/zju-result-1-5.png
  )

**Fig 1-5** A: The cultures after induction. B: Filtered and dried sediment
products. C: CaCO3 productions by CA1 and CA2 for 3d. D:Picture of precipitation under scanning
electron microscope.

### 1.2 Expression and function verification of ACCBP

To verify the function of **ACCBP**, we constructed the plasmid **Pet28a-His
tag-ACCBP** and **Pet28a-His
tag-Δ16 ACCBP**(Δ16 refers to the deletion of 16 amino acid of Nterminus) , and transformed them into
BL21(DE3). The ACCBP can be induced under specific conditions . After purification, we carried out the
extracellular crystallization experiment to prove the function of ACCBP.

1.2.1 Construction of Pet28a-His tag-ACCBP

We obtained the CDS and protein sequence of ACCBP and SacB secretion signal sequence of
*Bacillus subtilis*
from NCBI database, and designed the fusion protein SP-ACCBP. The codon of SP-ACCBP was optimized for
*Bacillus
subtilis* and synthesized by GenScript, and the plasmid PHY-300PLK-SP-ACCBP(PHY-ACCBP) was constructed.

Considering the large quantity of protein we needed for function verification, we constructed the expression
plasmid PET-28a(+)-ACCBP for *E.coli* in order to obtain the purified protein of high concentration for
later
experiments. Besides, we also constructed the palsmid Pet28a(+)-Δ16 ACCBP to explore the influence of N-terminal
amino acids to protein folding.

1.2.2 Protrein expression verification

To confirm that the optimized sequence could be produced by *E. coli*, we transformed
the plasmid pET-28a
(+)-ACCBP into BL21 (DE3) strain and cultured them in kanamycin resistant LB plates at 37 o C
overnight. The next day, the overnight-cultured solution was transferred into 5 ml kanamycin resistant LB medium
at a ratio of 1:20. The solution was cultured at 37°C, 220 rpm until OD600 reached 0.6-0.8. Then 0.1
mM
IPTG, 10% glycerol and 1mM CaCl2 was added to induce the protein expression at 8oC

At the end of induction, the bacterial solution was centrifuged at 12000 rpm at 4°C and the
bacteria were
collected. After washing the precipitation once with PBS, the precipitation were resuspended in a 1.5ml EP tube
with 1mL PBS (appropriate amount of PMSF can be added).The bacteria were disrupted by ultrasonic disruption
until
the suspension was clear. The products are used for SDS-PAGE.

**Result**: Acorrding to the result of SDS-PAGE, we can find that the protein were
both in the
supernatant and precipitation. And the more protein are in the precipitation, indicating that under this
condition
majority of the protein are insoluble.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-res-p-1.png)

**Fig 1-6** SDS-PAGE and Coomassie brilliant blue staining results of supernatant and
sediment of BL21(DE3) strain with PET-28a(+)-ACCBP. The lanes have been indicated .

1.2.3 Protein expression and purification

The overnight-cultured bacterial fluid was inoculated into 500 ml Kanamycin resistant LB medium at the ratio of
1:100. The solution was cultured at 37 ° C , 220rpm until OD600 reached 0.6-0.8 and
protein
was induced at 8 oC with 1mM IPTG, 1% glycerol and 1mM CaCl2 for 48h. Then we collect the bacterium
and
resuspend in a special protein extract buffer (containing 10% glycerol, 500 mM PH 7.5 Tris-HCl buffer, and
0.01mM
PMSF ). The next steps are same as 1.2.2 above .

Then we used His-tag Protein Purification Kit(Beyotime) to purify our protein (refered to [protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D))

Notes: In order to get a better protein expression efficiency, we also use palsmids Pet28a(+)-ACCBP,
Pet28a(+)-Δ16
ACCBP, strains BL21(DE3) and shuffle T7 for this experiment .

**Result**: The purified ACCBP and Δ16 ACCBP protein were obtained by multiple combination of plasmids and
strains. The protein will be used in further experiments.

![](https://static.igem.wiki/teams/4202/wiki/result-1-7.png)

**Fig 1-7** SDS-PAGE and Coomassie brilliant blue staining results of protein
purification.
A: The purification
result of ACCBP; B: The purification result of Δ16 ACCBP. The results show that both proteins have a
considerable
abundance in eluent 2 and 3 although there are some non-objective bands shown due to proteolysis.

