iGEM 2019 — Team ECUST_China

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# Team:ECUST China/Notebook

* TEAM

  [Team](https://2019.igem.org/Team%3AECUST_China/Team)
  [Collaborations](https://2019.igem.org/Team%3AECUST_China/Collaborations)
* PROJECT

  [Description](https://2019.igem.org/Team%3AECUST_China/Description)
  [Design](https://2019.igem.org/Team%3AECUST_China/Design)
  [Experiments](https://2019.igem.org/Team%3AECUST_China/Experiments)
  [Notebook](https://2019.igem.org/Team%3AECUST_China/Notebook)
  [Contribution](https://2019.igem.org/Team%3AECUST_China/Contribution)
  [Attributions](https://2019.igem.org/Team%3AECUST_China/Attributions)
  [Safety](https://2019.igem.org/Team%3AECUST_China/Safety)
* RESULTS

  [Results](https://2019.igem.org/Team%3AECUST_China/Results)
  [Demonstrate](https://2019.igem.org/Team%3AECUST_China/Demonstrate)
  [Improve](https://2019.igem.org/Team%3AECUST_China/Improve)
[![HOME](https://static.igem.org/mediawiki/2019/7/76/T--ECUST_China--white_logo.png)](https://2019.igem.org/Team%3AECUST_China)
* [PARTS](https://2019.igem.org/Team%3AECUST_China/Parts)
* HUMAN

  [Human Practices](https://2019.igem.org/Team%3AECUST_China/Human_Practices)
  [Education & Engagement](https://2019.igem.org/Team%3AECUST_China/Public_Engagement)
* AWARDS

  [Awards](https://2019.igem.org/Team%3AECUST_China/Awards)
  [Entrepreneurship](https://2019.igem.org/Team%3AECUST_China/Entrepreneurship)
  [Hardware](https://2019.igem.org/Team%3AECUST_China/Hardware)
  [Model](https://2019.igem.org/Team%3AECUST_China/Model)

![](https://static.igem.org/mediawiki/2019/b/be/T--ECUST_China--project_main.png)
![](https://static.igem.org/mediawiki/2019/5/5a/T--ECUST_China--notebok_mask.png)

* 01
  [Introduction](#main1)
* 02
  [Progress](#main2)

# Introduction

Paper Transformer is a superhero we made to change the paper world, and several of team members devoted most of their time and energy this season.

We assigned four iGEMers to achieve three abilities of our Paper Transformer. In detail, we assigned Hu Shijia for cellulose degrading module, Xiang Lan for cellulose producing module, Miao Zongjie and Jin Qianwei for inverter module.

# Progress

## —January 2019

The senior students publicized the iGEM and recruited candidates aspiring to participate iGEM representing ECUST-2019 iGEM team.

## —February 2019

We had a brain storm within the iGEM club of ECUST.

## —April 2019

We participated a meetup held at New York University Shanghai.

## —May 2019

We decided to focus on Paper Transformer as our project out of three others.

We were invited to Jiangnan University in Wuxi, Jiangsu province to discuss our project. We communicated with three teams of Jiangnan University on human practices and gene circuit design.

## —June 2019

We started to plan our wet lab arrangement and construction work.

## —July 2019

Our experiment didn’t start until this month, but we quickly assigned the tasks into several team member. In detail, shows as follows:

* **Cellulose degrading ability**

  This part was mostly undertaken by Hushijia, and later assisted by Xianglan. The results could be seen at  [here![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](https://2019.igem.org/Team%3AECUST_China/Results).

  2nd, July
  —We firstly attempted to construct the cex-hlyA-hlyBD and cenA-hlyA-hlyBD on pET-28b separately, since the dual inverter plasmid has not been constructed and the pET-28b is a reliable plasmid to express heterologous protein.

  9th, July
  —For cenA, we got desired parts from kit by PCR very easily. And through Gibson-assembly method, we ligated the parts we purified. For cex, firstly gene synthesis took nearly a month, and we finally found a suitable polymerase for obtaining clean PCR. And through Gibson-assembly method, we ligated the parts we purified.

  20th,July
  —When we tested the ligation section by test primer and first generation sequencing, nonetheless, there were not a single clone comprise all the fragments we expected to be ligated.So, we attempted for a simpler construction: cex-hlyA/pET and cenA-hlyA/pET, and this was achieved much easier. And we used cells lysis for enzyme activity measurement.

