iGEM 2023 — Team wuhan-hubei-china

![ notebooks](https://static.igem.wiki/teams/4795/wiki/notebook.png)

## catalogue

* [March](#march)
* [April](#april)
* [May](#may)
* [June](#june)
* [July](#july)
* [August](#august)
* [September](#september)
* [October](#october)

## March

**Week 1 (March 1 to 4)**

On March 2nd, our team was officially established. Although we didn't have a formal team name at this time,
our initial team was still actively brainstorming all night for our project.In the initial meeting on March
2, we also had a preliminary division of labor.On March 4, under active discussion, we produced our initial
logo and the first official account tweet. Although we are not good at painting in biology, everyone's
enthusiasm is enough to support us to move forward.

![picture 1](https://static.igem.wiki/teams/4795/wiki/notebook-1.png)

**Week 2 (March 5 to 11)**

On March 6th, we held our first large formal meeting with other teams and instructors from inside and outside
the school, and everyone was in high spirits! This is an expected debriefing on the work in the coming
months. We had the opportunity to listen to teachers from Chen Weizhao, Yang Jian and Zhang Lijun talk about
their team's experience in participating in iGEM over the years, and showed us all the rigorous and
interesting activities of the team. Teacher Yang Shihui provided us with a global perspective on what it
means to become iGEMmer. We also reported the products of our own brainstorming, where the teachers
evaluated and guided our ideas and answered our questions. It was a very interesting event and all of us
were happy to be part of the iGEM world.

![picture 2](https://static.igem.wiki/teams/4795/wiki/notebook-2.png)

**Week 3 (March 12 to 18)**

We held several group discussions this week, brainstorming for our project and analyzing and interpreting the
competition information on the official website.On March 18th, the second large-scale exchange and guidance
meeting was held. After listening to the competition experience of several instructors, we were greatly
touched. After discussion and research, we sorted out the general framework of the competition and had a
deeper understanding of the specific project arrangement.

![picture 3](https://static.igem.wiki/teams/4795/wiki/notebook-3.png)

**Week 4 (March 19 to 25)**

We began to discuss and analyze the excellent teams and projects of previous years, hoping to learn from
their work that are missing in the initial plan.Our project is taking shape. From the fallen leaves on the
campus, we think of these plant "waste" piled up in the land, to transform them. Although the idea was
questioned by the feasibility of the PI, it overall provided ideas for our project.

**Week 5 (March 26 to April 1)**

This week, we organized the knowledge sharing about laboratory safety in the group. The PI shared the
important safety knowledge in the laboratory with all the members of our team. Each of us has recorded these
knowledge carefully, and studied the "Biosafety Law of the People's Republic of China" and "Hubei University
Laboratory Safety Guidelines".

## April

**Week 6 (April 2 to 8)**

This week we set out the project theme and focus on the synthetic biology part of the project and how to use
molecular biology strategies to implement these ideas.We propose the idea of anchoring multiple cellulase
constituent fibrosomes to the cell surface.

**Week 7 (April 9 to 15)**

This week, we officially entered the lab for experiments.We mainly completed the surface display of two
cellulases in S. cerevisiae.The third cellulase did not exhibit enzymatic activity on the surface display
system.

 Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun

**Week 8 (April 16 to 22)**

We modified the inducible expression medium after the literature review and repeated the experiment again.

Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun.We also proposed to produce
popular science articles to publish on our team account to promote our team project while spreading
biological knowledge.On April 22, he published its first popular science article, "Have you ever heard of
synthetic biology?".
Participant:Wang Xinlingjun, Luan Ziyi, Chen Yanjing

**Week 9 (April 23 to 29)**

Last week's repeat experiment continued due to the low recovery of gene fragments.This week to complete the
routine transformation of E. coli.
Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun
We published the second popular science article "Laboratory safety Code" after organizing the knowledge of
the last safety sharing meeting.
Participant:Xiao Yuhe, Luan Ziyi, Chen Yanjing

**Week 10 (April 30 to May 6)**

After validation of colony PCR, plasmids were extracted for linearization, enzymatically linked, and
electrotransformed.
Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun
We published this week's popular science article, "Animal rumination".
Participant:Chen Kexin, Luan Ziyi, Chen Yanjing

## May

**Week 11 (May 7 to 13)**

Given the low concentration of the recovered fragments after linearization, the last week's experimental
work was repeated this week.The electrotransformed work was completed this week.
Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun
We published this week's popular science article, "Understanding cellulase".

 Participant:Tian Chao, Luan Ziyi, Chen Yanjing
A new member of our team, Liu Xiongxiao, who is responsible for the design of art resources. He designed a
whole new logo for our team.And he also provided support for cover production for our popular science
articles.

**Week 12 (May 14 to 20)**

After electrotransformation, colony PCR was performed to verify the target fragment, and the results were
not ideal, with no band after repetition, and electroconversion was redone.The electroconversion process was
successfully completed and verified by colony PCR.

Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun
We published this week's popular science article, "History of molecular biology development".

Participant:Wang Xinyu, Luan Ziyi, Chen Yanjing

**Week 13 (May 21 to 27)**

The electrotransformed yeast were induced expression, timed sampled, and enzymatic
assays.
Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun
We published this week's popular science article, "What is polysaccharides".

 Participant:Wang Xinlingjun, Luan Ziyi, Chen Yanjing, Liu Xiongxiao

**Week 14 (May 28 to June 3)**

The results of enzyme measurement are not good, find literature and analyze the reasons for failure.

Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun

 We discussed proposing to introduce our project in the form of animation.Chen Kexin, Wang Xinling Jun
proposed script; Liu Xiong Xiao made art resources; Wang Xinling Jun made animation; Wang Xinling Jun, Chen
Kexin, Xiao Yuhe, He Yinqi dubbing; we co-production.

 Participant:Wang Xinlingjun, Chen Kexin, Xiao Yuhe, He Yinqi, Liu Xiongxiao

## June

**Week 15 (June 4 to 10)**

We modified the promoter for galactose-induced expression according to the summarized reasons for failure,
while modifying the corresponding experimental protocol. Start back from scratch and complete the routine
transformation this week.

Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun
We published this week's article, "Straw single-cell protein feed".We hope to show our project to everybody
through this article.

Participant:Xiao Yuhe, Wang Xinlingjun, Luan Ziyi, Chen Yanjing

**Week 16 (June 11 to 17)**

This week, we discussed that the existing experimental scheme is too difficult to continue. After reviewing a
large amount of literature and consulting the PI, we proposed to change the plan. The original protocol was
changed to use gene fusion technology to fuse three cellulase genes for expression in yeast.

Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun
Wang Xinlingjun contacted and made the team's brochures, team uniforms and team flags. The drawings of the
uniforms and flags were drawn by Liu Xiongxiao, and the brochure was drawn by Wang Xinlingjun.

**Week 17 (June 18 to 24)**

We discuss and propose that the content other than the cellulose degradation pathway can also be designed to
enhance the straw degradation.We started to collect the xylan degradation protocol and tried to incorporate
it into our experiments.After initially establishing the experimental protocol, we first performed primer
design to amplify the target fragment and screened them for mutations using error-prone PCR.

Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun, Li Huanan
We officially started the questionnaire survey on the project this week. This week, we have compiled a set
of questionnaires for retail investors and farms, respectively.

Participant:Zhang Tian, Luan Ziyi, Xiao Yuhe, Chen Yanjing, He Yinqi, Huang Zining
We published this week's popular science article,"Xylosic alcohol".

Participant:Chen Kexin, Wang Xinlingjun, Luan Ziyi

**Week 18 (June 25 to July 1)**

After processing the gene fragments of xylanase, considering the possibility of forming the inclusion, we
also set up the experimental group adding lyolytic fragments for manipulation.

Participant:Wang Xinyu, Chen Kexin, Xiao Yuhe, Tian Chao, Wang Xinlingjun
We published this week's popular science article,"Cell surface display system".

 Participant:Tian Chao, Wang Xinlingjun, Luan Ziyi

## July

**Week 19 (July 2 to 8)**

This week, the enzyme connection and routine transformation work were performed.Furthermore, we also tried
to optimize the utilization of xylose by yeast.

Participant:Wang Xinyu, Chen Kexin, Tian Chao, Wang Xinlingjun
We have started our volunteer teaching activities since this week. We introduced our project to the
children, and explained the biological knowledge to them carefully.

Participant:Xiao Yuhe
Together with the other four teams from Hubei University, we hosted the Hubei Synthetic Biology Open
Conference. Gather a number of teams to report and communicate, and set up stalls to publicize the science
related to the project.
Participant:Wang Xinlingjun, Xiao Yuhe, Wang Xinyu
We published this week's popular science article,"Xylanase".
Participant:Wang Xinyu, Wang Xinlingjun, Luan Ziyi

**Week 20 (July 9 to 15)**

This week we electrotransferred the xylanase fraction, but it was not verified by colony PCR.After replacing
the promoter of xylose isomerase, the utilization efficiency of xylose and glucose has been greatly
improved.

Participant:Wang Xinyu, Chen Kexin, Wang Xinlingjun
We published this week's popular science article,"Know S. cerevisiae".