1.2.4 Verification of ACCBP structure

In order to verify the structure of active ACCBP protein, we conducted chemical crosslinking experiment. Add
0.1%
and 0.5% glutaraldehyde to 25μl ACCBP protein eluent, mix and let it stand at room temperature. Take samples at
regular intervals. After terminating the reaction with loading buffer, run 12% SDS-PAGE to detect the
cross-linking structure.

**Result:** According to the result of SDS-PAGE, we found that that the band of 140kDa was the most
significant
one of all samples. As the apparent molecular weight of ACCBP monomer is about 25kDa,we predicted that ACCBP
probably assembles as pentamers in the solution. And the cross-linking results were better with less
non-objective
bands when higher glutaraldehyde concentration was used.

![](https://static.igem.wiki/teams/4202/wiki/result-1-8.png)

**Fig 1-8** SDS-PAGE and Coomassie brilliant blue staining results of ACCBP chemical
crosslinking experiment.

1.2.5 Extracellular crystallization assay

Add an appropriate amount of ACCBP solution, water and MgCl2 solution into the
newly prepared
saturated Ca(HCO3)2**[4]** to prepare 20μl solution containing 0, 8.0, 16.0, 40.0 mM
MgCl2. Drip the 20μl reaction system onto the cover glass. After 24 hours of reaction, observe the
calcium carbonate crystals on the slides were studied by SEM.

**Result:** For better images, the calcium carbonate crystals were observed in different magnifications. It
was
obvious that when the concentration of Mg2+ was low, ACCBP induced the formation of typical square
calcite crystals. When the concentration of Mg2+ reached 40mM, ACCBP induced the formation of more
aragonite particles. Our results also indicated that the deletion of 16 N-terminal amino acids of ACCBP had no
significant effect on the protein function.

![](https://static.igem.wiki/teams/4202/wiki/result-1-9.png)

**Fig 1-9** The influence of Mg2+ to the crystallization results of ACCBP

## Module 2: Biological Scaffold

In this module, our experiment design mainly focuses on the structural validation and functional feasibility of biological scaffolds, and the proof of the function.

### 2.1 Function verification of Spytag-SpyCatcher

2.1.1 Fusion protein design and plasmid construction

The engineered SpyTag and Spycatcher protein sequences and CDS sequences were obtained from NCBI database. We
designed two fusion proteins, **HA Tag-GS-mRFP-GS-SpyTag** and **6\*HisTag-GS-Spycatcher-GS-mRFP**, and
commissioned GenScrip to synthesize the DNA sequences of the above two proteins. The sequences were inserted
into
Pet28a(+) to obtain new recombinant plasmids.

2.1.2 Pre-experiment: exploration of suitable conditions for protein
expression

The above plasmids were transformed into BL21(DE3) strain respectively. After cultured in kanamycin-resistant
LB
plates at 37°C overnight, a single colony was picked and inoculated into kanamycin resistant LB medium for
overnight culture. The next day, the overnight-cultured solution was transferred into 5ml kanamycin resistant
LB
medium at a ratio of 1:20. The solution was cultured at 37°C, 220 rpm until OD600 reached 0.6-0.8.
Then
1 mM IPTG was added to induce protein expression under following conditions. And we will also set up a control
group .Then we obtained crude protein solution by centrifugation and ultrasonic disruption.The products were
used for SDS-PAGE .

![](https://static.igem.wiki/teams/4202/wiki/yzhxx-condition.png
)

**Results**: Acorrding to the result of SDS-PAGE, we found that the more protein were
in
the supernatant .
Considering the concentration and quantity of the targeted protein ,we chose the condition of group 2 for the
purification experiment.

![](https://static.igem.wiki/teams/4202/wiki/yzh1-spy-pre-experiment.png)

**Fig 2-1** SDS-PAGE and Coomassie brilliant blue staining results of supernatant and
sediment of BL21(DE3) strain. A: Pet28a(+)-HA tag-GS-mRFP-GS-Spytag B: Pet28a(+)-His-tag-GS-spycatcher-GS-mRFP
.
The lanes and the target bands have been indicated.

2.1.3 Protein purification and function verfication

The protein mRFP-SpyTag was induced at 20oC for 10h, and then used HA-tag
Protein
IP Assay Kit with
Magnetic Beads (Beyotime) to purify our protein (refered to [protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)). The protein SpyCatcer-mRFP was induced at
20oC for 10h, and then used His-tag Protein Purification Kit(Beyotime) to purify our protein (
refered
to [protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)). The products were used for SDS-PAGE
analysis
and later experiments.