  [**Protocols**](https://static.igem.org/mediawiki/2019/8/8d/T--ECUST_China--experiments_protocols.pdf)
* **Cellulose producing ability**

  This part was firstly undertaken by Xianglan, and then taken over by Jinqianwei and Miaozongjie. The results could be seen at [here![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](https://2019.igem.org/Team%3AECUST_China/Results).

  1nd, July
  —We tried to purchase Gluconacetobacter xylinus from China General Microbiological Culture Collection Center, but we decided not to finish this order since we didn’t have the protocols and available plasmid in our school, making it almost impossible for us to set it as our chasis.

  7th, July
  —We chose E.coli DH5alpha as our chasis.

  18th,July
  —We met some problems about acquiring the acsAB and acsCD from the kit, since they were both about 5500bp.

  26th,July
  —Even though we occasionally got the desired fragments, problems still exist in the ligation procedure. We couldn’t ligate the acsAB, and acsCD with neither pET-28b backbone nor pSB1C3 by Gibson-assembly and T4-ligation respectively.
* **Inversion-control ability**

  This part was mostly undertaken by Jinqianwei and Miaozongjie. The results could be seen at [here![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](https://2019.igem.org/Team%3AECUST_China/Results).

  4nd, July
  —We successfully got all the fragments required to construct inverter dual plasmid system.

  10th, July
  —We constructed the inverter system and verified its sequence by first generation sequencing..

  25th,July
  —We got the preliminary results that the plasmid pIN2 worked as expected since the fluorescence of mRFP was detectable even with eyes.

## —August 2019

23th, August
—We went to ShenZhen to participate CCiC 2019.

28th, August
—We visited to Fulun, Hangzhou, a paper recycling company to investigate the value and application of our project.

* **Cellulose degrading ability**

  1st, August
  —We successfully constructed cex / cenA / eCFP with hemolysin system on pET-28b backbone, but saw not expression of either protein.

  9th, August
  —We ran a SDS-PAGE to detect protein expression, the eCFP and cex/cenA were all not expressed.

  12th, August
  —We suspected the failure of expression was because we changed the RBS of original pET-28b backbone during primer design. This time, we didn’t change any sequence of the backbone and chose a dual promoter to control the expression of cex/cenA and hlyABD.

  21th, August
  —We met some problems constructing this dual promoter plasmid, since there were always some trouble existing in the linkage section.
* **Cellulose producing ability**

  1st, August
  —We were still trying hard to get the acsAB and acsCD fragments

  12th, August
  —We sequenced the plasmid we miniprepared, the results showed there were no problem about the template plasmid, indicating the problem was on PCR level.
  We tried several high fidelity enzymes from different company, but only the acsAB and acsCD could be cloned.

  24th, August
  —We designed a much longer pair of primers to get the pET-28b backbone.

  26th, August
  —We finally got a clear band of pET-28b backbone on electrophoresis.

  28th, August
  —We purified all the fragments together by Gibson-assembly, yet all the colonies grown on the plate were proved to be negative.
* **Inversion-control ability**

  1st, August
  —We started to develop the cellobiose responsive element.

  5th, August
  —We acquired the reference sequence successfully form E.coli MG1455.

  13th, August
  —We point-mutated the sequence to impart it with cellobiose responsive capability.

  19th, August
  —We introduced the modified sequence on pIN2 plasmid and tried to induced it with different amount of cellobiose.

  27th, August
  —Sadly, the cellobiose responsive element didn’t work out because no downstream GFP fluorescence could be detected or detected in an unwanted way.

## —September 2019

* **Cellulose degrading ability**

  14th, September
  —We suspected the Gibson-assembly efficiency was not high enough, so we set a plan B to construct plasmid using traditional restriction-ligation method.

  26th, September
  —We finished the construction of the plasmid, but we still can’t detect desired protein on SDS-PAGE.
* **Cellulose producing ability**

  7th, September
  —We also tried construct our bacterial cellulose producing plasmid by restriction enzyme digest and T4 ligation. We chose the enzyme of prefix and suffix to digest acsAB plasmid and acsCD plasmid. The electrophoresis results were very good, since the pSB1C3 backbone was successful cleaved and the acsAB was released.