 Participant:Wang Xinlingjun, Luan Ziyi

**Week 21 (July 16 to 22)**

After repeating the experiment and having failed.In the experiment, it was found very unlikely that the
target protein was displayed exocytotically, so we chose to extract it and add it directly to our products.

Participant:Wang Xinyu, Chen Kexin, Tian Chao, Wang Xinlingjun
We published this week's popular science article,"Gene fusion expression".

Participant:Xiao Yuhe, Wang Xinlingjun, Luan Ziyi

**Week 22 (July 23 to 29)**

We spent this week trying to purify xylanases and measured enzyme viability.

Participant:Wang Xinyu, Chen Kexin, Tian Chao, Wang Xinlingjun
We published this week's popular science article,"Error-pronePCR".

Participant:Chen Kexin, Wang Xinlingjun, Luan Ziyi

**Week 23 (July 30 to August 5)**

We started building the fusion gene fragments this week.
Participant:Wang Xinyu, Chen Kexin, Tian Chao, Wang Xinlingjun
We published this week's popular science article,"Risk of sugar addiction".

 Participant:Tian Chao, Wang Xinlingjun, Luan Ziyi

## August

**Week 24 (August 6 to 12)**

We transferred the constructed fusion expression vector into the yeast used for surface display and found
that the yeast was unable to grow on the defective plates.

Participant:Wang Xinyu, Chen Kexin,Xiao Yuhe, Tian Chao, Wang Xinlingjun
We published this week's popular science article,"Why do you like eating spicy food".

Participant:Wang Xinyu, Wang Xinlingjun, Luan Ziyi

**Week 25 (August 13 to 19)**

We propose to discard the surface display technique and directly use the fusion gene for intracellular
expression. The yeast species were replaced and the electrotransfer experiment was repeated.

Participant:Wang Xinyu, Chen Kexin,Xiao Yuhe, Tian Chao, Wang Xinlingjun
Once again, we compiled a questionnaire for the public to collect public opinion.

Participant:Luan Ziyi, He Yinqi, Xiao Yuhe, Zhang Tian, Wang Xinlingjun
Xiao Yuhe helped us to contact the feed manufacturers in Yueyang area of Hunan Province, and we went to the
field investigation on August 18.We also made a field visit to the rural breeding retail investors in Hubei
province on the project.

Participant:Xiao Yuhe, Wang Xinlingjun, Huang Zining
We published this week's popular science article,"Ahypnosis".
Participant:Wang Xinlingjun, Luan Ziyi

**Week 26 (August 20 to 26)**

Made the yeast receptor of the new species, linearized the plasmid, reconstructed the vector, and
electrotransformed it.

 Participant:Wang Xinyu, Chen Kexin,Xiao Yuhe, Wang Xinlingjun
Compile online and offline practice research reports.

Participant:Zhang Tian, Xiao Yuhe, Huang Zining, He Yinqi, Luan Ziyi, Chen Yanjing

 We published this week's popular science article,"About mosquitoes".

Participant:Xiao Yuhe, Wang Xinlingjun, Luan Ziyi

**Week 27 (August 27 to September 2)**

Electroconversion plates were infected with miscellaneous bacteria, reworked the receptor state, and raised
the linearized carrier concentration.

Participant:Wang Xinyu, Chen Kexin,Xiao Yuhe, Wang Xinlingjun
We published this week's popular science article,"Sprouted potato".

Participant:Chen Kexin, Wang Xinlingjun, Luan Ziyi

## September

**Week 28 (September 3 to 9)**

After electrotransformation, it was transferred to the screening plate and found a hydrolysis circle around
the bacteria, which was verified by colony PCR.
Participant:Wang Xinyu, Chen Kexin,Xiao Yuhe, Wang Xinlingjun
We published this week's popular science article,"Sea fire".
Participant:Tian Chao, Wang Xinlingjun, Luan Ziyi

**Week 29 (September 10 to 16)**

Pick the bacteria with larger hydrolyzized ones and culture them in a shake flask. The enzyme viability of
the three cellulases and the total filter paper enzymes were determined.The enzyme viability of the three
cellulases was successfully determined, but the filter paper enzyme activity was abnormal.

Participant:Wang Xinyu, Xiao Yuhe, Wang Xinlingjun
Our team held a charity fundraising event with HBUT-China,HUBU-HUBEI-HUINA,HUBU-HUNA,
HUBU-SKY-China,HUBU-HUNA-WHUAN-CHINA,HUBU-CHNA, simultaneously at Hubei University and Hubei University of
Technology from September 16 to September 27.

 Participant: Xiao Yuhe, Wang Xinlingjun
On September 16, we also went to the Moon Bay community in Wuhan, Hubei province for fundraising activities
and science popularization lectures.

Participant: Xiao Yuhe, Chen Kexin, Wang Xinlingjun
We published this week's popular science article,"Why do you have allergies?".

Participant:Wang Xinyu, Wang Xinlingjun, Luan Ziyi

Week 30 (September 17 to 23)
Redo the electroconversion and remeasure the enzyme activity.The experimental task is thus fully completed.

Participant:Wang Xinyu, Xiao Yuhe, Wang Xinlingjun
On September 20th, Wuhan-Hubei-China and HUBU-China went to Wuhan University together and had an in-depth
project exchange with WHU-China team. This exchange played a certain role in promoting the improvement of
our project.

Participant:Chen Kexin, Luan Ziyi
We published this week's popular science article,"Why do you leave a scar?".

Participant:Wang Xinlingjun, Luan Ziyi

**Week 31 (September 24 to 30)**

This week,we wrote the wiki-related text and designed the web layout.

Participant:Tian Chao, He Yinqi, Xiao Yuhe, Huang Zining, Wang Xinlingjun
Together with four other teams from Hubei University, we held an exchange and science popularization meeting
on the life science competition.

Participant:Chen Kexin, Wang Xinlingjun
We published this week's popular science article,"Mango Little Knowledge".

Participant:Xiao Yuhe, Wang Xinlingjun, Luan Ziyi

## October

**Week 32 (October 1 to 7)**

This week, we optimized the wiki page, and make presentation videos.

 Participant:Tian Chao, Chen Kexin, He Yinqi, Xiao Yuhe, Huang Zining, Wang Xinlingjun

We published this week's popular science article.
Participant:Chen Kexin, Wang Xinlingjun, Luan Ziyi

**Week 33 (October 8 to 12)**

This week, we optimized the wiki page, and make presentation videos.

 Participant:Tian Chao, Chen Kexin, He Yinqi, Xiao Yuhe, Huang Zining, Wang Xinlingjun

{% extends "layout.html" %}
{% block title %}engineering{% endblock %}
{% block css %}
{% endblock %}
{% block page\_content %}

![ engineering](https://static.igem.wiki/teams/4795/wiki/engineering.png)

## catalogue

* [Summarize](#eng1)
* [Idea](#eng2)
* [Stage 1: Construction of high expression engineering strains](#eng3)
* [Stage 2: Construction of fibrosomes and assembly](#eng4)
* [Stage 3: The expression strain of saccharomyces cerevisiae was constructed by gene fusion expression](#eng5)
* [Stage 4: Building degradable xylan module](#eng6)
* [Stage 5: The transformation of xylose into xylulose into yeast intracellular carbon cycle](#eng7)

## Summarize

Based on the problem that food and feed in the planting industry can not meet the needs of breeding, we turned our attention to non-food crops, and finally locked in the straw. Our goal is to construct recombinant saccharomyces cerevisiae strains that can degrade cellulose and hemicellulose in straw and ferment to produce high-protein straw feed.

## Idea

1.Why straw is used as feed raw material: straw is the remaining part of many common crops after seed harvest, and the number is large. Direct incineration will increase the inhalable particles in the air, reduce the visibility of the air, and pollute the environment. At the same time, straw is composed of cellulose, hemicellulose and lignin, which is rich in nitrogen, phosphorus, potassium, calcium, magnesium and organic matter. It is a biological resource with rich nutritional value.

2.Why Saccharomyces cerevisiae is used as a chassis cell: Compared to many bacteria and mycelium fungi, the relatively large spherical cells of yeast are often easy to bioprocess. In addition, they can grow at ambient temperatures and their nutritional requirements are relatively simple, and the yeast's ability to multiply at low pH helps reduce the likelihood of contamination in medium and bioreactors in large-scale industrial operations. The two most widely used for recombinant membrane protein production among more than 1,500 yeast species are Saccharomyces cerevisiae and Pichia Pasteuris.

## Stage 1 : Construction of high expression engineering strains

### Design

It is planned to realize the surface display of three cellulases in yeast respectively, and then carry out molecular transformation of β-glucosidase mainly through genetic engineering technology to optimize enzyme performance, so as to obtain strains that can express β-glucosidase efficiently.

### Build

1.Linear PYD1 was used as the carrier, the target gene and vector were cloned with ExnaseII and then mixed with the receptor DH5α and coated on a plate. The colonies with good growth were selected, the green enzyme GreenTaqMix was used for PCR, and the three groups with the most obvious target bands were cultured in liquid by running the adhesive test, and the plasmid was extracted for enzyme digestion verification.

