iGEM 2019 — Team WHU-China

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

# Team:WHU-China/Notebook

* [Home](https://2019.igem.org/Team%3AWHU-China)
* Project
  + [Inspiration](https://2019.igem.org/Team%3AWHU-China/Inspiration)
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* Experiment
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  + [Notebook](https://2019.igem.org/Team%3AWHU-China/Notebook)
  + [Protocol](https://2019.igem.org/Team%3AWHU-China/Protocol)
* Hardware
  + [Introduction](https://2019.igem.org/Team%3AWHU-China/Hardware)
  + [Evolution](https://2019.igem.org/Team%3AWHU-China/Evolution)
* Human Practices
  + [HP overview](https://2019.igem.org/Team%3AWHU-China/Human_Practices)
  + [Integrated HP](https://2019.igem.org/Team%3AWHU-China/IntegratedHP)
  + [Public HP](https://2019.igem.org/Team%3AWHU-China/Public_Engagement)
* [Safety](https://2019.igem.org/Team%3AWHU-China/Safety)
* Parts
  + [Parts overview](https://2019.igem.org/Team%3AWHU-China/Parts)
  + [Contribution](https://2019.igem.org/Team%3AWHU-China/Contribution)
  + [Improve](https://2019.igem.org/Team%3AWHU-China/Improve)
* Team
  + [Team member](https://2019.igem.org/Team%3AWHU-China/Team)
  + [Collaborations](https://2019.igem.org/Team%3AWHU-China/Collaborations)
  + [Attribution](https://2019.igem.org/Team%3AWHU-China/Attributions)

![](https://static.igem.org/mediawiki/2019/3/3b/T--WHU-China--notebook0.jpeg)

[BC Group](https://static.igem.org/mediawiki/2019/d/d7/T--WHU-China--Notebook2.pdf)

[Converter Group](https://static.igem.org/mediawiki/2019/0/01/T--WHU-China--Notebook3.pdf)

[Functional Peptides Group](https://static.igem.org/mediawiki/2019/2/23/T--WHU-China--Notebook1.pdf)


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

# Team:WHU-China/Evolution

* [Home](https://2019.igem.org/Team%3AWHU-China)
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  + [Design](https://2019.igem.org/Team%3AWHU-China/Design)
  + [Model](https://2019.igem.org/Team%3AWHU-China/Model)
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* Experiment
  + [Results](https://2019.igem.org/Team%3AWHU-China/Results)
  + [Notebook](https://2019.igem.org/Team%3AWHU-China/Notebook)
  + [Protocol](https://2019.igem.org/Team%3AWHU-China/Protocol)
* Hardware
  + [Introduction](https://2019.igem.org/Team%3AWHU-China/Hardware)
  + [Evolution](https://2019.igem.org/Team%3AWHU-China/Evolution)
* Human Practices
  + [HP overview](https://2019.igem.org/Team%3AWHU-China/Human_Practices)
  + [Integrated HP](https://2019.igem.org/Team%3AWHU-China/IntegratedHP)
  + [Public HP](https://2019.igem.org/Team%3AWHU-China/Public_Engagement)
* [Safety](https://2019.igem.org/Team%3AWHU-China/Safety)
* Parts
  + [Parts overview](https://2019.igem.org/Team%3AWHU-China/Parts)
  + [Contribution](https://2019.igem.org/Team%3AWHU-China/Contribution)
  + [Improve](https://2019.igem.org/Team%3AWHU-China/Improve)
* Team
  + [Team member](https://2019.igem.org/Team%3AWHU-China/Team)
  + [Collaborations](https://2019.igem.org/Team%3AWHU-China/Collaborations)
  + [Attribution](https://2019.igem.org/Team%3AWHU-China/Attributions)

![](https://static.igem.org/mediawiki/2019/f/fe/T--WHU-China--hardwaree00.jpeg)

## *Hardware-evolution*

We got the original idea from [Toulouse\_France 2016.](https://2016.igem.org/Team%3AToulouse_France/Model) They created a device to solve the problem of fungal
contamination on cave murals. Inspired by them and to suit to our project, we thought our hardware should meet:

* Barrier for *safety*

* Installation for fixing fabrics

* Equipment for monitoring the process

* Device for *adding engineered bacteria and substances*

So we designed these basic components:

* Sealed housing to achieve internal and external isolation

* Magnetic chucks to fix the fabrics

* Temperature and pH detector

* Rotatable injector to add a specific sample in specific time

It led to the drafts shown below:

![](https://static.igem.org/mediawiki/2019/a/a2/T--WHU-China--Hardware1.png)