  16th, September
  —We purified the acsCD backbone and acsAB fragment and ligated them together. The colonies tested to be positive was actually not right because the sequencing could only be reached in acsAB plasmid cleavage site but not acsCD any further.

  25th, September
  —We decided to divide the whole plasmid into 6 fragments to ease the PCR of fragment. We immediately got all 6 fragments and ligated them through Gibson-ligation. Sadly, there were always some connection sites were missed, since we could get the desired band on the electrophoresis gel.
* **Inversion-control ability**

  3rd, September
  —We further our verification of inverter system by co-transferring the pIN1 and pIN2 plasmid in a single cell employing electro-transformation after chemical transformation, but we failed to prepare a vigorous competent cell since the voltage and precessing time were not appropriate and the E.coli were all dead after treatment.

  15th, September
  —We adopted chemical transformation to introduce two plasmid into the host and succeeded, because the cell turned purple after different concentration of IPTG induced.

## —October 2019

* **Cellulose degrading ability**

  1st, October
  —We started to shift our construction work on pIN2 plasmid we’ve developed.

  1st, October
  —We also started the linker optimization between the target gene and hlyA-tag by introducing three alternative linker.

  8th, October
  —We successfully tested the linkers’ functionality by SDS-PAGE. Further, we introduced his-tag to the fusion protein to further detect the functionality.

  10th, October
  —Later, we constructed cex-hlyA/hlyBD/pIN2 and cenA-hlyA/hlyBD/pIN2 just before deadline, we only got the time for secreted enzyme activity measurement and haven’t got the time for more experiments like western blot and gave FPA a second try. We successfully examined the enzyme activity using CMCNa assay an FPA assay by disrupting cex- and cenA-containing E.coli.

  14th, October
  —We successfully established the whole plasmid with cex-hlyABD or cenA-hlyABD, but the Western Blot results showed no secretion of Cex or CenA at all.

  October
  —We are still testing the secretion system efficiency by Western Blot.
* **Cellulose producing ability**

  1st, October
  —We decided to work together to conquer this problem.

  6th, October
  —Finally, we decided to express acsAB and acsCD separately before expressing them together, which might be a heavy burden to the host. And the pIN1 was our optional plasmid since it’s smaller than pET-28b and already proved to be functional.

  10th, October
  —We successfully constructed two plasmids harboring acsAB and acsCD respectively.

  12th, October
  —We prepared the protein sample and ran a SDS-PAGE to verify the expression of acsAB, acsC and acsD. The acsD was expressed clearly after IPTG induction. But no desired band could be seen for acsAB and acsD.

  16th, October
  —We used Congo Red Binding assay to test the minimal production of bacterial cellulose, and the results turned to satisfying since the there was a noticeable drop of absorbance on 490nm, indicating an binding of BC to free Congo Red dyes.

  October
  —We are still constructing acsAB with acsCD, attempting an eye-visible production of bacterial cellulose.
* **Inversion-control ability**

  2nd, October
  —We asked Professor Zhang of our school for help. He offered a lot precious suggestion for us to optimize the promoter.

  5th, October
  —We focused on another chbR mutated protein construction and characterization.

  14th, October
  —We tried to induce the promoter with an excess of cellobiose, but the results didn’t appear to be ideal.

  18th, October
  —We introduced chitobiose to test whether the ChbR could respond to its natural inducer, it appeared the chitobiose could indeed bind to ChbR and allowed weak activation of reporter gene expression.

  October
  —We are still testing the efficiency of cellobiose responsive element.

More details about our notebook please click

[**Notebook**](https://static.igem.org/mediawiki/parts/a/ac/T--ECUST_China--notebook.pdf)

#### ECUST\_China

EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY

Shanghai, China

[![](https://static.igem.org/mediawiki/2019/2/27/T--ECUST_China--ecust_school-w.png)](https://www.ecust.edu.cn/)
[![](https://static.igem.org/mediawiki/2019/3/33/T--ECUST_China--bioenger_logo-w.png)](http://biotech.ecust.edu.cn/)

#### GET IN TOUCH

+86 021-64253306

ecust\_igem\_2019@163.com

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# Team:ECUST China/Experiments

* TEAM

  [Team](https://2019.igem.org/Team%3AECUST_China/Team)
  [Collaborations](https://2019.igem.org/Team%3AECUST_China/Collaborations)
* PROJECT

