iGEM 2021 — Team UESTC-China

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# Team:UESTC-China/Notebook

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![logo](https://static.igem.org/mediawiki/2021/d/d7/T--UESTC-China--touming.jpg)

* [June](#title_1)
* [July](#title_2)
* [August](#title_3)
* [September](#title_4)
* [October](#title_5)

![logo](https://static.igem.org/mediawiki/2021/7/7d/T--UESTC-China--up.png)

June

Based on the available synthetic plasmids, we arranged the work of each member and carried out preliminary preparations such as purchasing necessary equipment and drugs. In addition, according to the pichia pastoris expression book and the actual laboratory
situation, we have designed a complete set of experimental flow schemes and set a weekly schedule to push our experiments forward.

July

## 7.1-7.7

We prepared competent escherichia coli and transfered our six target plasmids EG1, EGL7, XynB, LipLac, Scaford, SdbA synthesized by the company into E.coil. Then we use low-salt(LB) medium to culture our E. coli in order to get enough bacterial deposits
to extract our plasmids.

At the same time, we consulted a great number of literatures concerning modified enzymic preparations and bacterial preparations to determine which preparation we should use for subsequent deinking validation.

## 7.8-7.14

We extracted target plasmids from our escherichia coli bacterial deposits and we obtained a large number of target plasmids. We used agarose gel electrophoresis to verify our target plasmids and we used ethanol to precipitate the successful plasmids.

At the same time, we began to explore the conditions for the cultivation of Pichia pastoris and then cultivated Pichia pastoris. We finally obtained a batch of pure Pichia pastoris which was found to be free from bacterial contamination by microscopic
examination.

## 7.15-7.21

We began to explore the electrotransformation conditions, and tried to transfer our EG1, EGL7, XynB, LipLac, Scaford, SdbA plasmids into a new batch of Pichia pastoris and verified their successful expression of related proteins.

At the same time, we began to explore methods to detect the deinking activity of commercial enzymes.

## 7.22-7.31

We were stuck in our experiment for the reason that every time we cultured pichia pastoris after electrotransformation, miscellaneous bacteria always appeared. We finally decided to eliminate our lab completely by using potassium permanganate and formaldehyde.

During formaldehyde elimination in the lab, we started writing the framework of our wiki and drew up subsequent experimental arrangements. In addition, we also used the laboratory downstairs to conduct normal deinking verification experiments and successfully
verified that cellulase and xylanase have a decent deinking effect.

August

## 8.1-8.7

We were able to re-enter our old lab after testing formaldehyde levels to meet national standards and we used our original plasmids and restarted to culture a new batch of pichia pastoris in order to restart the electrotransformation. We finally succeeded
in avoiding contamination by miscellaneous bacteria and we successfully verified the target bands by colony PCR.

At the same time, we began to verify the deinking effect of laccase and lipase on paper and explore the synergistic effect between enzymes. However, we found that the deinking effect of laccase and lipase was not satisfactory.

## 8.15-8.21

We consulted our teacher and learned that the transformant would produce a large number of false positives and our teacher suggested that we could use three-rounds 96-well plates for screening. We adopted our teacher's suggestion and screened a batch
of new pichia pastoris through electrotransformation in 96-well plate.

At the same time, we have improved our deinking verification method and began to process a new round of deinking data. We used the line chart and histogram to show our processed data and successfully verified the correlation between the enzymes and deinking
effects.

## 8.22-8.28

We took our positive transformants screened by 96-well plates and fermented them. As before, we collected the bacterial solution from 1 to 4 days of fermentation. But this time we first used dot blot to verify that our protein was successfully expressed
and then detected the enzyme activity of the target enzymes. This time we successfully verified the enzyme activity of our cellulase and lipase.

## 8.29-8.31

We began continuous fermentation and tried to detect laccase and xylanase activity at the same time we designed a new experimental method to verify the deinking effect of laccase and lipase. At the same time, we successfully produced the second generation
of deinking machine.

September

## 9.1-9.7

For those former pichia pastoris that dot blot tested successfully, we used SDS-PAGE and Western Blot to further verify our protein expression results, but it didn't work out so well. Our analysis is that the target protein of the previously preserved
fermentation liquor has been degraded. Therefore, we started to re-ferment a new batch of pichia pastoris.

## 9.8-9.14

After a lot of trial and error, we finally verified the target protein bands with western Blot and we finally determined the optimal deinking time of the core enzymes cellulase and xylanse and successfully produced the third generation of deinking machine.