2.The plasmid containing the target fragment was electrically transferred into the receptive yeast cell EBY100, coated with a plate, and then colony PCR verification was performed. The recombinant saccharomyces cerevisiae with the most obvious rubber strip was cultured for two days, and the enzyme activity was measured with an enzyme marker.

### Test

Glucose and PNPG standard curves were measured first, and then extracellular enzyme liquid was added to different substrates to measure enzyme activity using enzyme marker. For β-glucosidase (BG), substrate PNPG was added to determine OD405. For exodextranase (CBH), substrate MCC was added and OD540 was determined. For endoglucanase (EG), substrate CMC was added to determine OD540.

### Learning

For the plan of molecular modification of β-glucosidase, after conducting an experiment, we found that the original strain in the laboratory could express β-glucosidase efficiently, so we directly used the existing strain in the laboratory. However, the problem in the experiment was endoglucanase: the first time NheI enzyme cut the glue, it was found that the strip was too small, and the enzyme cutting was considered unsuccessful. The strip was normal when repeated, but the glue recovery concentration was low, and the experiment was repeated many times.The surface display experiment of endoglucanase is still not ideal.

## Stage 2 : Construction of fibrosomes and assembly

### Design

It is planned to combine the highly expressed β-glucosidase, endocellulase and exocellulase through a protein scaffold, anchor them by AGglutinin Aga on the surface of yeast cells, and induce their expression through galactose.

### Build

The highly expressed fungal free enzymes β-glucosidase, endocellulase and exocellulase were fused into the corresponding docking module to participate in the construction of fibrosomes. A heterozygous scaffold was formed by connecting the adhesive modules from different sources, and the docking module from the corresponding source was fused with the different cellulase components. According to the specific binding characteristics of the docking module and the adhesion module, cellulase was quantitatively and locatively assembled on the fiber body. Then each unit of the fibrosome is expressed in the same cell, and finally self-assembly is realized in the extracellular. The resulting fibrosomes were anchored on the cell surface by AGglutinin Aga and induced by galactose.

### Test

The expression of fibrous bodies was induced by galactose, and two small pieces of WhatmanNo.1 filter paper (about 0.5mg) were added, and OD540 was determined after reaction at 45℃.

### Learning

In the advance experiment, it was found that the surface display scheme was more difficult than expected and difficult to carry out. After discussion, we chose to adopt the method of gene fusion expression to achieve the simultaneous expression of three kinds of cellulose.

## Stage 3 : The expression strain of saccharomyces cerevisiae was constructed by gene fusion expression

### Design

The plan is to use fusion strategies to construct a single gene with three different cellulase functions to reduce complex operations during the expression of multifunctional cellulase, then express the gene in Saccharomyces cerevisiae and screen strains with three cellulase functions.

### Build

Using PYD1-BG, PYD1-EG and PYD1-CBH as templates, the CD coding sequences of the catalytic domains of cellulase BG, CBH and EG were obtained by PCR reaction. The resulting PCR products were treated with restriction endonuclides XbaⅠ and EcoRⅠ, and mixed into the linking reaction system according to the mass concentration of the same substance. Using the enzyme-linked mixture as template, BG-SnaB I-F and EG-Eco81 I-R primers were used to amplify the fragment about 3.0kb in length by PCR, which was the fusion gene bce. The fusion gene was verified by enzyme digestion, and then seamlessly cloned and electrocuted into receptive yeast cell EBY100. The plate was coated, colony PCR verification was performed, and the recombinant saccharomyces cerevisiae with the most obvious rubber strip was cultured for two days, and the enzyme activity was determined by enzyme marker.

### Test

Glucose and PNPG standard curves were measured first, and then extracellular enzyme liquid was added to different substrates to measure enzyme activity using enzyme marker. β-glucosidase (BG) activity determination: adding substrate PNPG, determination of OD405; The activity of exodextranase (CBH) was determined by adding substrate MCC to determine OD540. Determination of endoglucanase (EG) activity: adding substrate CMC to determine OD540. Determination of enzyme activity of filter paper: Add two small pieces of WhatmanNo.1 filter paper (about 0.5mg) to determine OD540. The activity of three kinds of cellulase enzyme was compared with that of filter paper enzyme.

### Learning

In the specific experiment, the electric transfer failure occurred when saccharomyces cerevisiae EBY100 was used as the receptive cell, and the experiment still failed after repeated for many times. To solve this problem, we changed the type of saccharomyces cerevisiae to INVSc1, continued the experiment, and finally measured the enzyme activity.

## Stage 4 : Building degradable xylan module

### Design

While building highly efficient cellulose degrading strains, we plan to recombine the gene expressing xylanase into saccharomyces cerevisiae cells, so that saccharomyces cerevisiae can not only degrade cellulose, but also degrade 20% hemicellulost in straw.

### Build

1.Linear PYD1 was used as the carrier, the target gene and vector were cloned with ExnaseII and then mixed with the receptor DH5α and coated on a plate. The colonies with good growth were selected, the green enzyme GreenTaqMix was used for PCR, and the three groups with the most obvious target bands were cultured in liquid by running the adhesive test, and the plasmid was extracted for enzyme digestion verification.

2.The plasmid containing the target fragment was electrically transferred into the receptive yeast cell EBY100, coated with a plate, and then colony PCR verification was performed. The recombinant saccharomyces cerevisiae with the most obvious rubber strip was cultured for two days, and the enzyme activity was measured with an enzyme marker.

### Learning

In the specific experiment, after the expression of xylanase gene was transferred to saccharomyces cerevisiae INVSc1, the recombinant saccharomyces cerevisiae did not produce hydrolytic rings on a plate containing xylans, and no enzyme activity was detected by enzymoscope. After colony PCR was performed to confirm the size of the target gene fragment and determine the sequence of the gene fragment, it was concluded that xylanase was not expressed in the yeast. We decided to use Escherichia coli to produce inclusion bodies containing xylanase, and at the initial stage we had already constructed Escherichia coli containing the xylanase gene.After extracting the plasmid, the plasmid was transferred to Escherichia coli by errory-prone PCR. The purified protein was obtained after bacteria breaking, Ni column regeneration, Ni column separation and purification, and protein ultrafiltration. The xylanase was purified for protein glue detection, and it was found that the band size was correct and the band corresponding to the purified protein was more obvious. It has high activity.

## Stage 5 : The transformation of xylose into xylulose into yeast intracellular carbon cycle

### Design

Xylose is obtained after the degradation of xylan, but saccharomyces cerevisiae lacks the ability to fermentation xylose although it has a strong glucose metabolism capacity. We plan to use xylose isomerase to convert xylose to xylulose, which can enter the pentose phosphate pathway and be utilized by yeast. At the same time, by optimizing the promoter of xylose isomerase, we transferred the constructed plasmid into saccharomyces cerevisiae after optimization, and the enzyme was expressed in the cell. After xylose was converted into xylose, it could enter the carbon cycle and be utilized by yeast. In this way, straw resources can be maximized.

### Build

Xylose isomerase xylA gene fragment was cut with XbaI and NotI enzymes and cloned into pHM368-PGK-CBH-Ura cut with the same enzyme to obtain pHM368-PPGK-xylA-Ura. The promoters of ADH1, GAPDH, PDC1 and TEF1 were amplified by corresponding primers and inserted into pHM368-PPGK-xylA-Ura cut by NdeI and NotI by seamless and link-independent cloning. The expression plasmids pHM368-PADH1-xylA-Ura, pHM368-PGAPDH-xylA-Ura, pHM368-PPDC1-xylA-Ura and pHM368-PTEF1-xylA-Ura were constructed. The constructed plasmid was linearized with HpaⅠ and transferred into saccharomyces cerevisiae INVSc1 by lithium acetate method. Engineered strains INVSc1/pHM368-PPGK-xylA,INVSc1/pHM368-PADH1-xylA,INVSc1/pHM368-PGAPDH-xylA,INVSc1/pHM368-PPDC1-xylA and INVSc1/pHM36 were obtained, respectively 8-PTEF1-xylA. Strains were selected on an SD board without Ura and then verified by PCR. Finally, the activity of xylose isomerase was determined.

### Test

The engineered strains of Saccharomyces cerevisiae containing different promoters were incubated in YPD medium for 48h to obtain crude enzymes. The reaction mixture containing 10mMMnCl2, 100mM xylose and 0.1mL crude enzyme was incubated at 75℃ for 20min, stopped with 50% trichloroacetic acid (TCA) and incubated at room temperature for 20min. Then cysteine-carbazol-sulfuric acid reagent containing 1.5% cysteine, 0.12% carbazole and 13MH2SO4 were added and incubated at 37℃ for 20min. Measure OD540 to determine the amount of xylose released. The most active xylose isomerase was obtained.

### Learning

Through xylose isomerase activity detection, strain INVSc1/pHM368-PADH1-xylA was found to be the most effective. It has been confirmed that glucose can protect cells under high stress, and glucose may activate the expression of xylA gene involved in xylose catabolism. Low glucose concentrations promote co-utilization of glucose and xylose by easing catabolism and lead to greatly increased fluxes in different steps of the pentose phosphate pathway.