**Results**:After repeated trials, we adjusted the composition of lysis buffer to
obtain
better purification
efficiency. Finally, we successfully obtained the purified proteins. For mRFP-SpyTag, we found that the
targeted
band can be observed in Fig2-2 the lane 3, though the most of the targeted protein still reserve in the
magnetic
separation of supernatant after adsorption. For SpyCatcer-mRFP, we obtained a good purification effect
according
to the results of SDS-PAGE analysis. It was worth noting that in order to obtain better purification effect,
we
increased the content of imidazole in the eluation buffer (eluent(150 mM imidazole) ). The final results
indicated
that the protein with high purity was obtained in the eluent(150 mM imidazole).

![](https://static.igem.wiki/teams/4202/wiki/yzh2-mrfp-spytag-purification.png
)

**Fig 2-2** SDS-PAGE and Coomassie brilliant blue staining results of HA-tag protein
purification. Lane 1: Bacterial lysate(Input); Lane 2: Magnetic separation of supernatant after adsorption;
Lane
3:Elution with loading buffer.

![](https://static.igem.wiki/teams/4202/wiki/yzh3-spycatcher-mrfp-purification.png
)

**Fig 2-3** SDS-PAGE and Coomassie brilliant blue staining results of His-tag protein
purification. The lanes and the target bands have been indicated.

2.1.4 Purified proteins quantification

The purified proteins were quantified by the BCA method (refered to [protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)).Limited to
laboratory equipment, we
chose OD578(the best wavelength is 562) for quantitative protein measurement. Then, we test the
purified protein mRFP-spytag and spycatcher-mRFP, and get the concertration of protein by the standard curve
roughly. It`s worthy to note that, we utilize the HA-peptide solution to elute the mRFP-spytag, so the result
is
not quite accurate.

**Result:**

![](https://static.igem.wiki/teams/4202/wiki/yzh4-bca-standard-curve.png
)

**Fig 2-4** The BCA-standard curve

the correlation between OD578nm and the protein content(μg):

$OD578-μg: Y = 0.0446X R\_2=0.98$

$OD578(\frac{1}{2} SpyCatcher-mRFP)=13.22$

$OD578(mRFP-SpyTag)=0.418$

$Protein concentration: $

$SpyCatcher-mRFP=1.3 mg/μl$

$mRFP-SpyTag=0.02 mg/μl$

2.1.5 SpyTag-SpyCatcher combination capability verification

The mRFP-spytag and spycatcher-mRFP protein samples were diluted to appropriate fold. We
collocted 200μl of each,
mixed and added 100μl 5×PBS. The mixture was incubated at 37 oC for 2h. Sample was collected for
SDS-PAGE and WesternBlot analysis.It`s worthy to note that we choose exhibit the whole PDVF to abtain a visual
outcome.

**Result** :In the lane of spy, we clearly found the signal of Spycatcher-SpyTag
complex
and corresponding
protein. However, in the lane of Spycatcher-mRFP and mRFP-SpyTag, we only saw the signal of the protein
possessing
the corresponding tag. So proof the function of SpyTag-SpyCatcher.

![](https://static.igem.wiki/teams/4202/wiki/yzh5-spy-wb.png
  )

**Fig 2-5** The result of Western Blot by two kinds of antibody . Spy:The mixture of two
protein after
incubation spy

### 2.2 Expression and function verification of EutM-SpyCatcher

For the validation of EutM-SpyCatcher, we constructed plasmids PHY-PsacB-EutM-SpyCatcher
(following as
PsacB-EutM-SpyCatcher) and PHY-P43-EutM-SpyCatcher (following as P43-EutM-SpyCatcher). The two plasmids were
transformed into *Bacillus subtilis* and *E. coli* for protein production and purification.
Subsequently, the purified proteins were functionally verified by protein analysis experiments such as SDS-PAGE
and Transmission Electron Microscope（TEM）.

2.2.1 Fusion protein design

We obtained the CDS and protein sequence of EutM, engineered SpyCatcher and SacB
secretion
signal sequence of
*Bacillus subtilis* from NCBI database, and designed the fusion protein SP-EutM-spycatcher. To ensure
that
the designed fusion protein still has the ability to assemble, I-TASSER was used for homology modeling. The
results showed that the recombinant protein could still form a reasonable spatial structure.