## 9.15-9.21

The work of deinking verification has basically been completed and we set out to verify the binding of scaffold proteins to enzymes but until now we have not detected the xylanase and laccase enzyme activity.

## 9.22-9.31

We carried out some supplementary experiments for our deinking work. We tried to use fermented supernatant for deinking verification, but the result was not good. At the same time, we carried out a lot of fermentation to get enough target protein.

October

## 10.1-10.5

We finally measured the xylanase activity by ammonium sulfate precipitation method of protein concentration and we began to verify the promoting effect of scaffolding on targeted protein.

## 10.6-10.8

We ultrasonically verified the expression and the activity of all our key enzymes and successfully tested their function.

## 10.9-10.21

We did some supplementary experiments to verify our design and theory and we began to write wiki and prepare for the final presentation.

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# Team:UESTC-China/Engineering

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![LOGO](https://static.igem.org/mediawiki/2021/7/7a/T--UESTC-China--duibiao.png)

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![logo](https://static.igem.org/mediawiki/2021/d/d7/T--UESTC-China--touming.jpg)

* [Parts Engineering](#title_1)

+ [1.Enzymes Expression And Activity Test](#stitle_1)
+ [2.Scaffoldin Expression And Definition](#stitle_2)

* [Biodeink system engineering](#title_2)
* [Experimental Method engineering](#title_3)

+ [Force](#stitle_4)
+ [Features design](#stitle_5)

* [Hardware engineering](#title_4)

+ [1st iteration](#stitle_6)
+ [2nd iteration](#stitle_7)
+ [3rd iteration](#stitle_8)
+ [4th iteration](#stitle_9)

![logo](https://static.igem.org/mediawiki/2021/7/7d/T--UESTC-China--up.png)

![logo](https://static.igem.org/mediawiki/2021/a/ab/T--UESTC-China--DDD.png)

**Figure1. Circle of engineering**

As Judgingbook goes *'Engineering is **creative, rigorous application of knowledge** about a system to solve problems or develop new technologies and products. It is a mindset and a framework that enables systematic thought about the assumption and approximations in a design, defining both what is known and what is unknown in order to gain a view on the expected performance of a design'*,
we got a profound understading about **ENGINEERING** that we never have.

About the question we brought up--**How can we realize in-situ regeneration of Office waste paper?** There was no doubt that we were not the only ones who came up with this question, for example, PaperLab designed by EPSON. However,
its whopping price makes it impossible to step into everyone's daily life. We are **the pioneering group** trying to apply **biomethods** on this field, but at the same time, we are **facing numerous problems as all innovators may meet** too.

We shaped and carried forward our strategy with **several** research-imagine-design-build-learn and redesign loops. We had a lot of thoughts **in pieces** at first. But as the knowledge and experience accumulating, **a whole, integral, enviromental-friendly system** of engineering not only including biology but also **connecting all aspects of works** were established! Besides, we aimed to design a safe and humanized product, finally we got **DEINKER**!

To engineer Deinker successfully, we separated apart the things we knew, and the things needed be created, then made tailored plans to complete then as much as we could. Given the COVID-19 and short of time, we have already tried our best.

Our experimental part and hardware part are accomplished in strict accordance with the idea of engineering. We propose the idea of in-situ deinking, which requires hardware devices and biological enzymes.

We screened and designed the enzymes needed in the deinking process and we tried our best to acquire proteins with high expression in Pichia pastoris. At the same time, we used the same type of commercial enzyme to explore the deinking method. Finally,
we found that laccase had no effect on deinking and lipase didn’t play a significant role in deinking either. The best deinking efficiency could be obtained when cellulose and xylanase were mixed in a proper proportion. Based on these
results, we further improved the design of the cellulosome and used it for the next round of deinking exploration.

The hardware needs to be continously revised and refined from the birth of an idea to the establishment of working modules. Through precise assembly, the different working modules eventually constitute an organic whole, so that the initial deinking function
can be barely completed. In this process, we found and documented problems with the device, corrected its original design, and invested in the development of the next generation of hardware. In this way, we have accomplished three
iterations of Deinker and are ready to develop a fourth generation Deinker that is more similar to the printer.

Parts Engineering

We registered a total of 29 parts this year, including 21 basic parts and 8 composite parts, which are derived from 6 plasmids designed by us, and most of them have been tested to insure their reliability. Click [here](https://2021.igem.org/Team%3AUESTC-China/Demonstrate) for more details.