{% endblock %}

![Experiments](https://static.igem.wiki/teams/4795/wiki/experiments.png)

catalogue

* 1 Cellulose utilization module
  + [1.1 Preparation of competent cells of escherichia coli by TB method](#a11)
  + [1.2 Transformation of e. coli](#a12)
  + [1.3 Extraction plasmid](#a13)
  + [1.4 Recovery of linearize DNA fragments by ethanol precipitation](#a14)
  + [1.5 Recovery of linearize DNA fragments by gel electrophoresis](#a15)
  + [1.6 PCR reaction amplified DNA](#a16)
  + [1.7 Enzyme digestion of DNA fragments](#a17)
  + [1.8 Connection of DNA fragments](#a18)
  + [1.9 Construction of expression strain of saccharomyces cerevisiae](#a19)
  + [1.10 Screening and identification of recombinant saccharomyces cerevisiae](#a110)
  + [1.11 Determination of standard curve](#a111)
  + [1.12 Determination of enzyme activity](#a112)
* 2 Xylan degradation module
  + [2.1 sequentialermr pronepcr](#a21)
  + [2.2 Cell disruption](#a22)
  + [2.3 Extraction and purification of protease](#a23)
  + [2.4 Determination of xylanase activity](#a24)
* 3 Xylose utilization module
  + [3.1 Construction of engineered S. cerevisiae strains](#a31)
  + [3.2 Xylose isomerase activity assay](#a32)
  + [3.3 Functions of various promoters in engineered S. cerevisiae strains](#a33)

## 1 Cellulose utilization module

### 1.1 Preparation of competent cells of escherichia coli by TB method

1. The preserved E. coli strain was taken out, scribed on LB solid medium, and activated cultured at 37 ℃ for about 12 hours;
2. The activated single colonies were picked and inoculated into NZY liquid medium at 18 ℃and 250 rpm until OD 600 ≈ 0.7.
3. The bacterial liquid was transferred into a centrifuge tube, allowed to stand on ice for 15 minutes, and centrifuged at 4 ℃and 6000 rpm for 5 minutes to collect bacterial cells;
4. Re-suspending bacteria with pre-cooled TB buffer solution, standing on ice for 30 minutes, and vigorously mixing the bacteria solution at intervals of 5 minutes;
5. Collect thallus by centrifuge at 6000 rpm for 5 minutes at 4° c;
6. The cells were re-suspended in pre-cooled TB buffer, and DMSO was added to a final concentration of 7% and mixed gently.
7. Subpackaging into pre-cooled lyophilized tube, and storing at-80 C.

### 1.2 Transformation of e. coli

#### 1.2.1 Conventional conversion

1. Then, 100 μL freshly melted competent E. coli cells and 10 μL ligation products were mixed gently in a pre-cooled centrifuge tube and allowed to stand on ice for 30 minutes.
2. 42℃, water bath for 45 seconds, quickly put back on the ice let stand for 2 minutes;
3. Then 200 μL pre-cooled NZY liquid medium was added and mixed gently.
4. Culture at 37 ℃ at 250 rpm for 1 hour;
5. A proper amount of bacterial liquid was applied to LB solid medium containing the corresponding antibiotics and cultured at 37 ℃for about 12 hours.

![img](https://static.igem.wiki/teams/4795/wiki/wps1.png)

#### 1.2.2 Fast conversion

1. Then 20 μL freshly melted competent E. coli cells and 2 μL plasmid were mixed gently in a pre-cooled centrifuge tube and allowed to stand on ice for 6 minutes.
2. A proper amount of bacterial liquid was applied to LB solid medium containing the corresponding antibiotics and cultured at 37 ℃for about 12 hours.

![img](https://static.igem.wiki/teams/4795/wiki/wps2.png)

### 1.3 Extraction plasmid

1. Collecting bacterial liquid;
2. Centrifuge at 12000rpm for 2min, and discarding that supernatant;
3. Add 250μl buffers (pre-cooled) to the centrifuge tube, and swirl until the bacterial liquid is fully suspended;
4. Add 250μl BufferLS to the suspension, and mix gently upside down for 6-8 times, and the solution becomes transparent and relatively viscous.
5. Then 350μlBuffer BS was added to obtain a white mass, which was then mixed upside down gently for 6 to 8 times.
6. Standing for 2min, centrifuging at 12000rpm for 10 min at room temperature, and collecting the supernatant;
7. Transfer the above solution to Piasmid DNA Mini Column, allow to stand for 1min, centrifuge at 12000rpm for 1min, and discard the filtrate.
8. Add 500μl of Buffer WA to Mini Column, centrifuge at 12000rpm for 1min, and discard the filtrate.
9. Add 750μl Buffer WB to Mini Column, centrifuge at 12000rpm for 1min, and discard the filtrate.
10. Repeat 9;
11. Centrifuge the minicolumn at 12000 rpm for 2 min;
12. Place Mini Column in a new 1.5ml centrifuge tube, add 50μl sterilized water to the center of the filter membrane, and allow to stand for 1min (no elution filtrate was observed basically; the filtrate appeared after centrifuging at 12000rpm for 2min).
13. The lower filtrate was added to the center of the membrane, allowed to stand for 2min at room temperature, and DNA was eluted by centrifuging at 12000rpm for 2min.

![img](https://static.igem.wiki/teams/4795/wiki/wps3.png)

### 1.4 Recovery of linearize DNA fragments by ethanol precipitation

1. Diluting the DNA to be purified to an appropriate volume, adding an equal volume of chloroform, fully mixing, and standing for 10 minutes;
2. Centrifuge at 12000 rpm for 10 minutes and slowly draw the supernatant into a new centrifuge tube;
3. Adde 2 times of volume ethanol, mix, standing for 30 minutes at that temperature of minus 20 ℃;
4. Centrifuge at 12000 rpm for 15 minutes and discard that supernatant;
5. After 600 μL pre-cooled 75% ethanol was added, it was mixed evenly, centrifuged at 12000 rpm for 5 minutes, the supernatant was discarded, and 200 μL spear head was used to blot out as much as possible.
6. The centrifuge tube was placed at 37 ℃for drying and appropriate amount of double distilled water was added for dissolution and storage at-20 C.

### 1.5 Recovery of linearize DNA fragments by gel electrophoresis

1. After DNA electrophoresis, the gel containing the DNA fragment of interest was quickly cut off under an ultraviolet lamp. It is recommended that the liquid on the surface of the gel be sucked up and cut up with a paper towel, and that any excess gel be removed as far as possible. The gel was weighed (excluding the empty tube weight) and 100mg of gel was equivalent to 100μL of volume as one gel volume.
2. Add an equal volume of Buffer GDP. Water bath at 50–55 ℃for 7–10 min, and adjust the time appropriately according to the gel size to ensure the complete dissolution of the gel block. During the water bath, the sols were accelerated by reversing and blending twice. (1–3 times the volume of Buffer GDP does not affect DNA recovery. If the recovery of DNA fragments less than or equal to 100 bp, add 3 times the volume of Buffer GDP, water bath after sol, add 1 time the gel volume of isopropanol, after mixing again according to the step 3.
3. Collect the droplets on the tube wall by briefly centrifuging. The FastPure DNA Mini Columns-G adsorption column was placed in a 2 mL collection tube of Collection Tubes, and ≤700μL of sol-gel was transferred to the adsorption column and centrifuged at 12000rpm(13400×g) for 30-60sec. If the sol volume was greater than 700 μL, the adsorption column was returned to the collection tube, and the remaining sol liquid was transferred to the adsorption column and centrifuged at 12,000 rpm (13,400 × g) for 30 to 60 sec.
4. Discarding the filtrate, and placing the adsorption column into a collection tube. Add 300 μL Buffer GDP to the adsorption column. Allow to stand for 1min. Centrifuge at 12000rpm(13400×g) for 30-60sec.
5. Discarding the filtrate, and placing the adsorption column into a collection tube. Add 700 μL Buffer GW (anhydrous ethanol has been added) to the adsorption column. Centrifuge at 12000rpm(13400×g) for 30-60sec. (Please add Buffer GW along the periphery of the adsorption column wall, or add Buffer GW and mix with the cover upside down for 2 to 3 times to help completely rinse the salt adhered to the tube wall. )
6. Repeat step 5. (the use of Buffer GW for two flushes can ensure the complete removal of salts and eliminate the influence on subsequent experiments.) )
7. Discard the filtrate and put the adsorption column back into the collection tube. Centrifuge at 12000rpm(13400×g) for 2min.
8. Place the adsorption column in a sterilized 1.5 mL centrifuge tube, add 20-30 μ L of Elution Buffer to the center of the column, place for 2 min, and centrifuge at 12 000 rpm (13400 × g) for 1min. The adsorption column was discard and that DNA was stored at ℃ .. (If maximum throughput is required, it is recommended that the resulting solution be reintroduced into the centrifuge column and repeat Step 8 for a second elution. When fragments larger than 3kb are recovered, it is recommended that the Elution Buffer be preheated to 55 ℃to increase the recovery efficiency. )

![img](https://static.igem.wiki/teams/4795/wiki/wps4.png)

### 1.6 PCR reaction amplified DNA

The PCR reaction system and procedures were performed according to the following table.