![](https://static.igem.wiki/teams/4202/wiki/result/m21.png
)

**Fig 2-6** The result of homologous modeling by I-TASSER

2.2.2 Plasmid construction

The codon of SP-EutM-spycatcher was optimized for *Bacillus subtilis* and
synthesized
by
GenScript, and the
plasmid PHY-300PLK-SP-EutM-spycatcher (PHY-EutM-spycatcher) was constructed. We ligated appropriate promoter
and
terminator sequence with CDS sequences by In-Fusion Clone strategy to construct plasmids,
PHY-PsacB-SP-EutM-SpyCatcher-DT (DT means Double Terminator) and PHY-P43-SP-EutM-SpyCatcher-DT, respectively.

2.2.3 Pre-experiment: exploration of suitable conditions for protein
expression

To screen the best conditions for the protein expression of *B.subtilis*, we
employed
SMM medium and LB
medium for pre-expreiment. We transformed the plasmid PHY-P43-EutM-spycatcher-DT into *Bacillus subtilis*
WB600 by chemical transformation (refered to [protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)),
inoculated in tetracycline-resistant LB plates and
cultured it at 37oC overnight, picked single colonies, and cultured them in tetracycline resistant
LB
or SMM medium at 37oC overnight.

**Result:**  As shown in the table, SMM medium maintained a relatively stable pH
value,
and the final
concentration of the bacteria reached 5 times that of LB medium, which is thought to be an increase in pH to
inhibit the growth of bacteria in LB. Subsequent experiments will be cultured with SMM medium.

![](https://static.igem.wiki/teams/4202/wiki/result/t1.png
)

2.2.4 Protein characterization of EutM-SpyCatcher

*Bacillus subtilis* transferred with plasmids was cultured in SMM for 48h according
to
the above culture
method. 14. Having harvested the cells, we analyzed the total protein in supernatant and pellet. However, a
very
low amount of secreted protein was observed in the supernatant compared to
visible bands in the pellet, and we suspected that secreted and scaffolded EutM proteins were pelleting
together
with the cells.

After reviewing the literature, we conducted numerous tests to eventually discover the
protein expression
validation technique **[5]**. Extracellular secretion and intracellular accumulation of scaffold building
blocks by recombinant Bacillus strains was analyzed by preparing four different fractions from cultures for
SDS-PAGE analysis.The preparation of all four protein fractions is shown as a flow chart. The products
obtained
were used for SDS-PAGE analysis.

![](https://static.igem.wiki/teams/4202/wiki/result/m2.png)

**Fig 2-7** Experimental workflow used for the analysis of EutM scaffold building block
secretion and expression by engineered *Bacillus. subtilis* .(i) culture supernatant, (ii) urea
supernatant
containing urea solubilized scaffolds that co-precipitated with cells, (iii) lysed cell pellets after
solubilization of co-precipitated scaffolds, and (iv) total protein (secreted-precipitated and intracellular)
from
completely lysed cells with sonication followed by lysozyme treatment.

Besides, we utilized the purified mRFP-SpyTag to verify the function of SpyCatcher domain in EutM-SpyCatcher.
The
details of this experiment could be found in 2.1.5 .

**Result**: A significant change in the bacterial fluid properties was observed, which
turned into a viscous
liquid and the proteins attached to the bacteriophage could be pulled. It was thought to be a change brought
about
by EutM. According the outcome of SDS-PAGE , we clearly found the band of complex of
EutM-SpyCatcher-mRFP-SpyTag(35 kD).

![](https://static.igem.wiki/teams/4202/wiki/result/m23.jpg
  )
![](https://static.igem.wiki/teams/4202/wiki/result/m24.png
  )

**Fig 2-8** Observed EutM secretion property and SDS-PAGE results of EutM-SpyCatcher
protein

2.2.5 EutM-SpyCatcher protein purification

Since the protein expressed by *B.subtilis* may be difficult to purify using Ni-NTA
resin
, due to protein
folding, etc, we transformed PHY-P43-EutM-SpyCatcher-DT into *E. coli* strain in the hope of obtaining
purified target protein for subsequent analysis. We transformed the above plasmid into BL21 (DE3) strain and
cultured them in ampicillin-resistant LB medium at 37  ° C for 24h. Product would be analysis by
work
flow in 1.2. His-tag Protein Purification Kit(Beyotime) was used for protein purification(refered to [protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)),
and the products would be used for SDS-PAGE and following experiments.