  [Description](https://2019.igem.org/Team%3AECUST_China/Description)
  [Design](https://2019.igem.org/Team%3AECUST_China/Design)
  [Experiments](https://2019.igem.org/Team%3AECUST_China/Experiments)
  [Notebook](https://2019.igem.org/Team%3AECUST_China/Notebook)
  [Contribution](https://2019.igem.org/Team%3AECUST_China/Contribution)
  [Attributions](https://2019.igem.org/Team%3AECUST_China/Attributions)
  [Safety](https://2019.igem.org/Team%3AECUST_China/Safety)
* RESULTS

  [Results](https://2019.igem.org/Team%3AECUST_China/Results)
  [Demonstrate](https://2019.igem.org/Team%3AECUST_China/Demonstrate)
  [Improve](https://2019.igem.org/Team%3AECUST_China/Improve)
[![HOME](https://static.igem.org/mediawiki/2019/7/76/T--ECUST_China--white_logo.png)](https://2019.igem.org/Team%3AECUST_China)
* [PARTS](https://2019.igem.org/Team%3AECUST_China/Parts)
* HUMAN

  [Human Practices](https://2019.igem.org/Team%3AECUST_China/Human_Practices)
  [Education & Engagement](https://2019.igem.org/Team%3AECUST_China/Public_Engagement)
* AWARDS

  [Awards](https://2019.igem.org/Team%3AECUST_China/Awards)
  [Entrepreneurship](https://2019.igem.org/Team%3AECUST_China/Entrepreneurship)
  [Hardware](https://2019.igem.org/Team%3AECUST_China/Hardware)
  [Model](https://2019.igem.org/Team%3AECUST_China/Model)

![](https://static.igem.org/mediawiki/2019/b/be/T--ECUST_China--project_main.png)
![](https://static.igem.org/mediawiki/2019/5/55/T--ECUST_China--experiment_mask.png)

* 01
  [Cellulose degradation](#main1)
* 02
  [Bacterial cellulose synthesis](#main2)
* 03
  [Regulator](#main3)
* 04
  [Linker optimization](#main4)

# Cellulose degradation

For this part, we want our chassis organism degrade lignocellulose into cellobiose firstly, so we designed a plasmid which can express *C. fimi* endoglucanase CenA and *C. fimi* β-1,4-exoglucanase Cex and can secrete these two enzymes out of*E. coli*using hemolysin secretion system, which is the best-characterized and most widely used TISS is the ahemolysin (HlyA) secretion pathway from uropathogenic *E. coli*. The secretory machinery of this pathway consists of three components: HlyB, an ATP-binding cassette (ABC transporter); HlyD, a membrane fusion protein; and TolC, an outer membrane protein( [Su L. et al, 2012](https://linkinghub.elsevier.com/retrieve/pii/S0141-0229%2816%2930149-1%20)).

![](https://static.igem.org/mediawiki/2019/5/5a/T--ECUST_China--design_circuit-secre.png)
**Figure 1.1.** Gene circuit of cellulase secretion system
*cex*: Cellulase exoglucanase gene, *cenA*: Cellulase endoglucanase gene,*hlyA, hlyB, hlyD*: α-hemolysin system gene

In order to use *E. coli* DH5α to express recombinant protein into the growth medium through the a-hemolysin secretion pathway, the α-hemolysin specific signal peptide HlyAs, which corresponds to residues 965-1024 of α-hemolysin, was fused to the C-terminus of CenA and Cex, which genes was modified by overlapping PCR. In addition, the genes encoding the components of translocator, HlyB and HlyD, were amplified together by PCR using part:[BBa\_K1166002![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](http://parts.igem.org/Part%3ABBa_K1166002)as template.

### Enzyme activity of Cex-HlyA/CenA-HlyA

To make sure our final construction would work, we first analyzed the enzyme activities of the fusion protein CenA-HlyA and Cex-HlyA. *hlyA* was amplified from part:[BBa\_K1166002![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](http://parts.igem.org/Part%3ABBa_K1166002) and cenA was amplified from part:[BBa\_K523015![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](http://parts.igem.org/Part%3ABBa_K523015), while *cex* was amplified from the gene synthesized by Genewiz, and the sequence was obtained from NCBI. The PCR products were cloned into a modified pET28b (+) plasmid pIN2 and enzyme activity within cells harboring cloned genes were tested qualitatively using carboxymethyl cellulose (CMC) substrates and FPA (Filtered Paper Assay) substrates( [Duedu KO, French CE, 2016](https://linkinghub.elsevier.com/retrieve/pii/S0141-0229%2816%2930149-1%20) ) .