Based on this original idea, we drew our hardware by *3DMax* and keep optimizing, here are our all prior generations:

### *Hardware 1.0*

|  |  |  |  |
| --- | --- | --- | --- |
| |  |  | | --- | --- | | ![](https://static.igem.org/mediawiki/2019/a/a0/T--WHU-China--Hardware2.png) | ![](https://static.igem.org/mediawiki/2019/6/68/T--WHU-China--Hardware3.png) | |  |

In the 1.0 generation, we used *movable injector* and slide track to add the engineered bacteria. The injector moved to the specified location to add the storage,
so it could control the restoration region roughly. The fabrics would be fixed by the magnetic force and restored by the engineered bacteria in the work space under
monitoring.

### *Hardware 2.0*

Considering the *stability and maintenance risk* of movable injector, we changed it to a fixed one and installed the *control valve*. This change also improved
the efficiency of adding.

|  |  |  |  |
| --- | --- | --- | --- |
| |  |  | | --- | --- | | ![](https://static.igem.org/mediawiki/2019/8/86/T--WHU-China--Hardware4.png) | ![](https://static.igem.org/mediawiki/2019/5/54/T--WHU-China--Hardware5.png) | |  |

But there were still many defects in the adding device, so in 3.0 generation we had some tries on the *construction of pipes*.

### *Hardware 3.0*

Compared these four types, we chose *concentric pipes* for it have higher controllability and drew the 3.0 generation below. So far, our design
for the adding device had finalized.

![](https://static.igem.org/mediawiki/2019/8/8f/T--WHU-China--Hardware6.png)

But there were still many detects in the adding device, so in 3.0 generation we had some tries on the construction of pipes.

![](https://static.igem.org/mediawiki/2019/c/cd/T--WHU-China--Hardware7.png)

### *Hardware 4.0*

After the trip to [Hangzhou](https://2019.igem.org/Team%3AWHU-China/IntegratedHP), we knew the shape of weak area on the fabrics was *irregular*
in most situations, it inspired us to create a *light system* to control the BC reinforcement. Meanwhile, we needed to make some adjustments to the hardware.

So in 4.0 generation, we set up some components to correspond to the [light system design in gene-level](https://2019.igem.org/Team%3AWHU-China/Design).

We used the *baffle units* not light emitter units to control the shape of light. .

|  |  |  |  |
| --- | --- | --- | --- |
| |  |  | | --- | --- | | ![](https://static.igem.org/mediawiki/2019/c/c4/T--WHU-China--Hardware8.png) | ![](https://static.igem.org/mediawiki/2019/a/ad/T--WHU-China--Hardware9.png) | |  |

### *Hardware 5.0*

After repeated discussions and exchanges about the safety concerns, we thought only the sealed housing to ensure the engineered bacteria not to leakage is not
enough, so we designed [suicide circuit](https://2019.igem.org/Team%3AWHU-China/Safety) and added the *cleaning system* to clean the fabric and
the *infrared-thermal detector* to detect whether there are bacteria remain or not when the restoration is finished.

|  |  |  |  |
| --- | --- | --- | --- |
| |  |  | | --- | --- | | ![](https://static.igem.org/mediawiki/2019/2/26/T--WHU-China--Hardware10.png) | ![](https://static.igem.org/mediawiki/2019/8/8f/T--WHU-China--Hardware11.png) | |  |

We had shown our 5.0 generation to other teams, some thought highly of it and offered many advice. It is worth raising that [SZU-China](https://2019.igem.org/Team%3AWHU-China/Collaborations) suggested
us consider more about the cost, for example, we could cut the cost by replacing the emitter array in our scanning system maybe.

### *Hardware 6.0*

![](https://static.igem.org/mediawiki/2019/6/6e/T--WHU-China--Hardware12.png)

Inspired by SZU\_China, we designed horizontal rotary with *emitter row* and concentric tracks to replace the emitter array.
The emitter row rotates a circle along the track to finish a scanning cycle.

What’s more, we *estimated* the cost of our hardware for further application and made a [report](https://static.igem.org/mediawiki/2019/8/88/T--WHU-China--Hardwarepdf.pdf).

Thanks to all the help and effort from our teammates and other teams, the hardware evolved to the final generation.