**Results:**

![](https://static.igem.wiki/teams/4202/wiki/result/m25.png
)

**Fig 2-9** SDS-PAGE and Coomassie brilliant blue staining results of EutM-SpyCatcher
protein expression in  *E.coil*(left) and feature(right). Negative control was the result of empty *E.coli*.

![](https://static.igem.wiki/teams/4202/wiki/result/m26.png
)

**Fig 2-10** His label recombinant protein purification effect. SU: supernatant lysate
CL:
cell lysate; FT: flow through; W1-3: wash 1-3; E1-5: elution 1-5

2.2.6 TEM characterization of EutM-SpyCatcher

Concentrations of purified proteins were measured using the BCA Assay. For negative
staining,
10 μL of protein
was applied to the surface of a 200 μm formvar/carbon-coated copper grid . An equal volume of Trump’s fixative
was
added to the surface of the grid, and the protein/fixative drop was allowed to settle for 2 min. The surface
of
the grid was rinsed with 10 μL deionized water and excess fluid was removed. The protein on the grid was
stained
by applying 10 μL uranyl acetate (1%). Scaffolds were imaged on Phillips CM12 TEM with magnifications of x
20,000,
x 50,000 and x 100,000.

**Result**: The TEM image shows that when we overexpressed and purified EutM, the
isolated
protein readily
precipitated out of solution as large crystalline arrays, with obvious hexameric organization and symmetry.
These
results indicated that EutM could serve as a building block for the design of a protein-based scaffolding
system.

![](https://static.igem.wiki/teams/4202/wiki/result/m27.png
)

**Fig 2-11** Protein-based scaffolding system. Negative stain TEM of the white
precipitate
formed in the tube shows crystalline EutM scaffolds composed of hexameric tilesthat are assembled from
structurally ordered arrays of EutM hexamers (red line indicates ordered crystalline lattice). Insert
highlights
individual EutM hexamers.

### 2.3 Function verification of Hag-spytag

In the validation experiments of this part, we constructed the plasmid
PHY-P43-Hag::SpyTag588 and induced
the protein expression in *B.subtilis*WB600 and *E.coli* BL21 (DE3). The protein samples were analyzed
by SDS-PAGE, fluorescent staining and other methods.

2.3.1 Protein function verification

We obtained the CDS and protein sequence of Hag *Bacillus subtilis* from subtiwiki,
and
designed the
recombinant protein **Hag::SpyTag588**. We employed overlapping PCR and In-Fusion Clone to ligated
appropriate promoter and terminator sequence with CDS sequences to construct plasmids
PHY-P43-Hag::SpyTag588-DT. The plasmids were transformed into BL21(DE3) strain. Following step the same
as
2.1.3. The broken products were centrifuged, and the supernatant were collected and mixed with purified
Spycather-mRFP, incubated at 37 °C for 2h. His-tag Protein Purification Kit (Beyotime) was used for
protein purification and the products could be used for SDS-PAGE analysis.

**Results:**

![](https://static.igem.wiki/teams/4202/wiki/yzh14-hag-spy.png
)

**Fig 2-12** SDS-PAGE and Coomassie brilliant blue staining results of His-tag protein
purification. The target band of SpyCatcher-mRFP-Hag::SpyTag588 has been noted.

2.3.2 Verification of recombinant flagellum function using fluorescence
microscopy

We tranformed the plasmid PHY-P43-Hag::SpyTag588-DT into *Bacillus subtillis* WB600 strain and inoculated into
tetracycline resistant LB medium for overnight . Then we cultured the *Bacillus subtillis* WB600 containing
PHY-Hag::SpyTag588 and PHY-EutM-SpyCatcher in the tetracycline resistance SMM medium. After culturing 36h, we added
the 0.2 mg purified SpyCatcher-mRFP(The fluorescence probe can bind with Hag::spytag to charcterize the presence of the
engineered Hag) per 5 ml medium into the medium and cultured the bacteria for 12h.Besides, we settled WB600 group and
calcium carbonate group for control. Then we utilized the confocal laser scanning microscope (FLUOVIEW FV3000) to detect
the cultures treated by SYTO .

**Result:** We can clearly observe the green fluorescence at 507 nm and red
fluorescence
at 610nm, while the
group for control showed negative outcome. So we could conclude that the biological scafforld can be assembled
reasonably.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-result-233.png)

**Fig 2-14** Confocal laser scanning microscope of different groups. The cells were
stained
by SYTO for
indicated.

## Module 3: Quorum Sensing

In this part, we mainly learned from the previous iGEM teams. However, we tend to make our
experiments more
complete and more radical. Therefore, the three basic modules of quorum sensing, including subtilisin
production,
signaling and immunity, were verified separately.