![](https://static.igem.org/mediawiki/2019/a/a9/T--ECUST_China--experiment_cex.cenA-pET.png)
**Figure 1.2.** Plasmids for Cex-HlyA and CenA-HlyA expression
*cex*: Cellulase exoglucanase gene, *cenA*: Cellulase endoglucanase gene,*hlyA, hlyB, hlyD*: α-hemolysin system gene

![](https://static.igem.org/mediawiki/2019/3/31/T--ECUST_China--experiment_pETpcr.png)
**Figure 1.3.** PCR amplification results by 1% Agarose Gel
Lane 1: linearized pET-cenA backbone; Lane 3: pET-cex Lane 4: hlyA-cenA; Lane 6: hlyA-cex; Lane 8: CenA; Lane 9: hlyB&hlyD

![](https://static.igem.org/mediawiki/2019/3/3c/T--ECUST_China--experiment_pETplate.png)
**Figure 1.4.** Plates of DH5α transformants (cenA-hlyA/pIN2 and cex-hlyA/pIN2)

Due to mRFP had been inserted into our backbone pIN2 for the construction of the invertor system, the negative colonies would show red for expressing mRFP, and white colonies were mostly positive. We conducted colony PCR and Sanger sequencing for further confirmation.

![](https://static.igem.org/mediawiki/2019/9/9f/T--ECUST_China--experiment_pETcolony.png)
**Figure 1.5.** Colony PCR of cenA-hlyA/pIN2 plate of cenA gene

![](https://static.igem.org/mediawiki/2019/5/5b/T--ECUST_China--experiment_pETseq.png)
**Figure 1.6.** Sanger sequencing results for cenA-hlyA/pIN2 and cex-hlyA/pIN2 plasmids

After successfully constructed both clones, we followed Crude enzyme extraction protocol and cellulase enzyme activity assay protocol to obtained the enzyme activity of Cex-HlyA and CenA-HlyA, and the results are shown below. We also measured the enzyme activity curve of CenA alone for the characterization of Part: [BBa\_K523015![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](http://parts.igem.org/Part%3ABBa_K523015).

![](https://static.igem.org/mediawiki/2019/8/8f/T--ECUST_China--experiment_pETCMC.png)
**Figure 1.7.** Enzyme activity of different temperature and pH, measured by CMCNa assay

![](https://static.igem.org/mediawiki/2019/5/58/T--ECUST_China--experiment_FPA.png)
**Figure 1.8.** Enzyme activity of measured by CMCNa assay and FPA assay (pH=7, temp.=37℃)

To fully verify our cellulase hydrolysis products were mostly cellobiose for the activation of cellobiose promoter and as a result turning off cellulase expression and turning on bacterial cellulose synthase, we performed TLC on silica gel plates using a ethyl acetate/ methanol /acetic acid/distilled water (12:3:3:2, V/V/V/V) mixture as the mobile phase for our FPA assay products and CMCNa assay products and results are as follows:

![](https://static.igem.org/mediawiki/2019/1/1a/T--ECUST_China--experiment_TLC.png)
**Figure 1.9.** TLC results for FPA assay products and CMCNa assay products
Our products show spots at the same horizontal position as cellobiose standard solutions, revealing that the reaction products have the component of cellobiose

### Secretion efficiency of Cex-HlyA/CenA-HlyA

After validating the secretion ability of a-hemolysin system by HlyA fused with RFP, we also want to make sure that Cex and CenA can be secreted and to analyze the secretion efficiency separately, we planned to construct pCDFDuet-MCS1-cenA-HlyA-MCS2-HlyBD and pCDFDuet-MCS1-cex-HlyA-MCS2-HlyBD.