After linerization, electrotransfection and colony PCR, we successfully inserted plasmids: pEGL7, pXynB, pLiplac and pScaf into chassis P.pastoris GS115. Dot-Blot and enzyme activity definition tests help us verify their expression. Here are some important
results：

## 1.Enzymes Expression And Activity Test

### a.XynB-Doc

![logo](https://static.igem.org/mediawiki/2021/9/9a/T--UESTC-China--result-xynb.png)

**Figure2. Plasmids Linearization.** pGAPZa-XynB digested by BglⅡ, produces a 3974bp band; Controls are the circular (not linearized) plasmids, which will give ≥2 bands: a DNA supercoil band (the shorter one), and a DNA helix band
(the longer one). Markers are Trans 2K Plus Ⅱ from Transgen.

![logo](https://static.igem.org/mediawiki/2021/2/2b/T--UESTC-China--result-xynb1.png)

**Figure3. Colony PCR results of recombined GS115(pXynB),** The left arrow refers to specific primers' product (662bp), and the right one refers to non-specific primers' product(1448bp).

![logo](https://static.igem.org/mediawiki/2021/6/6e/T--UESTC-China--part.22.png)

**Figure4. The Dot-Blot results of recombined GS115(pXynB) fermentation broth.** The sample was gained after fermentation in YPD for 96h. XynB(His-Tag) was tested by adding 2ul fermentation supernatant on NC membrane and exposing
60s.

![logo](https://static.igem.org/mediawiki/2021/2/28/T--UESTC-China--result-xynb3.png)

**Figure5. The SDS-PAGE results of recombined GS115(pXynB)** fermentation broths. Line 1 and 2 are GS115(pXynB) supernate and GS115 supernate respectively after 75-fold concentration. And at 43.4KDa, which is XynB with Ctdockerin's
size, the expected band is not clear enough by naked eyes.

![logo](https://static.igem.org/mediawiki/2021/3/3c/T--UESTC-China--result-xynb4.png)

**Figure6. The Western-Blot results of recombined GS115(pXynB) fermentation broth.** Control is GS115's concentrated fermentation supernatant. XynB is 43.4KDa. Line 1, 2, 3 refer to sediment, supernatant and concentrated supernatant.

![logo](https://static.igem.org/mediawiki/2021/9/97/T--UESTC-China--result-xynb5.png)

**Figure7. The DNS results of XynB reflects enzymes' activity.** The solution was gained after 96h of fermentation and concentrated by ammonium sulfate deposition method. 2ml enzyme solution reacted with 2ml 1% Xylan solution for
30min, then added 5ml DNS to shut down the process. During boiling in water bath, DNS combined with reducing sugar and produced dark brown, while control just shown unobvious color change.

### b. EGL7

![logo](https://static.igem.org/mediawiki/2021/a/a3/T--UESTC-China--engineering-PCRE7.png)

**Figure8. Plasmids Linearization.** pGAPZa-EGL7 digested by BglⅡ, produces a 4460bp band;

![logo](https://static.igem.org/mediawiki/2021/5/54/T--UESTC-China--emgineering-E71.png)

**Figure9. Colony PCR results of recombined GS115(pEGL7).** The left arrow refers to non-specific primers' product(2207bp), and the right one refers to specific primers' product(694bp).

![logo](https://static.igem.org/mediawiki/2021/4/48/T--UESTC-China--result-egl7-3.png)

**Figure10. The Dot-Blot results of recombined GS115(pEGL7) fermentation broth.** The sample was gained after fermentation in YPD for 96h. EGL7(His-Tag) was tested by adding 2ul fermentation supernatant on NC membrane and exposing
60s.

![logo](https://static.igem.org/mediawiki/2021/c/c9/T--UESTC-China--result-egl7-4.png)

**Figure11. The DNS results of EGL7 reflects enzymes' activity.** The solution was gained after 96h of fermentation. 2ml fermentation supernatant reacted with 2ml 1% Xylan solution for 30min, then added 5ml DNS to shut down the
process. During boiling in water bath, DNS combined with reducing sugar and produced dark brown, while the control just showed unobvious color change.

### c.Lipase

![logo](https://static.igem.org/mediawiki/2021/9/9c/T--UESTC-China--lip-1.png)

**Figure12. Plasmids Linearization.** pGAPZa-Liplac digested by AvrⅡ, produces a 6386bp band. Controls are the circular (not linearized) plasmids, which will give ≥2 bands: a DNA supercoil band (the shorter one), and a DNA helix
band (the longer one). Markers are Trans 8K from Transgen.