3.1 Immunity part

Initially, we confirmed the function of immune part (NO.[BBa\_K302018](http://parts.igem.org/Part%3ABBa_K302018)) and the inhibitory concentration of
subtilisin by zone of inhibition experiment and mixing subtilisin into the solid culture medium.

We first tested the inhibitory concentration of subtilisin by zone of inhibition
experiment,
using BL21
transformed by PHY-Immunity plasmid or PHY-Empty plasmid as control. The concentration of subtilisin were
100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml.

**Results:** We could see from the radius of inhibition zone that the subtilisin needed
quite a high
concentration to work as an efficient antibiotic. Meanwhile, there was no obvious difference between the
Immunity
group and the control group.

![](https://static.igem.wiki/teams/4202/wiki/gll-immunity-1.png)

**Fig 3-1** Zone of inhibition experiment for subtilisin

Then, we prepared normal LB culture plates with different concentration of subtilisin,
including 0.1mg/ml,
0.2mg/ml, 1mg/ml and 10mg/ml, in individual dishes. Then we inoculated BL21 transformed by immunity plasmid or
PHY-empty plasmid as control in the plates.

**Results:**For the immunity group, some colonies grew on the surface of the media with
1mg/ml subtilisin,
while the control group didn’t. This suggested that the PHY-Immunity plasmid worked well in BL21.

![](https://static.igem.wiki/teams/4202/wiki/gll-immunity-2.png)

**Fig 3-2**The colonies growth condition in plates with different concentration of
subtilisin

3.2 Signal part

For signal module, we used GFP as the reporter gene to show the trigger of subtilisin by
observing the bacterial
colony’s phenotype and running SDS-PAGE.Therefore, we constructed PHY-Signal-GFP plasmid, using the biobrick
[BBa\_K104001](http://parts.igem.org/Part%3ABBa_K104001). Then we inoculated WB600 transformed by
PHY-signaling-GFP plasmid on the media with or without
0.1mg/ml subtilisin.

**Results:**It’s quite clear that GFP was induced by 100μg/ml subtilisin, even though
the
growth of WB600 was
moderately blocked by subtilisin.

![](https://static.igem.wiki/teams/4202/wiki/gll-signal-1.png)

**Fig 3-3**Phenotype of GFP-expressing colonies campared with the control group

In order to confirm the expression of GFP, we ran the SDS-PAGE later.

**Results:**Compared with the control group, the WB600 cultured with 0.1mg/ml subtilisin
extended
significantly strengthened expression of GFP.

![](https://static.igem.wiki/teams/4202/wiki/gll-signal-2.png)

**Fig 3-4** SDS-PAGE to confirm the GFP expression

In general, although the promotor of signal module showed a little leakiness, it still
worked
pretty well in
response to the subtilisin, especially in WB600.

3.3 Subtilisin production part

Based on our design, a large population density led to low-oxygen environment, thus
resulting
in the expression
of subtilisin production gene. Therefore, we compared the bacteria population and gene expression level between
culturing in 15ml tubes and 50ml flask by measuring the OD600 and running SDS-PAGE.

Different from the previous part [BBa\_K302021](http://parts.igem.org/Part%3ABBa_K302021), we built a new plasmid using the original
Spa-box
of strain
ATCC6633, which was another  *Bacillus subtilis* strain producing the subtilisin. Then we ligated the
fragment with the linear vector containing Oxygen-limitation induced promotor and its RBS.

![](https://static.igem.wiki/teams/4202/wiki/gll-production-1.png)

**Fig 3-5**Plasmid construction of Oxygen-limitation-SpaBCTS

Succeeding in constructing the plasmid(see new parts [BBa\_K4202007](http://parts.igem.org/Part%3ABBa_K4202007) ), we transformed it into our chassis for
further
protein expression. 50ml shaking flasks with 5ml culture media were introduced to reach an extra-large bacterial
population while the normal 12ml shaking bacteria tubes were used as control.

**Results:**From the SDS-PAGE image, we could see a clear strip locating in 120kDa’s
area
for the 50ml shaking
flasks group, revealing the excellent function of Oxygen-limitation induced promotor. However, it seemed that
only
the first gene (SpaB,120kDa) had expressed under the condition, suggesting that the original sequence didn’t
match
well to the promotor.