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

# Team:WHU-China/Results

* [Home](https://2019.igem.org/Team%3AWHU-China)
* Project
  + [Inspiration](https://2019.igem.org/Team%3AWHU-China/Inspiration)
  + [Description](https://2019.igem.org/Team%3AWHU-China/Description)
  + [Design](https://2019.igem.org/Team%3AWHU-China/Design)
  + [Model](https://2019.igem.org/Team%3AWHU-China/Model)
  + [Demonstrate](https://2019.igem.org/Team%3AWHU-China/Demonstrate)
  + [Future](https://2019.igem.org/Team%3AWHU-China/Future)
* Experiment
  + [Results](https://2019.igem.org/Team%3AWHU-China/Results)
  + [Notebook](https://2019.igem.org/Team%3AWHU-China/Notebook)
  + [Protocol](https://2019.igem.org/Team%3AWHU-China/Protocol)
* Hardware
  + [Introduction](https://2019.igem.org/Team%3AWHU-China/Hardware)
  + [Evolution](https://2019.igem.org/Team%3AWHU-China/Evolution)
* Human Practices
  + [HP overview](https://2019.igem.org/Team%3AWHU-China/Human_Practices)
  + [Integrated HP](https://2019.igem.org/Team%3AWHU-China/IntegratedHP)
  + [Public HP](https://2019.igem.org/Team%3AWHU-China/Public_Engagement)
* [Safety](https://2019.igem.org/Team%3AWHU-China/Safety)
* Parts
  + [Parts overview](https://2019.igem.org/Team%3AWHU-China/Parts)
  + [Contribution](https://2019.igem.org/Team%3AWHU-China/Contribution)
  + [Improve](https://2019.igem.org/Team%3AWHU-China/Improve)
* Team
  + [Team member](https://2019.igem.org/Team%3AWHU-China/Team)
  + [Collaborations](https://2019.igem.org/Team%3AWHU-China/Collaborations)
  + [Attribution](https://2019.igem.org/Team%3AWHU-China/Attributions)

BC reinforcements
BC production
Light system
CBD detect
Streptavidin detect
Biotinylation detect
DSAOP detect
AMP detect

![](https://static.igem.org/mediawiki/2019/f/f6/T--WHU-China--Result0.jpeg)

## *Result*

#### *Bacterial Cellulose (BC) Reinforcements*

* Microscopic Examination

**1.Optical Microscopic Images**

The effect of aging and ex-situ reinforcement is clear enough under the optical microscope while in-situ reinforcement is less
obvious. Although further observation needs to be done by microscope with higher resolution, it is believed that in-situ reinforcement
would not disturb the silk’s texture.

![](https://static.igem.org/mediawiki/2019/0/03/T--WHU-China--result100.png)

2.SEM (Scanning Electron Microscope) Images

The images vividly indicate the effect of the aging, ex-situ reinforcement and in-situ reinforcement on the silk samples. The
images proves that in-situ reinforcement is a more appropriate way to do reinforcement based on the effective aging.

![](https://static.igem.org/mediawiki/2019/5/59/T--WHU-China--Result2.png)

![](https://static.igem.org/mediawiki/parts/9/95/T--WHU-China--Result3.png)

![](https://static.igem.org/mediawiki/parts/7/72/T--WHU-China--Result4.png)

* Functional Test

1. Mechanical strength is improved. In-situ reinforced silk samples is developed in tensile strength to some degree, which stands
 for mechanical strength.

![](https://static.igem.org/mediawiki/2019/c/cd/T--WHU-China--Result5.png)

2. Softness is improved. In-situ reinforced silk samples become softer than the aged ones because they have larger elongation at break,
which helps the silk perform better facing mechanical pressure.

![](https://static.igem.org/mediawiki/2019/c/cf/T--WHU-China--Result6.png)

* Chromatic Aberration Analysis

Since the bacteria cellulose is transparent, it is believed that the pattern of the silk would not be affected. Besides, we also
managed a color shift experiment to test whether the pattern of the colored silk samples would be affected.

![](https://static.igem.org/mediawiki/2019/9/99/T--WHU-China--Result7.png)

![](https://static.igem.org/mediawiki/2019/6/61/T--WHU-China--Result8.png)

#### *BC Production*

We were lucky to get the optimized set of plasmids to produce BC in *E. coli* from Professor Tomoya Imai and successfully expressed
BC in *E. coli* BL21(DE3) after exploring the conditions for a period of time.

1.Observation with Naked Eyes.

After inducing the expression of CesA, CesB and DGC by IPTG and arabinose for 36 hours, we found flocculent substances were similar to BC in the medium, which made the medium more viscous.

![](https://static.igem.org/mediawiki/parts/b/be/T--WHU-China--Result9.png)

2.In-situ Observation under Scanning Electron Microscope (SEM)

We also sprayed *E. coli* on the silk and observed with SEM (without cleaning). It is proved that engineered *E. coli* BL21(DE3) strain has the ability to synthesize and secrete bacterial cellulose, and to reinforce the silk. The secreted BC can attach to and connect between silk fibroins to achieve reinforcement.