![](https://static.igem.org/mediawiki/2019/8/81/T--ECUST_China--experiment_pCDF.png)
**Figure 1.10.** Plasmids for cex-hlyA-hlyBhlyD and cenA-hlyA-hlyBhlyD expression

![](https://static.igem.org/mediawiki/2019/c/c3/T--ECUST_China--experiment_pCDFpcr.png)
**Figure 1.11.** PCR amplification results by 1% Agarose Gel
Lane 1: linearized pCDFDuet-MCS2 backbone; Lane 2: HlyBHlyD Lane 7: CenA; Lane 8: HlyA-CenA; Lane 9: HlyA-Cex

![](https://static.igem.org/mediawiki/2019/1/1d/T--ECUST_China--experiment_pCDFcolony.png)
**Figure 1.12.** Colony PCR pCDFDuet-MCS1-MCS2-HlyBD plate (HlyBHlyD gene and nick 1/2)

![](https://static.igem.org/mediawiki/2019/8/8f/T--ECUST_China--experiment_pCDFoverlap.png)
**Figure 1.13.** Schematic presentation of overlapping PCR drawn by Iris Hu

Firstly, the *hlyBD* was cloned into MCS2 by overlapping PCR. After obtaining pCDFDuet-MCS1-MCS2-hlyBD, we tried multiple methods to insert *cenA-hlyA* into MCS1, such as seamless cloning, double enzymes digestion and ligation, and overlapping PCR, but all these attempts failed for a mysterious missing of a part of *hlyBD* in the final construction while the sequence of pCDFDuet-MCS1-MCS2-HlyBD was proved to be correct by Sanger sequencing of the whole sequence.

![](https://static.igem.org/mediawiki/2019/3/33/T--ECUST_China--experiment_pCDF1.png)
**Figure 1.14.***cenA-hlyA* fragments and pCDFDuet backbone fragments after Double Enzyme Digestion & cex fragments prepared for overlapping PCR

![](https://static.igem.org/mediawiki/2019/9/90/T--ECUST_China--experiment_pCDF2.png)
**Figure 1.15.** Colony PCR of pCDFDuet-MCS1-cenA-HlyA-MCS2-HlyBD for cenA gene and pCDFDuet-MCS1-cex-HlyA-MCS2-HlyBD for nick 1/2/3.

![](https://static.igem.org/mediawiki/2019/c/c9/T--ECUST_China--experiment_pCDFseq.png)
**Figure 1.16.** Sanger sequencing results of pCDFDuet-MCS1-MCS2-HlyBD, pCDFDuet-MCS1-cenA-HlyA-MCS2-HlyBD, pCDFDuet-MCS1-cenA-HlyA-MCS2-HlyBD
 The results shows that the sequence of the first plasmid is correct while the sequence of later two plasmids are incorrect.

At last, due to the intense deadline, we couldn’t finish this construction on time, nevertheless the results of Enzyme activity within cells and secretion efficiency demonstrated by mRFP can shed light on the feasibility of our ultimate goal.

### Supernatant enzyme activity of Cex-HlyA/CenA-HlyA

Interestingly, we inserted *cenA-hlyA*, *cex-hlyA*, *hlyBhlyD* into the plasmid pIN2 of our invertor system just before deadline. After consulting former iGEM characterizations, we learned that the secretion efficiency is relatively low, so the supernatant was concentrated by 20 times for better visualization.

Also, we used the above two plasmids: cex-hlyA/pIN2 and cenA-hlyA/pIN2 to measure to secreted enzyme activity for Cex and CenA, and the results are as below.

![](https://static.igem.org/mediawiki/2019/b/be/T--ECUST_China--super1.png)
**Figure 1.17.** Supernatant enzyme activity of CenA measured by CMC-Na assay (pH=7.0 temp.=37℃)

### Collaboration with Hubei University

Due to the fact that all of these proteins were fused with Flag-tag, we were able to detect their presence by Western Blot using a mouse-derived anti-Flag primary antibody and detection antibody probed with fluorescence.

![](https://static.igem.org/mediawiki/2019/a/aa/T--ECUST_China--experiment_WSET1.png)
**Figure 1.18.** Western blot of *ZM4* and *E. coli* T1 different secretion system

### Collaboration with Hubei University

From the Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot, we can conclude that the Secretion Efficiency of PhoD-CelA outshines other two types, while *ZM4* showed higher expression efficiency of GAP promoter than *E. coli* T1.

