Team:UESTC-China/Engineering
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 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
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.
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).
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.
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.
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.
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
Figure8. Plasmids Linearization. pGAPZa-EGL7 digested by BglⅡ, produces a 4460bp band;
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).
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.
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
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.
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).
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.
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
Figure16. Plasmids Linearization. pGAPZa-Scaffoldin digested by AvrⅡ, produces a 5582bp band;
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).
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.
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 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 to view more.
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 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.
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.
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.
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
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 to view more.