![logo](https://static.igem.org/mediawiki/2021/6/66/T--UESTC-China--engineering-lila1.png)

**Figure13. Colony PCR results of recombined GS115(pLiplac).** The left arrow refers to non-specific primers' product(4124bp), and the right one refers to specific primers' product (861bp).

![logo](https://static.igem.org/mediawiki/2021/e/eb/T--UESTC-China--lip-3.png)

**Figure14. The Dot-Blot results of recombined GS115(pLiplac) fermentation broth.** The sample was gained after fermentation in YPD for 96h. Lipase(Myc-Tag) was tested by adding 2ul fermentation supernatant on NC membrane and exposing
60s.

![logo](https://static.igem.org/mediawiki/2021/5/50/T--UESTC-China--multi-lipase1.png)

**Figure15. Alkali Titration results of Lipase.** 1ml concentrated protein solution mixed with 5ml 50mM, PH=5.0 phosphate buffer and 4ml emulsion, then reacted at 40℃ for 15min. In the experiment system. Produced fatty acid neutralizes
100mM NaOH solution so experiment system's color was stable during titration, while system turn red directly in control.

## 2.Scaffoldin Expression And Definition

![logo](https://static.igem.org/mediawiki/2021/8/8f/T--UESTC-China--engineering-scaf1.png)

**Figure16. Plasmids Linearization.** pGAPZa-Scaffoldin digested by AvrⅡ, produces a 5582bp band;

![logo](https://static.igem.org/mediawiki/2021/b/b3/T--UESTC-China--engineering-scaf2-1.png)

**Figure17. Colony PCR results of recombined GS115(pScaf).** The left arrow refers to specific primers' product(1901bp), and the right one refers to non-specific primers' product(3047bp).

![logo](https://static.igem.org/mediawiki/2021/4/4a/T--UESTC-China--engineering-scafdot.png)

**Figure18. The Dot-Blot results of recombined GS115(pScaf) fermentation broth.** The sample was gained after fermentation in YPD for 96h. Scaffoldin(Myc-Tag) was tested by adding 2ul fermentation supernatant on NC membrane and
exposing 60s.

![logo](https://static.igem.org/mediawiki/2021/0/04/T--UESTC-China--engineering-scafwb.png)

**Figure19. The western blot results of recombined GS115(pScaf) fermentation broth.** Control is GS115's concentrated fermentation supernatant. Scaffoldin is 98.5KDa in theory. Line 1, 2, 3 refer to sediment, supernatant and concentrated
supernatant.

Biodeink system engineering

At first, we selected 4 kinds of enzymes to realize our goal. (Click [here](https://2021.igem.org/Team%3AUESTC-China/Demonstrate#title_3) for more details.)
Because we could not get our enzymes by expression in a short
time, we bought commercial enzymes which are similar to the enzymes we have chosen. We used those enzymes to create a brandnew measurement (see below) and a series of experiments to verify and provide information for our redesign.
-
Through tests, we found that laccase would make paper yellow for its special buffer, so we removed laccase from our design.
- Cellulase and xynalase had a good synergy between the two, and it was proved that the deinking effect
had been the best when the concentration ratio of the two was 2:1
- Considering that we had introduced cellulosome to improve deinking efficiency (it was also the first time that cellulosome had been used in the field of deinking),
which could control the ratio of enzymes by regulating the number of doc-cohs, so we also adopted the ratio 2:1 to design domains. Click [here](https://2021.igem.org/Team%3AUESTC-China/Design) to view more.

Experimental Method engineering

During biodeink system engineering, a question arose: How to assess the residual ink on A4 paper? We did a lot of research and had a lot of brainstorming, but this problem still puzzled us for a long time. There is a tailored guidance published by TAPPI,
Technical Association of the Pulp and Paper Industry, but just for papermaking from the pulp. And we found that their methods are difficult to measure our deinking efficiency.
The design of effective deinking
method is inseparable from a complete set of scientific identification indicators. As a new technology, the effect of non-pulping deinking has not yet been scientifically universally measured. In order to ensure the scientificity and
reliability of the experiment, through multiple iterations, we have originally created the dot grid verification method which is a scientific measure of the deinking effect.
The core problems are how to achieve variable control
in terms of forces and how we define indicators that objectively describe deinking effects.