![](https://static.igem.org/mediawiki/2019/c/c0/T--ECUST_China--experiment_SDS.png)
**Figure 1.19.** SDS-PAGE of *ZM4* and *E. coli* T1 different secretion system

# Bacterial cellulose synthesis

This part consists of two parts:

* Cellobiose to glucose
* Glucose to bacterial cellulose

Since the substrate of BC synthesis is UDP-Glc, cellobiose should be degraded to glucose or glucose-phosphate firstly by Cep94A and then be transformed to bacterial cellulose via AcsABCD.

![](https://static.igem.org/mediawiki/2019/d/d6/T--ECUST_China--bc_plasmid.png)
**Figure 2.1** Gene circuit of bacterial cellulose synthesis

### Cellobiose to glucose

Short cellulose from pulp fibers were initially degraded to cellobiose which could accumulate in the cytosol, then the cellobiose needed the further degradation by cellobiose phosphorylase (Cep94A).

Cellobiose phosphorylase is an enzyme that catalyzes the chemical reaction cellobiose + phosphate → alpha-D-glucose 1-phosphate + D-glucose. We obtained the sequence of cellobiose phosphorylase from Part:[BBa\_K2449003![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](http://parts.igem.org/Part%3ABBa_K2449003). Unfortunately, this part was not contained in iGEM distribution kit, so we synthesized this gene (*cep94A*) from Genewiz.

![](https://static.igem.org/mediawiki/2019/e/e5/T--ECUST_China--bc_cep94a-pla.png)
**Figure 2.2** Gene circuit of cep94A

In order to characterize the function of Cep94A specifically, we cloned the *cep94A* into p15a. Firstly, we successfully got the target gene and backbone through PCR. Because of the genes were all flanked with homologous end sequence, we used the Gibson assembly to construct the final plasmid.

![](https://static.igem.org/mediawiki/2019/d/d1/T--ECUST_China--bc_pcr-cep94a.png)
**Figure 2.3** The PCR results of *cep94A* and p15a backbone

Ligated plasmids were transferred into *E.coli* DH5α competent cell, then applied onto the Kan-containing LB plate. After incubating the cells at 37℃ for 12 hr, we selected 8 colonies of each plate and used the test primer to verify whether the fragments linked properly.

![](https://static.igem.org/mediawiki/2019/c/c4/T--ECUST_China--bc_cPCR2.png)
**Figure 2.4** Colony PCR results shown on 1% agarose gel

We chose the positive colonies (number1,4,7) which were also sequenced successfully to perform the further characterization experiment.

![](https://static.igem.org/mediawiki/2019/9/91/T--ECUST_China--bc_sdspage1.png)
**Figure 2.5** SDS-PAGE results of Cep94A (about 91kDa)

To make out whether the Cep94A protein was expressed, SDS-PAGE was performed. The result showed that Cep94A was actually expressed (about 91kDa) after induction.

After verifying the expression of Cep94A, we decided to measure the Cep94A enzyme activity to perform the quantitative experimental characterization. After induction for 16 hours, the cells were collected and resuspended in 10 ml of PBS buffer, afterwards sonicated to obtain 10 ml supernatant. The enzyme activity of Cep94A was measured by the DNS method: 2 μL cellobiose +1 μL enzyme solution + 17 μL citrate buffer (pH =4.8) was reacted at 50 °C for 30 min. Then 30 μL DNS reagent was added, boiling for 10 min. The reacton mixture was diluted 5 times and transferred to 96-well microplate to measure the OD540 with a microplate reader.

![](https://static.igem.org/mediawiki/2019/f/fe/T--ECUST_China--bc_dns.png)
**Figure 2.6** The DNS assay of Cep94A

Enzyme activity = moles of substrate converted per unit time. So we could measure the activity of Cep94A by monitoring the rate at which a product (glucose) formed. Firstly, we drew the glucose standard curve: y = 0.6746x - 0.0491，R² = 0.9775 (y represents OD540, x represents glucose concentration), then the enzyme activity was calculated based on the glucose standard curve. Pluging OD540=0.093 into y, we worked out that x (glucose concentration) = 0.211mg/ml. According to the formula: Cep94A enzyme activity (U/mL) =enzyme dilution factor×moles of glucose /reaction time, we calculated that the enzyme activity of Cep94A was 0.781U/ml.