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

3.Quantitative Determination

We also did quantitative determination of BC content by anthrone method after being treated by strong acid. We set bacteria cellulose produced by *G.xylinus* as positive control, the uninduced *E. coli* as negative control. By comparing with these two groups, we can confirm that the flocculent substances in the medium are exactly bacteria cellulose produced by *E. coli*.

![](https://static.igem.org/mediawiki/2019/3/3f/T--WHU-China--Result10.png)

#### *Verification of Light System*

To realize the irregular reinforcement, we designed a light system to control the *E. coli* to secret BC in specific site.

*1.Light System Validation*

1.0-Blue light system

At the beginning of our project, we planned to use the 1.0 light system--blue light system. Thus, we collaborated with
[ShanghaiTech\_China](https://2019.igem.org/Team%3AWHU-China/Collaborations) and got a blue light system from them.
We tested their system’s feasibility and effectiveness. The results are as follows:

![](https://static.igem.org/mediawiki/2019/6/6f/T--WHU-China--Result11.png)

From the comparison of the results between the non-induced group and the induced group, it is clear that more fluorescent
proteins can be produced when induced by blue light. There were extremely significant differences. Therefore, we can estimate that
the blue light system can achieve the purpose of regulating protein expression. This provided a very reliable light control element
for our project.

2.0-Red light system

When we designed the red light system, we considered that the promoter pOmpC may have osmotic noise. Thus we did improvement on it by adding a riboswitch controlled by theophylline. We did experiments to verify that the riboswitch can realize a tight control of this promoter. The following is the results:

![](https://static.igem.org/mediawiki/parts/5/5e/T--WHU-China--Result-ompc%2Bribo.png)

What’s more, we did experiments to confirm that the promoter pOmpC with riboswitch can open via theophylline induction under certain concentration.

![](https://static.igem.org/mediawiki/2019/3/38/T--WHU-China--theopphylline0000.png)

Thus, the improved pOmpC can be tight controlled as well as be induced effectively. As for the whole system, we constructed complete pathway in E.coli successfully. But time limited, the function verification experiments did not get significant results, we hope we can verify it in the future.

*2.Irregular Shaped Controlling*

When we got a reliable light system, we can use it to verify our idea of irregular shaped controlling. We created several
measurement methods to confirm that idea. Unfortunately, because of the limits of time and experimental conditions, we did not get
good results up to now. However, we will try to improve our methods to confirm this idea until the last minute.

![](https://static.igem.org/mediawiki/2019/a/ab/T--WHU-China--Result12.png)

#### *Verification of The Cellulose Binding Ability*

To verify the cellulose binding ability of the cellulose binding domain, we chose dCBM3a and CBM3a. We set three groups in
this experiment, experimental groups of dCBM3a, CBM3a and the negative control--sfGFP. Then the protein samples were mixed with
bacteria cellulose homogenate.

The fluorescent residues were measured after incubating the bacterial lysate with BC membrane (pre-dried in a black 96-well
plate) and washing for a few times. We confirmed that both of dCBM3a and CBM3a had good cellulose binding ability compared with
sfGFP. What’s more, the dCBM3a we improved had higher affinity with bacteria cellulose than CBM3a.
(Learn more details [Improve](https://2019.igem.org/Team%3AWHU-China/Improve))

![](https://static.igem.org/mediawiki/2019/3/3c/T--WHU-China--Result13.png)

#### *Streptavidin Detection*

Streptavidin can specifically bind to biotin, so we decided to use biotin magnetic beads to detect the existence of streptavidin
domain of dCBM3a. After incubating the biotin magnetic beads with engineered bacteria lysate and washing, it was found that the
target protein was successfully attached to the biotin magnetic beads by SDS-PAGE detection. We can see that the supernatant after
incubation has obviously lower amount of the dCBM3a proteins, while there were a lot of dCBM3a bound to the biotin magnetic beads.
Thus, the function of streptavidin domain of dCBM3a had been confirmed to bind to biotin successfully.

![](https://static.igem.org/mediawiki/2019/b/b6/T--WHU-China--improve03.png)

#### *Biotinylation Detection*

To realize the complete assembly of converter we designed, we had to verify the enzyme BirA can successfully biotinylate target protein with Avi-Tag .