Table 1-1 PCR reaction system table

| material | Volume (μL) |
| --- | --- |
| Ex Taq DNA polymerase (5 U/μL) | 0.3 |
| Template DNA | 0.7 |
| Forward primer (10 mM) | 2.5 |
| Reverse primer (10 mM) | 2.5 |
| dNTP（2.5 mM each） | four |
| 10×Ex Taq Buffer（Mg2+ Plus, 20 mM） | five |
| Double distilled water | 35 |

Table 1-2 PCR reaction program table

| procedure | temperature | time | Number of cycles |
| --- | --- | --- | --- |
| Predenaturation | 95℃ | 5 minutes | 1 time |
| Cycle: denaturation | 95℃ | 30 seconds | 30 times |
| Cycle: annealing | The value of that prim Tm determines a | 30 seconds |  |
| Cycles: extension | 72℃ | Amplified fragment length decision b |  |
| polishing | 72℃ | 5 minutes | 1 time |
| preserve | 4 ℃ |  | 1 time |
Note in the table: A: annealing temperature is determined by the Tm value of primers, and the temperature with small Tm value in two primers is generally taken as the annealing temperature; b：
The extension time is determine by that length of the amplified fragment and is generally calculated at an extension rate of 1 kb/ minute.

![img](https://static.igem.wiki/teams/4795/wiki/wps5.png)

### 1.7 Enzyme digestion of DNA fragments

1. The first enzyme digestion was conducted as follows: reaction temperature 37℃ and reaction time 1 h;

| plasmid | buffer 10×H | SalⅠ | water |
| --- | --- | --- | --- |
| 5μL | 5μL | 1μL | 39μL |

2. Carry out gum recovery on that enzyme-cut product;
3. The first enzyme digestion was conducted according to the following system, with the reaction temperature of 25℃ and time of 20min.

| plasmid | NE Buffer | SWAⅠ | water |
| --- | --- | --- | --- |
| 12μL | 5μL | 1μL | 32μL |

### 1.8 Connection of DNA fragments

The enzyme linkage reaction system was 20 μL, and the enzyme linkage was operated as follows: reaction temperature 37℃, and reaction time 30min.

| carrier | 5×CEⅡ Buffer | ExnaseⅡ | BCE | water |
| --- | --- | --- | --- | --- |
| 2μL | 4μL | 2μL | 1μL | 11μL |

### 1.9 Construction of expression strain of saccharomyces cerevisiae

#### 1.9.1 Construction of fusion gene bce

Using the vectors pHBM368-pgk-bg, pHBM368-pgk-cbh and pHBM368-pgk-eg as templates, the CD coding sequences of the catalytic domains of cellulase BG, CBH and EG were obtained by PCR reaction, with the primers shown below.

Table 1-3 primer summary table for constructing saccharomyces cerevisiae expression vector

| Amplified sequence | Primer name | Primer sequence (5'→3') | Tm value (c) | Length (kb) |
| --- | --- | --- | --- | --- |
| BG | BG-SnaB I-F | ATGTACGTAAGTAATCCGTTCCCCGAC | 61.1 | 1.40 |
| BG-L-Xba I-R | ATCTAGACGAGCCACCGCCACCCGACCCACCACCGCCCGAGCCACCGCCACCCCCCAGGCACGCCCCATT | 82.6 |  |  |
| CBH | CBH-Xba I-F | ACTTCTAGACAGGGAAATCAGGATTTC | 58.1 | 0.97 |
| CBH-L-EcoR I-R | AGAATTCCGAGCCACCGCCACCCGACCCACCACCGCCCGAGCCACCGCCACCATAAGTGCTATCAATCGGA | 78.8 |  |  |
| EG | EG-EcoR I-F | ATCGAATTCCAGTCGCTTTGCGACCAAT | 61.1 | 0.69 |
| EG-Eco81 I-R | ACTCCTGAGGCTAGTTGTTTTGTTGGGCGGA | 64.9 |  |  |
Table note: bold for the enzyme cutting site; The italicized sequence is the reverse-coded sequence for flex connector (G4S)3 (i.e., GGGGSGGGGSGGGGS).

The PCR product was recovered by ethanol precipitation and treated with the corresponding restriction endonuclease. BG was digested by Xba I, CBH was digested by Xba I and EcoR I, and CBH was digested by ECOR I. The fragments after enzyme digestion were recovered by gel electrophoresis, and mixed according to the mass concentration of equivalents and added into the ligation reaction system. Using the enzyme linkage mixture as the template, and using the primers BG-SnaB I-F and EG-Eco81 I-R, a fragment with a length of about 3.0 kb was obtained by PCR amplification, namely, the fusion gene bce.

#### 1.9.2 Cloning and verification

1. Performing enzyme cutting treatment on the fusion gene bce fragment and the vector, performing agarose gel electrophoresis detection and performing gel recovery;
2. The recovered fragments were mixed according to a certain mass concentration ratio (DNA fragments: linearized vector = 10: 1) and added into a linking reaction system;
3. After completion of the ligation reaction, E. coli XL10-Gold competent cells were routinely transformed, coated into LB solid medium (with 100 mg/mL ampicillin added) and incubated at 37 ℃ for approximately 12 hours;
4. The selected individual colonies were incubated in a centrifuge tube containing 1 mL of LB liquid medium (with 100 mg/mL ampicillin). The plasmid of E. coli transformant was extracted, digested with the restriction endonuclease EcoR I, and detected by agarose gel electrophoresis.

#### 1.9.3 Preparation of competent cells of saccharomyces cerevisiae

1. The preserved *S. cerevisiae* INVSc1 strain was taken out, scribed on YPD solid medium, and activated and cultured at 28 ℃ for about 24 hours.
2. The activated single colonies were picked and inoculated into YPD liquid medium at 28 ℃ and 220 rpm until OD 600 ≈ 1.0.
3. The bacterial liquid was transferred to a centrifuge tube, allowed to stand on ice for 15 minutes, and centrifuged at 4 ℃ at 4000 rpm for 1 minute to collect bacterial cells;
4. Re-suspending the thalli with pre-cooled double distilled water, centrifuging at 4 ℃ for 1 minute at 4000 rpm to collect the thalli;
5. Repeat the previous step;
6. The cells were resuspended with pre-cooled 1 M sorbitol solution, allowed to stand on ice for 15 minutes, and centrifuged at 4 ℃ at 4000 rpm for 30 seconds to collect the cells.
7. Re-suspending thallus with pre-cooled 1 M sorbitol solution, and uniformly mixing the bacterial solution to obtain saccharomyces cerevisiae competent cells; Low temperature storage, time should not be too long, it is best to prepare immediately after the conversion.

![img](https://static.igem.wiki/teams/4795/wiki/wps6.png)

#### 1.9.4 Electric shock transformation of saccharomyces cerevisiae

1. After 100 μL of the carrier to be converted was treated with restriction endonuclease Hpa I, it was recovered by ethanol precipitation method and dissolved in 10 μL double distilled water.
2. Mix 100 μL freshly prepared *S. cerevisiae* INVSc1 competent cells with the recovered linearized carrier gently in a pre-cooled centrifuge tube, and allow to stand on ice for 5 minutes.
3. Setting the parameters of the electric rotator to 1800V and 5ms, adding the mixture into a pre-cooled electric rotator cup, connecting electrodes, and performing electric shock;
4. Quickly taking out the electric rotor, slowly adding 1 mL of pre-cooled 1 M sorbitol solution, gently mixing, immediately placing on ice, and standing for 5 minutes;
5. A proper amount of bacterial liquid was applied to SC solid medium and cultured at 28 ℃ for about 4 days.

### 1.10 Screening and identification of recombinant saccharomyces cerevisiae

#### 1.10.1 Plate screening of recombinant saccharomyces cerevisiae

1. Single colonies of *S. cerevisiae* which could grow on SC solid medium were selected and inoculated on YPD solid medium, and the original strain of *S. cerevisiae* INVSc1 was selected as the control at the same time and cultured at 28 ℃ for about one day.
2. The activated single colonies were picked and inoculated on YPC- phenol blue solid medium for culture at 28 ℃ for about 4 days.
3. The growth of colonies and the size of the hydrolysis circle were observed, and the colonies that grew normally and formed with the hydrolysis circle were the recombinant *S. cerevisiae* strain.

![img](https://static.igem.wiki/teams/4795/wiki/wps7.png)![img](https://static.igem.wiki/teams/4795/wiki/wps8.png)

#### 1.10.2 Expression of cellulase in saccharomyces cerevisiae

1. The single colony of recombinant *S. cerevisiae* obtained after screening was picked and inoculated into a 250 mL conical flask fitted with 50 mL of YPD liquid culture medium, and cultured at 28 ℃ and 220 rpm until OD 600 ≈ 1.0;
2. The activated bacterial solutions were inoculated into 500 mL conical flasks filled with 100 mL of YPD liquid medium, and the inoculation amount (about 5 mL) was controlled to maintain the same initial concentration of each group of bacterial solutions. Then, the samples were cultured at 28 ℃ and 220 rpm for 2 days.
3. Then the mixture was centrifuged at 8000 rpm for 10 min at 4 ℃ and the supernatant was taken out as extracellular enzyme solution which was stored at 4 C.