## Force

**1st iteration**
 We can control the adhesion and friction between the brush and the surface of paper by adjusting the height of the two rows of toothbrushes. However, because the variables cannot be controlled, the
two separate experiments cannot be compared horizontally. In addition, the brushing method is less efficient and easily damages the paper.
**2nd iteration**
 In the subsequent experiments, we determined the adhesive roller as the tool for deinking, and the effect of the adhesive roller was extremely sensitive to pressure so we found that the results obtained
by manually pushing the adhesive roller were unreliable. In the experiment, in order to take good use of this tool and obtain relevant data of the machine design, we initially designed a transmission method by combining the sticky
roller with rubber roller to adjust the stress of the springs suspended on both sides of the two shafts and obtain the corresponding tensile force to the sticky roller so as to accurately control the pressure between the adhesive roller
and the paper. Then the paper can be loaded on a thin plate and passed between the two rollers to realize deinking.
Although the original intention of our design was replacing manual work with a simple machinery in order to obtain
reliable reproducible data but this device has the problem of complicated operation and inconvenient maintenance. Even though it has been greatly improved compared with manual methods, it is still difficult to satisfy the requirement
for precise measurement.
In order to further improve the accuracy, we redesigned the way of deinking on the inclined surface of the drum. Compared with the previous method, the environmental variables in the experiment are only
slope angle and sticky roller weight. In theory, to ensure that these two variables are the same, the results obtained can be consistent. We also performed a series of repeated deinking experiments with specific concentrations of commercial
enzymes to confirm the feasibility of our design.

## Features design

**1st iteration**
Initially, our team chose A4 paper filled with Chinese character "tian" as the experimental material for deinking, yet the experimental results showed that although using A4 paper filled with Chinese
character "tian" as the experimental material can visually show the deinked effect. However, this method makes it difficult to quantify the effect of deinking, to accurately record the experimental results and to carry out subsequent
more detailed experiments like optimization of the ratio of the enzyme. In order to quantify the deinking effect, we designed the dot grid verification method as an indicator to measure the deinking effect.
**2nd iteration**
We
divide the paper into several different rectangular regions, and there are several small ink dots in each rectangular region. By measuring the number of residual ink dots after deinking, not only the quality of the deinking effect
can be quantified, but also multiple groups of repeated experiments can be carried out at the same time, so as to test the stability of deinking effect. The results obtained by this method are easy to record and do statistical analysis.

Through many iterations, we finally determined that the grid point verification method and the drum slope method were used as the method to test the effect of non-pulping deinking in the experiment. This method is original, accurate
and convenient, which promotes further development of the experiment. It also has reference value for formulating more accurate non-pulping deinking industry standard in the future.

Hardware engineering

## 1st iteration

According to the practical problems to be solved, we quickly designed and manufactured the initial Deinker. It is composed of enzyme spraying part and paper drying part.

The initial Deinker can complete four functions :

1. Paper transmission
2. Enzyme spraying
3. Fiber separation
4. Paper drying

## 2nd iteration

In the actual operation process, we found that the initial Deinker had some problems such as large volume, insufficient stability, unsatisfactory drying effect and deinking effect. So we need to design a second generation Deinker.

Through systematic redesign and reselection of components, the problems of the initial Deinker were solved.

This generation of Deinker can basically meet all the requirements for hardware functions in the early days of the project.

## 3rd iteration

However, in the process of deinking, we found that the second generation of Deinker tends to damage the integrity of the paper. Therefore, the third generation design of Deinker focuses on optimizing the detachment of ink spots from paper fiber after
the enzyme reaction, trying to find an ideal range of interaction that can not only avoids paper damage but promote the effect of deinking as better as it can.

After a period of redesign, we finally solved this problem by using a sticky roller as the fiber separation part. The subsequent results show that the paper damage is very little in the process of deinking, and the paper treated with enzyme solution can
be easily separated from the paper fiber to complete the deinking of the paper.

Finally, the third-generation Deinker perfectly fulfilled several functional requirements proposed in the initial stage of the project. Compared with the previous two generations, we have also carried out all-round optimization, including case packaging,
manual interaction function optimization and systematic optimization, making the third generation device closer to DEINKER in our mind.

## 4th iteration

In the process of displaying the third generation Deinker, we found that there was still a certain gap between ours and the ideal device in people's eyes, so I also continuously designed the fourth generation Deinker.

Click [here](https://2021.igem.org/Team%3AUESTC-China/Hardware1) to view more.

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# Team:UESTC-China/Experiments

# Experiments

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).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [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)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:UESTC-China/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)