![](https://static.igem.wiki/teams/4202/wiki/gll-production-2.png)

**Fig 3-6**Characterization of Oxygen-limitation induced promotor by SDS-PAGE

Unfortunately, due to the tight time schedule, we lacked time rebuilding the plasmid and
producing the
subtilisin.

## Module 4: Biosafety

In this module, our experimental design focuses primarily on:

1. Characterize the sucrose-inducible promoters and make improvement on it.

2. Characterize the kill switch we designed.

### 4.1 Characterization of the sucrose-induced promoter PsacB

4.1.1 Construction of characterization plasmid

In order to characterize the sucrose inducible activity of the sucrose inducible
promoter
PsacB, we extracted
genomic DNA of  *Bacillus subtilis* WB600, and amplified the PsacB sequence by PCR. The plasmid
pHY300PLK-PsacB was constructed using In-Fusion clone. The mRFP:rrnB terminator:T7 terminator sequence coming
from
**BBa\_K143082** element was inserted into the 6bp
position
downstream of PsacB to obtain the pHY300PLK-PsacB-mRFP plasmid. At the same time, we also obtained the
expression
element containing Pveg-spoVG-RFP-rrnB T1 terminator-T7 terminator from BBa\_K143082. The promoter Pveg is a
constitutive promoter with medium activity. We also constructed it onto the pHY300PLK plasmid and obtained the
pHY300PLK-Pveg-mRFP plasmid as a control of PsacB promoter strength.

**Results:**
![](https://static.igem.wiki/teams/4202/wiki/zju-china-result-4-1.png
)

**Fig 4-1** A.the atlas of pHY300PLK-PsacB-mRFP plasmid. B.the atlas of
pHY300PLK-Pveg-mRFP
plasmid

 4.1.2 Characterization experiment

Refering to the [protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D) provided
by
LMU, we transferred the pHY300PLK-PsacB-mRFP plasmid and
the
pHY300PLK-Pveg-mRFP plasmid into  *Bacillus subtilis* WB600. Two positive clones were inoculated into 5ml
tetracycline resistant LB medium and cultured overnight. The next day, the culture was transferred to 10mlLB
medium at a ratio of 1:100. For both Pveg and PsacB promoter, characterization was carried out in LB medium
containing 0%, 1%, 3%, 5% and 10% (w/v) sucrose.

The culture was incubated in shaking flask at 220rpm and 30 oC for 12h and
centrifuged at
8000g, RT for 2min to
harvest the cell. The expression of mRFP protein in harvested cells was first determined by naked eyes to
roughly
reflect the promoter strength.

**Results:**

It could been seen that the transcription initiation activity of Pveg promoter was much
higher than that of PsacB
promoter, and the PsacB promoter did not produce a visible mRFP accumulation.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-result-4-2.png)

**Fig 4-2**: A rough comparison of mRFP expression levels driven by different promoters
under different induction
conditions. A: the inducion result of Pveg promoter. B: the induction result of PsacB promoter.

The ultrasonic disruption product of the harvest cell was used to run the SDS-PAGE.

**Results:**It can be seen that the transcription initiation activity of Pveg promoter
is
much higer than
PsacB promoter.The intensity of the mRFP band produced by PsacB promoter is very weak, and there is a certain
degree of leakage expression when the induction activity do not exist under the condition of 5% sucrose
induction.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-result-4-3.png)

**Fig 4-3** the SDS-PAGE result of mRFP protein driven by different promoters under
different induction
conditions. CK: Bacillus subtilis with plasmid PsacB:EutM

To investigate the correlation between promoter activity and inducer concentration, we
measured the bacterial
fluorescence intensity at different inducer concentrations using sucrose and sucralose as inducers,
respectively.
The fluorescence producted by GFP were detected using flow cytometer.

**Results:**  The flow cytometry results indicate that both sucrose and sucralose can
positively regulate the
activity of promoter. The activity of the promoter can increase up to ~5 times compared to the control when
using
sucrose as the inducer. However, due to the detection limit of flow cytometer, the data may not be very
accurate.
This may be due to the low activity of this promoter, too.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-result-4-4.png
)

**Fig 4-4** The fluorescence output after inducing using different types and
concentrations
of inducers. SUC:
sucrose, SUL: suralose.