#### 1.10.3 Validation of recombinant strain by colony PCR

The screened recombinant *S. cerevisiae* was selected with a clean toothpick into a PCR tube, and re-suspended in 20 μL double distilled water. Heat at 98 ℃ for 5 min by PCR instrument, transferred to-80 ℃ and allow to stand for 5 min. After three replicates, 0.5 μL was used as the template for PCR using the primers BG-SnaB I-F and EG-Eco81 I-R.

![img](https://static.igem.wiki/teams/4795/wiki/wps9.png)

#### 1.10.4 SDS- polyacrylamide gel electrophoresis

**Preparation of polyacrylamide gel**

Slowly add the prepared separation gel (see Table 1-4) into a 1.0 mm glass bath along the edge until the liquid level is 0.5-1.0 cm away from the comb teeth. Add the double distilled water seal gum and allow to stand for 30-60 minutes until the gel polymerizes.

After the aqueous layer is removed, quickly add the prepared concentrated gel (see Table 1-5) to a glass bath, insert the comb teeth in parallel, and allow to stand for about 30 minutes at room temperature until the gel polymerizes. Gently pull out the comb, and put the rubber plate into the electrophoresis tank, and add the electrophoresis buffer, so that the upper and lower ends of the rubber are immersed in the buffer.

Table 1-4 SDS-PAGE 8% Separation Rubber Formulation Table

| component | Volume mL |
| --- | --- |
| Double distilled water | 4.6 |
| 30% Acr-Bis （29:1） | 2.7 |
| 1.5 M Tris-HCl （pH 8.8） | 2.5 |
| 10% SDS | 0.1 |
| 10% ammonium persulfate | 0.1 |
| TEMED | 0.006 |

Table 1-5 SDS-PAGE 5% concentrated gel formula table

| component | Volume mL |
| --- | --- |
| Double distilled water | 6.8 |
| 30% Acr-Bis （29:1） | 1.7 |
| 1.0 M Tris-HCl （pH 8.8） | 1.25 |
| 10% SDS | 0.1 |
| 10% ammonium persulfate | 0.1 |
| TEMED | 0.006 |

**Processing, loading and electrophoresis**

The extracellular enzyme solution and 4× protein SDS-PAGE loading buffer were taken, mixed evenly in the ratio of 1:3, treated at 100 ℃ for 10 minutes, and then slowly added into the sample well after cooling on ice, namely, ready-to-use protein molecular weight standard (Broad) was used for simultaneous loading.

Turn on the power supply, starting current 20 mA. When bromophenol blue reached the separation gel, a current of 30 mA was set. When bromophenol blue is close to the bottom of the electrophoresis tank, disconnect the power supply.

**Dyeing and decolouring**

After electrophoresis, the separation gel was separated from the glass plate, put into Coomassie brilliant blue R-250 staining solution, and stained for 6–8 hours at room temperature; then, the separation gel was immersed into the decolorizing solution, and shaken for decolorization at 20 rpm until the bands on the gel became clear.

### 1.11 Determination of standard curve

#### 1.11.1 Determination of standard curve of glucose

1. Accurately weighing 10.0 mg of glucose, dissolving in double distilled water, and regulating the volume to 10 mL in a volumetric flask to obtain a 1 mg/mL glucose standard solution;
2. The standard solutions were diluted with double distilled water into glucose standard solutions at concentrations of 0 mg/mL , 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, and 1.0 mg/mL, respectively.
3. Separately, 200 μL of the above standard solutions were mixed with 400 μ L of DNS reagent, and then placed in a boiling water bath for 5 minutes.
4. After cooling on ice for 2 min, 200 μL reaction solution was added into a dry and clean ELISA plate, and OD540； of glucose solution at each concentration was measured with an ELISA reader.
5. Standard curves were drawn with the OD 540 value as the ordinate and the glucose concentration value as the abscissa.

![img](https://static.igem.wiki/teams/4795/wiki/wps10.png)![img](https://static.igem.wiki/teams/4795/wiki/wps11.jpg)

#### 1.11.2 Determination of PNPG standard curve

1. Accurately weighing 27.8 mg of PNP, dissolving in double distilled water, and regulating the volume to 10 mL in a volumetric flask to obtain 20 mmol/L PNP standard mother solution;
2. The mother liquor was diluted with double distilled water into PNP standard solutions at concentrations of 0 mmol/L (i.e., double distilled water), 0.2 mmol/L, 0.4 mmol/L, 0.6 mmol/L, 0.8 mmol/L and 1.0 mmol/L, respectively.
3. Then 500 μL of the standard solutions were added with 500 μ L of 1 M sodium carbonate solution, mixed evenly and reacted for 5 minutes at room temperature.
4. Then 200 μL reaction solutions were added into a dry and clean ELISA plate, and the absorbance of each group's reaction solutions at 405 nm was measured with an ELISA reader.
5. Standard curves were drawn with the OD405 value as the ordinate and the PNP concentration value as the abscissa.

![img](https://static.igem.wiki/teams/4795/wiki/wps12.jpg)

### 1.12 Determination of enzyme activity

#### 1.12.1 Determination of β -glucosidase activity

After 50 μL of extracellular enzyme solution was added with 950 μL 5mM PNPG for reaction at 45 ℃ for 15 minutes, 400 μL was immediately added into a centrifuge tube containing 400 μ L of 1 M sodium carbonate solution for thorough mixing and OD405 was determined.

Enzyme activity unit definition: The amount of enzyme required to produce 1 μmol PNP per hour of reaction is 1 U.

#### 1.12.2 Determination of exoglucanase activity

After 50 μL of extracellular enzyme solution was mixed with 950 μ L of 1% (w/v) MCC, the mixture was reacted at 45 ℃for 1 hour, and immediately after that, 200 μL of extracellular enzyme solution was mixed in a centrifuge tube containing 400 μ L of DNS reagent. After that, the mixture was immersed in a metal bath at 100 ℃ for 10 minutes, cooled on ice for 5 minutes, and OD540 was measured.

Enzyme activity unit definition: The amount of enzyme required to react to produce 1 μmol of reducing sugar per hour is 1 U.

#### 1.12.3 Determination of endoglucanase activity

After 50 μL of extracellular enzyme solution was added with 950 μ L of 1% (w/v) CMC for reaction at 45 ℃ for 30 minutes, 200 μL was immediately added into a centrifuge tube containing 400 μ L of DNS reagent for thorough mixing. After metal bath at 100 ℃ for 10 minutes and ice cooling for 5 minutes, OD540 was measured.

Enzyme activity unit definition: The amount of enzyme required to react to produce 1 μmol of reducing sugar per hour is 1 U.

#### 1.12.4 Determination of filter paper enzyme activity

To 50 μL of extracellular enzyme solution, 950 μL citric acid buffer was added, together with two small Whatman NO.1 filter papers (about 0.5 mg). After reacting for 1 hour at 45 ℃, 200 μL was immediately added into a centrifuge tube containing 400 μ L of DNS reagent and fully mixed. After the mixture was immersed in a metal bath at 100 ℃ for 10 minutes, it was cooled on ice for 5 minutes to determine OD540.

Definition of enzyme activity unit: The amount of enzyme required to produce 1 μmol of reducing sugar per hour of reaction is 1 U.

## 2 Xylan degradation module

### 2.1 sequentialermr pronepcr

Perform error-prone PCR and temperature gradient PCR as per the following table.

![img](https://static.igem.wiki/teams/4795/wiki/wps13.jpg)

Note: dATP: 10 mm; dCTP：10mM; dGTP：10mM； dTTP：100mM； MnCl2：5mM; MgCl2：50mM； Pri.F/Pri.R：20mM; BSA：0.1μg/μL

| Temp（℃） | 95 | 94 | fifty-two | seventy-two | seventy-two | 32cycles |
| --- | --- | --- | --- | --- | --- | --- |
| Time（min） | five | 0.5 | 50 s | one | 10 |  |

The PCR products were detected by agarose gel electrophoresis, and the target band was recovered by gel filtration and integrated with the vector, which was often transferred to the competence of Escherichia coli BL21. Screening was then performed using RBB- xylan plates.

![img](https://static.igem.wiki/teams/4795/wiki/wps14.png)

The bacteria with the largest and most obvious hydrolysis circle were selected and inoculated into 200mL LB liquid medium for culture in a 37 ℃ shake flask for about 12 h. The protein position was detected by SDS-polyacrylamide gel electrophoresis.