Thus, we co-expressed BirA enzyme and the reporter protein sfGFP with Avi-Tag. By using the method of HABA/Avidin, we detected the biotin level
of co-expression system and compared with single sfGFP expression system. We got the results as follows:

![](https://static.igem.org/mediawiki/2019/c/c8/T--WHU-China--Resul15.png)

According to the results, we know that the sfGFP produced by pBGa are biotinylated well. Thus, we directly proved that the
functional peptides with Avi-Tag can be successfully biotinylated via BirA enzyme in vivo in our project.
(Learn more details Contribution)

#### *DSAOP Function Detection*

To verify the antioxidant ability of DSAOP, we arranged two measurements.

*1.Measurement of DPPH Radical Scavenging Activity*

The DSAOP antioxidant activity was determined using the DPPH radical scavenging assay.  When free radical scavenger is added
to it, DPPH can be combined or replaced to reduce the number of free radicals and make the solution color become lighter. We can
use the reduction of absorbance to evaluate the ability of scavenging free radicals. In other words, the antioxidant capacity
was calculated by detecting the effect of removing DPPH from the sample at the wavelength of 517nm.The DPPH radical scavenging
activity was calculated as follows:

**DPPH radical scavenging activity (100%) = [1 − (Ai − Aj )/ A0] × 100.**

The following is the result of the DPPH radical scavenging activity of DSAOP compared with DSAOP+Avitag and Vitamin
E (positive control).

![](https://static.igem.org/mediawiki/2019/d/d1/T--WHU-China--Result16.png)

*2.Measurement of Hydroxyl Radical Scavenging Activity*

Fenton reaction to generate hydroxyl free radicals react with salicylic acid, generated at 510 nm with special absorption
of 2, 3 - dihydroxy benzoic acid. Reaction is as follows: if join in reaction system which has the function of removing hydroxyl
free radical was tested, it will reduce the number of generated hydroxyl free radicals. Therefore, it reduce the generation of
colored compounds corresponding. Reaction time was fixed. The absorbance of the reaction solution containing the measured substance
was measured at 510 nm and compared with the blank solution to determine the hydroxyl content of the measured substance clearance
of bases.

The following picture is the result of the Hydroxyl radical scavenging activity compared with DSAOP+Avitag and Vitamin E.

![](https://static.igem.org/mediawiki/2019/6/6c/T--WHU-China--Result17.png)

The DSAOP-Avitag has the best hydroxyl scavenging activity and is better than DSAOP. According to this result, we got that adding
Avitag may not cause damage on DSAOP’s hydroxyl scavenging activity, but can increase this activity.

#### *AMP Function Detection*

The expression and purification of antimicrobial peptides were the difficulties in our project. The expression of such short
peptides in E.coli is often prevented by their solubility and effectiveness. At the beginning of the project, we carried out a lot
of exploratory experiments, including the direct expression and purification with his-tag, the indirect expression with sumo-tag.
Finally, we successfully purified the antimicrobial peptides by using the GST tag system and proved its effectiveness to against
mold.(Learn more details [Notebook](https://2019.igem.org/Team%3AWHU-China/Notebook))

The results were as follows:

Firstly, to cleave the GST-tag from the fusion protein, we expressed and purified the Prescission Protease (PPase).

![](https://static.igem.org/mediawiki/parts/thumb/6/6e/T-WHU-China-GST-PPase-1.png/626px-T-WHU-China-GST-PPase-1.png)

We used the purified PPase to cleave the fusion protein in cleavage buffer hung in 4℃ for 16h. We did the SDS-PAGE electrophoresis.

![](https://static.igem.org/mediawiki/2019/1/18/T--WHU-China--Result19.png)

It was strange that, after cleavage, there was only the protein about 26kDa (may be GST) without the PgD5 in the SDS-PAGE gel.
After repeated the experiment many times and analyzed the process, we speculated that we had succeeded in the cleavage but the
concentration of PgD5 was too low to be stained by the Coomassie blue. As a result, we did silver staining. It proved our conjecture,
so we condensed the cleavage sample by PEG-20000. The SDS-PAGE gel showed that we got the PgD5 successfully!

![](https://static.igem.org/mediawiki/2019/6/62/T--WHU-China--Result20.png)

The next job was to test whether the PgD5 could inhibit the mold. We used the solvent (cleavage buffer in purification, pH~7.0)
as the negative control, 10% NaOH as positive control and Aspergillus japonicus as the test object. To test the function of
antimicrobial peptides in various pH environments, we adjusted the pH of the PDA medium (pH~5.5) to pH~7.5 and pH~8.0. The results
indicated that PgD5 had a strong antifungal effect under pH~7.5 and pH~8.0.

![](https://static.igem.org/mediawiki/2019/e/e5/T--WHU-China--Result21.png)