To enable higher promoter activity after induction, we also planned to engineered the
PsacB
promoter.

Through literature review, we found that the inducible activity of PsacB promoter comes
from
its ribonucleic
antagonist sequence and the sacY protein that can bind to this sequence and stabilize its structure. [6] Based
on
this, we plan to iterate on the existing PsacB.

### 4.2 Iteration of sucrose inducible promoters

4.2.1 Iterative design

We introduce a strong constitutive promoter Pveg to replace the constitutive promoter of
PsacB. To reduce the
possible leakage of expression caused by the replacement of the strong promoter, we designed multiple repeat
of
the leader RNA sequence separated by spacers, and added spoVG RBS to enhance its expression. We constructed
pHY300PLK-Pveg-N×LR vector containing 1× , 2× , 3× and 5× leader RNA sequence and used firefly luciferase or
GFP
as the reporter gene downstream of these promoters.

4.2.2 Characterization of promoter

The characterization method is similar to the characterization method of PsacB. We also
use
flow cytomoter to
measure the fluorescence produced by GFP.

**Result:**We characterized all our engineered promoters and found that the promoter
activity decayed less
under higher sucrose concentration conditions after replacing the constitutive promoter as well as multipling
leader RNA (only in Pveg-2×LR promoter). Also, promoters with different numbers of leader RNA repeats showed
different increases in activity after induction.The promoter Pveg-5×LR show the highest(more than 2 fold)
induction activity.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-result-4-5.png)

**Fig 4-5** The characterization result of our engineered promoters.

### 4.3 The construction and characterization of kill switch

4.3.1 The construction of our kill switch

Our suicide switch consists of a sucrose-sensitive promoter and the mazE-mazF
antitoxin-toxin
system. We
commissioned GenScript to synthesis the above sequence and construct it to the pHY300PLK plasmid.

**Result:**

![](https://static.igem.wiki/teams/4202/wiki/zju-china-result-4-6.jpg
      )

**Fig4-6** The structure of our kill switch

4.3.2 The characterization of our kill switch

To characterized our kill switch, we transformed both the empty vector and the vector
containing the kill
switch into Bacillus subtilis WB600 and cultured them in the LB medium containing 1% sucrose. The
OD600nm(L=1mm) of the cultures was measured every 40 minutes when the OD600nm(L=1mm) of
cultures reached approximately 0.1.

**Result:**

Compared to the control group, the OD600nm(L=1mm) of strains containing the
kill switch began to
decrease after the OD600nm(L=1mm) reached 0.12. The early decrease of OD600nm indicated
that the bacteria are dying, which means our kill switch is working.

![](https://static.igem.wiki/teams/4202/wiki/zju-china-result-4-7.png
        )

**Fig 4-7** The growth curve of two *Bacillus subtilis* strains. Blue curve
represents strains
containing suicide switches and the yellow line represents strains containing the empty plasmid(CK).

### Reference:

[1] Faridi, S., & Satyanarayana, T. (2016). Novel alkalistable α-carbonic anhydrase from
the
polyextremophilic
bacterium Bacillus halodurans: characteristics and applicability in flue gas CO2 sequestration. Environmental
Science and Pollution Research, 23(15), 15236-15249.

[2] Zheng, T., & Qian, C. (2020). Influencing factors and formation mechanism of CaCO3
precipitation induced by
microbial carbonic anhydrase. Process Biochemistry, 91, 271-281.

[3] Achal, V., & Pan, X. (2011). Characterization of urease and carbonic anhydrase
producing
bacteria and their
role in calcite precipitation. Current microbiology, 62(3), 894-902.

[4] Xu, G., Yao, N., Aksay, I. A., & Groves, J. T. (1998). Biomimetic synthesis of
macroscopic-scale calcium
carbonate thin films. Evidence for a multistep assembly process. Journal of the American Chemical
Society, 120(46), 11977-11985.

[5] Kang, S. Y., Pokhrel, A., Bratsch, S., Benson, J. J., Seo, S. O., Quin, M. B., ... &
Schmidt-Dannert, C.
(2021). Engineering Bacillus subtilis for the formation of a durable living biocomposite material. Nature
communications, 12(1), 1-17.

[6] Aymerich, S., & Steinmetz, M. (1992). Specificity determinants and structural features
in
the RNA target of
the bacterial antiterminator proteins of the BglG/Sacy family. Proceedings of the National Academy of Sciences,
89(21), 10410–10414.

{% endblock %}