![img](https://static.igem.wiki/teams/4795/wiki/wps15.png)

### 2.2 Cell disruption

1. Centrifuge collection The bacterial liquid was collected into a 50ml centrifuge tube and centrifuged at 8000rpm for 4min;
2. Bacterial wash PH7.0, 20ml of 0.2M PBS buffer was carried out twice;
3. Lysis buffer was prepared by adding. 200ml,20–30 mL lysis buffer was added into each centrifuge tube and mixed with shaking;
4. Pre-cooling on ice for 1–2 h (shaking at intervals);
5. Control the crusher. Change bar: 6 gear; ultrasonic on time: 1s; ultrasonic off time: 2s (switching time ratio is 1:2); alarm temperature: 50℃; power: 20%; duration: 35-40min; centrifuge crushing solution at 8000 rmp, take supernatant for 10min (save supernatant 4℃).

### 2.3 Extraction and purification of protease

#### 2.3.1 Ni column rebirth

1. Rinse the Ni column with 8 x the volume of stripping buffer until it turns white;
2. The Ni column was rinsed with a 10 x-volume of deionized water;
3. Wash the Ni column with 2 x the volume of deionized water;
4. The Ni column was washed with a 10 x volume of buffer (PH 7.0,20mM PBS).

#### 2.3.2 The Ni columns were isolated and purified

1. Pour the sample into the reborn Ni column and mix well;
2. Two lattice flow rate to the sample will be exhausted;
3. The miscellaneous proteins were eluted with 20 mM imidazole, 0.5M NaCl of PBS dissolved in PH 7.0;
4. The protein was eluted with 400 mM imidazole, 0.5M NaCl, and the eluted liquid was collected into the centrifuge tube; (the elution was determined by sampling and mixed with G250)
5. After elution, rinse twice with buffer, rinse twice with deionized water, and seal the column with 20% ethanol;
6. Rinse the Ni column with 10 x the volume of deionized water;
7. Wash the Ni column with 2 x the volume of deionized water;
8. The Ni column was washed with a 10 x volume of buffer (PH 7.0,20mM PBS).

![img](https://static.igem.wiki/teams/4795/wiki/wps16.png)

#### 2.3.3 Protein ultrafiltration

1. Pour the sample into the reborn Ni column, fully mix well and soak the ultrafiltration tube with 0.1M NaOH for 30min;
2. Two pure water washing centrifugation, 10-15min, 3000 rcf, down 3 gear;
3. Sample loading, collect and concentrate;
4. Rinsed with 1-1.5ml buffer for 30-40min, 2-3 times;
5. NaOH Soak the ultrafiltration tubes for 30min, washed and sealed with 20% ethanol.

![img](https://static.igem.wiki/teams/4795/wiki/wps17.png)

### 2.4 Determination of xylanase activity

1. The purified enzyme solution was divided into three groups:

①100μl substrate (containing 1% xylan mixed with water) +20μl enzyme solution;

②100μl water +20μl enzyme solution;

③100μl substrate +20μl water;

2. ①②③Three groups reacted for 5 minutes in a 60 ℃ water bath;
3. The samples were centrifuged for 1min;
4. 60ul of reaction solution+60μl of DNS was reacted at 100 ℃ for 10min, and 120 μl of each group was added into the grid;
5. Preparation of standard curve: 1mg/mL glucose mother solution was prepared and 60μl of solutions with different concentrations of 0, 0.2, 0.4, 0.6, 0.8 and 1.0mg/mL were reacted with 60μlDNS respectively at 100 ℃ for 10min. Then 120μl of reaction solution was added into the grid. Enzyme activity was measured by ELISA reader: single wavelength was 540nm.

## 3 Xylose utilization module

#### 3.1 Construction of engineered *S. cerevisiae* strains

Plasmids pHM368-PPGK-xylA-Ura, pHM368-PADH1-xylA-Ura, pHM368-PGAPDH-xylA-Ura, pHM368-PPDC1-xylA-Ura and pHM368-PTEF1-xylA-Ura were linearized with *HpaⅠ*and transformed into INV*Sc1* using the lithium acetate method to obtain engineered strains INV*Sc1*/pHM368-PPGK-xylA, INV*Sc1*/pHM368-PADH1-xylA, INV*Sc1*/pHM368-PGAPDH-xylA, INV*Sc1*/pHM368-PPDC1-xylA and INV*Sc1*/pHM368-PTEF1-xylA, respectively. The transformants were selected on SD plates without Ura and then validated by polymerase chain reaction (PCR).

#### 3.2 Xylose isomerase activity assay

The expression of *xyl*A *in* the engineered strains contained different promoters were evaluated. The crude enzyme was obtained after 48-h incubation of the engineered *S. cerevisiae* strains harboring different promoters in YPD medium. XI activity was measured as described by Tanino *et al.* with minor adjustments. Briefly, the reaction mixture containing 0.05 mL of 10 mM MnCl2, 0.05 mL of 100 mM xylose and 0.1 mL of crude enzyme was incubated at 75 °C for 20 min. The reaction was stopped by 0.05 mL of 50% (w/v) trichloroacetic-acid (TCA) and then incubated on room temperature for 20 min, followed by adding a dose of cysteine-carbazole-sulfuric acid reagent contained 0.2 mL of 1.5% (w/v) cysteine, 0.2 mL of 0.12% carbazole and 6 mL of 13 M H2SO4 and incubating at 37 °C for 20 min. OD540 was measured to determine the amount of the released xylulose. One unit of XI activity (U) was defined as the amount of enzyme required to produce 1 µg of xylulose per minute under conversion of 1 mL of crude enzyme solution.

#### 3.3 Functions of various promoters in engineered *S. cerevisiae* strains

Engineered *S. cerevisiae*strains harboring different promoters were fermented with the media containing glucose and xylose to evaluate the xylose utilization rates. Cells were collected through centrifugation at 5000 rpm for 2 min and washed twice after growing in YPD medium at 28 °C for 24 h. Then the cells were transferred into 100 mL of SD-G4X10 medium (0.4% Glucose, 1% xylose). Unless otherwise stated, OD600 of the cell culture was adjusted to 1 at the initial phase, and the assays were performed with 200 rpm shaking at 28 °C under aerobic conditions.

[^](#linking)

![RESULT](https://static.igem.wiki/teams/4795/wiki/result.png)

## catalogue

* [1 Part 1](#re1)
  + [1.1 Design and construction of the fusion genes](#re1-1)
  + [1.2Expression and identification of the fusion gene bce in S. cerevisiae](#re1-2)
  + [1.3Analysis of cellulase activity in recombinant strains of S. cerevisiae](#re1-3)
* [2 Part 2](#re2)
  + [2.1 Mutation-optimized target genes](#re2-1)
* [3 Part 3](#re3)
  + [3.1 Construction of the recombinant S. cerevisiae expressing xylA](#re3-1)

Our team through experiments successfully constructed three kinds of cellulase fusion expression
of S. cerevisiae
strains, through error-prone PCR mutation breeding and purification isolated the high enzyme live xylan ase,
and
the promoter of S. cerevisiae xylose utilization optimization, various mutual combination, realize our
expectations to improve the efficiency of straw degradation.

## 1 Part 1

## 1.1 Design and construction of the fusion genes

In this study, three cellulases from different sources, having similar reaction conditions, BG,
CBH and EG were
selected as shown in the Table 1-1. As predicted by the SignalP 4.1 tool
(http://www.cbs.dtu.dk/services/SignalP/), two clear signal peptide cleavage sites are present at G15 of
exoglucanase and A16 of endoglucanase; but no signal peptide in β-glucosidase. According to the analysis of
the
Carbohydrate Active Enzyme database (http://www.cazy.org/), the three cellulases are derived from three
different glycoside hydrolase families: BG from the GH 1 family, CBH from the GH 7 family, and EG from the
GH 12
family. According to the analysis of the Conserved Domain database (https://www.ncbi.nlm.nih.gov/cdd), none
of
the three cellulases has CBD regions.

In conclusion, the three selected enzymes have different sources, different classification, simple
structure and
compatible reaction conditions, which accord with the idea of the fusion strategy.

Table 1-1 Information tables of the three cellulases

| Cellulase | Sources | Family |
| --- | --- | --- |
| BG | Sphingomonas sp | GH1 |
| CBH | Yellow limb scattered termite | GH7 |
| EG | Stachybotrys atra | GH12 |

The reverse coding sequence of the flexible adaptor (G4S) 3 (sequence GGGGSGGGGSGGGGS) was added
to the reverse
primers BG-L-Xba I-R and CBH-L-EcoR I-R for CBG and CBH. Two segments (G4S) 3 serve as the domain CD of the
three cellulases. Meanwhile, appropriate restriction enzyme sites were added at the end of the primers to
facilitate the fusion DNA with the cloned gene.The specific operation process is shown in Figure 1-1.

![Figure 1-1](https://static.igem.wiki/teams/4795/wiki/result-1.png)

Figure 1-1 Flow chart of the fusion gene bce construction

PCR reactions were performed using primers BG-SnaB I-F and EG-Eco81 I-R using the enzyme ligation
mixture as a
template, and the results are shown in Figures 1-1. The PCR reaction mainly produces three bands, among
which
the main band with the highest electrophoresis brightness is about 3.0 kb, namely the fusion gene bce, while
the
band of about 2.4 kb is caused by the byproduct produced in the enzyme reaction as the template of the PCR
reaction.

![Figure 1-2](https://static.igem.wiki/teams/4795/wiki/result-2.png)

Figure Note: M: 250 bp DNA Ladder molecular weight standard, lane 1-6: PCR
reaction product.

Figure 1-2 PCR-amplified bce electropherograms of the fusion gene

The resulting fusion gene bce from PCR was purified and digested with restriction enzymes SnaB I
and Eco81 I and
subjected to gel electrophoresis to recover the DNA fragments. The vector pHBM368-pgk-bg was also subjected
to
the same treatment. After ligation of the two recovered reactions, E. coli XL10-Gold competent cells were
transformed. Single colonies were picked and inoculated in LB liquid medium containing ampicillin, and
plasmids
were extracted as described in Figures 1-3-a.

The restriction enzyme EcoR I was selected for enzyme digestion. The vector pHBM368-pgk and the
fusion gene bce
both contain a EcoR I restriction site, and the size of the two fragments is predicted to be 8.2 kb and 2.4
kb
after the recombinant restriction enzyme, respectively. Transformants with slower electrophoretic rates than
the
control pHBM368-pgk-bg were picked, digested with EcoR I, and then examined by electrophoresis. The
electrophoresis results are shown in Figures 1-3-b.

![Figure 1-3](https://static.igem.wiki/teams/4795/wiki/result-3.png)

Figure Note: a: Electrophoregram of plasmid. Lanes 1-5: transformant
pHBM368-pgk-bce; lane 6: control pHBM368-pgk-bg. b: The electropherogram of EcoR I digestion. M: 250 bp
DNA
Ladder molecular weight standard; lane 1-5: pHBM368-pgk-bce cut band for transformants; lane 6: control
pHBM368-pgk-bg cut band.

Figure 1-3 Verification plot of the fusion gene expression vector bce S. cerevisiae

Plasmids that met the predicted band size were selected for sequencing. The electrophoresis rate
of plasmid 1 was
slower than the control, and the size of EcoR I digestion met the prediction and the sequencing results met
the
design requirements. The recombinant vector was named pHBM368-pgk-bce. The schematic diagram of the vector
is
shown in Figure 1-4.

![Figure 1-4](https://static.igem.wiki/teams/4795/wiki/result-4.png)

Figure 1-4 Schematic representation of the expression vector of the fusion gene bce Saccharomyces cerevisiae

## 1.2 Expression and identification of the fusion gene bce in S. cerevisiae

Saccharomyces cerevisiae INVSc1 competent cells were prepared and the expression vector
pHBM368-pgk-bce was
digested with the restriction enzyme HpaI and recovered. The fusion expression vector was then introduced
into
competent cells using electroconversion. SC media without uracil was used for screening. The Saccharomyces
cerevisiae primitive INVSc1 is of the uracil-deficient type, while the expression vector contains the uracil
screening marker URA 3. The recombinant strain could grow on SC medium, indicating that the genome of the
strain
was integrated with the recombinant vector.Single colonies of S. cerevisiae that could grow normally on SC
solid
medium were picked, activated on YPD solid medium, inoculated onto YPC-phenol blue solid medium and grown
for
28℃ for approximately 4 days. The screening results of some recombinant strains are shown in Figure 1-5.

![Figure 1-5](https://static.igem.wiki/teams/4795/wiki/result-5.png)

Figure 1-5 Plate screening plots of recombinant Saccharomyces cerevisiae strains

The original bacteria INVSc1 grew very poorly on YPC-Taiwan phenol blue solid medium with CMC-Na
as the only
carbon source, while the recombinant strains grew normally, indicating that the recombinant bacteria have
cellulase (endoglucanase) activity. Because tryphenol blue can bind macromolecular polysaccharides and
cannot
bind small molecular polysaccharides, if the recombinant strain decomposes cellulose to produce small
molecules
of sugars, hydrolysis circle will appear around the colony. A clear hydrolysis circle can be seen around the
colonies in the figure, indicating that these recombinant strains achieved the secreted expression of
cellulase.

PCR was verified using total DNA from strains 7, the most prominent hydrolyzed ed, as template
using primers
BG-SnaB I-F and EG-Eco81 I-R, and the results are shown in Figures 1-6. Among them, the total DNA of strain
7 as
the template and the predicted 3.0 kb band was observed in the products of PCR of the recombinant plasmid
pHBM368-pgk-bce as the template. And not in the products of PCR using S. cerevisiae primitive INVSc1 total
DNA
as template. Therefore, strain 7 was selected as the S. cerevisiae expressing strain with single-gene
multifunctional cellulase BCE and named INVSc1-BCE.

![Figure 1-6](https://static.igem.wiki/teams/4795/wiki/result-6.png)

Note: M: DL5000 DNA Molecular weight standard; lane 1: product of PCR with
total
DNA of S. cerevisiae 7 as template; product of PCR with total DNA of S. cerevisiae INVSc1 as template;
product of PCR with recombinant plasmid pHBM368-pgk-bce as template.

Figure 1-6 PCR validation plots of recombinant S. cerevisiae

## 1.3 Analysis of cellulase activity in recombinant strains of S. cerevisiae

Three activities of cellulase-specific substrate enzymes and total cellulase activity were
determined, which were
analyzed and compared.

![Figure 1-7](https://static.igem.wiki/teams/4795/wiki/result-7.png)

Figure 1-7 Comparison of three cellulase-specific substrate enzyme activities and total cellulase activity

Figure 1-7 shows that the β -glucosidase activity of BCE reached 1144 U / L and the exoglucanase
activity of BCE
reached 950U / L, which was lower than the overall filter paper enzyme activity with an enzyme activity of
1278U
/ L. However, the endoglucanase activity of BCE was much higher than the overall filter paper enzyme
activity,
up to 4978U / L. The comparative results show that incorporating the expression of the CD region of the
three
cellulases can yield monopeptide proteins possessing the three cellulase activities. Moreover, the overall
filter paper enzyme activity of fusion expression was higher than the specific substrates of two of the
enzymes,
suggesting a synergistic effect of the three enzymes.

## 2 Part 2

## 2.1 Mutation-optimized target genes

Mutation of the xylanase gene fragment using error-prone PCR results in Figure 2-1.

![Figure 2-1](https://static.igem.wiki/teams/4795/wiki/result-8.png)

Note: M is 2,000 bp Marker. 1~5: The mutation rate was 0.2%, and the Tm
values
were 50,52,56,58, and 60℃, respectively.6~10: a mutation rate of 0.3% and Tm values of 50,52,56,58 and
60℃.11~15: The mutation rate was 0.5%, and the Tm values were 50,52,56,58, and 60℃, respectively.16~20:
with
a mutation rate of 1.0% and Tm values of 50,52,56,58, and 60℃, respectively.21~25: Tm values are
50,52,56,58
and 60℃ respectively.

Figure 2-1 Validation plot of the mutated genes

The target band size was 717 bp. They appears in part of conditions. The target bands of 56,58,
and 60℃ with 0.5%
and 1.0% mutation rate were recovered, integrated with the vector, and often transferred into BL21 receptor.
The
substrate plate was continued to screen for strains producing high enzyme live xylanase.The target fragments
of
the obtained strains were tested in Figure Figure 2-2.

![Figure 2-2](https://static.igem.wiki/teams/4795/wiki/result-9.png)

Note: M is 5,000 bp Marker. 1-2: Original fragment before double
digestion;3-4:
After the digested target fragment.

Figure 2-2 Validation of the target genes after the optimization

After the selected strains, the xylanase was isolated and purified, and the activity was up to
25980U / L.

## 3 Part 3

## 3.1 Construction of the recombinant S. cerevisiae expressing xylA

Five strong constitutive promoters were used to control the transcription of xylA. The recombinant
INVSc1 bearing
xylA driven by different promoters were named as INVSc1/pHM368-PPGK-xylA, INVSc1/pHM368-PADH1-xylA,
INVSc1/pHM368-PGAPDH-xylA, INVSc1/pHM368-PPDC1-xylA and INVSc1/pHM368-PTEF1-xylA, respectively. The XI
activities in engineered S. cerevisiae strains were evaluated. As shown in Figure 3-1, the XI activity of
INVSc1/pHM368-PADH1-xylA reached 0.179 U at 48 h, which was 2.7-, 3.1- and 2.4-fold higher than
INVSc1/pHM368-PPDC1-xylA (0.066 U), INVSc1/pHM368-PPGK-xylA (0.058 U) and INVSc1/pHM368-PTEF1-xylA (0.074
U),
respectively. No XI activity was detected in INVSc1/pHM368-PGAPDH-xylA. XI activity of the engineered
strains
improved by promoter optimization strategy. According to this result, ADH1 promoter was the most efficient
one
for the expression of XI among these five promoters.

![Figure 3-1](https://static.igem.wiki/teams/4795/wiki/result-10.png)

Figure 3-1 XI activity of S. cerevisiae strains INVSc1/pHM368-PADH1-xylA, INVSc1/pHM368-PGAPDH-xylA,
INVSc1/pHM368-PPDC1-xylA, INVSc1/pHM368-PPGK-xylA and INVSc1/pHM368-PTEF1-xylA containing different
promoters.
Error bars indicate the average deviation.