![](https://static.igem.org/mediawiki/2019/1/11/T--ECUST_China--bc_standard.jpg)
**Figure 2.7** Glucose standard curve

### Glucose to bacterial cellulose

After the degradation of cellulose into glucose by above-mentioned steps, our chasis was supposed to utilize the glucose immediately and produce bacterial cellulose. We chose the cellulose synthase from the *Gluconacetobacter. xylinus* —*acsA* (the catalytic domain), *acsB*( the cyclicdi-GMP-binding domain), *acsC* and *acsD* (two accessory protein). These four genes, invented by Imperial London 2014 iGEM team, were in the distribution kit the iGEM send to us.

Since the plasmid containing *acsAB, acsC,acsD* was too big to construct (almost 12kb), we decided to characterize *acsAB* and *acsC, acsD* separately. By using the mini-prepared plasmid as template, we got the desired fragments as follows:

![](https://static.igem.org/mediawiki/2019/0/05/T--ECUST_China--bc_pcr.png)
**Figure 2.8** The PCR results of target genes of BC synthesis

Since the fragments were all flanked with homologous end sequence, we used the Gibson assembly to obtain our final plasmid.Ligated plasmids were transferred into*E. coli*DH5α competent cell, then applied onto the Chl-containing LB plate and incubated at 37℃ for 12hr. After colony PCR and sequencing, we obtained the positive clones.

For *acsAB*, we verified its function using congo red binding assay. After inducing AcsAB-containing cells with 0.4mM IPTG in 100mL LB supplied with Chl at 37 °C, cells were pelleted at 8000rpm for 5minutes and the supernatant was kept for further analysis (both empty vector controls and un-induced controls were also assayed). Cells were resuspended in 1xPBS and sonicated for 3 minutes as on/off cycles of 15 seconds. Both the pellet and supernatant were kept for further analysis. Congo Red was then added to the samples up to a final concentration of 20μM. Samples were incubated for 2h at static conditions and at room temperature to allow for Congo Red binding. Then the samples were transferred to 96-well plate for absorbance measurements at 490nm. Only if the free congo red existed (not binding to bacterial cellulose), could the absorbance at 490nm be detected.

![](https://static.igem.org/mediawiki/2019/3/32/T--ECUST_China--bc_congo.png)
**Figure 2.9** The congo red binding assay of acsAB

The results revealed that the cells which expressed acsAB had the obviously lower absorbance at 490nm. Illustrating that there was bacterial cellulose produced in the cytosol.

In terms of *acsC* and *acsD*, it is difficult to characterize its function separately. So we just verified the expression of acsC and acsD via SDS-PAGE.

![](https://static.igem.org/mediawiki/2019/3/36/T--ECUST_China--bc_sdspage2.png)
**Figure 2.10** SDS-PAGE results of AcsC and AcsD

The SDS-PAGE results illustrated that AcsC and AcsD were exactly expressed(almost 138.8kDa and 17.4kDa respectively).

Having the prospective results of *acsAB* and *acsCD*, now we are working on constructing the whole plasmid which includes *acsAB* and *acsCD*. We hope that the cells with this plasmid could express functional cellulose synthase and synthesize bacterial cellulose.

# Regulator

In order to achieve the purpose of switching the procedure of cellulose hydrolysis and bacterial cellulose synthesis automatically. A novel regulator was designed, containing inverter system and cellobiose response element. Inverter just liked the switch of the two functional genes while cellobiose realized the automatic regulation of this switch.

### Inverter system

Inverter consists of lactose operon, CⅠprotein and *PλR012*. We constructed the inverter into a dual-plasmid (pIN1-pIN2) and used GFP and RFP to verify the effectiveness of inverter([Yokobayashi Y et al.,2002](https://www.pnas.org/content/99/26/16587)).

![](https://static.igem.org/mediawiki/2019/8/88/T--ECUST_China--experiment_inverter.png)
**Figure 3.1** Gene circuit of inverter system

All of the biobricks of the inverter were obtained from iGEM distributon kit via PCR while the plasmid backbones were stored in our laboratory.

![](https://static.igem.org/mediawiki/2019/0/09/T--ECUST_China--regulator_pcr-pIN1.png)
**Figure 3.2** The PCR results of the parts of pIN1

![](https://static.igem.org/mediawiki/2019/e/ec/T--ECUST_China--regulator_pcr-pIN2.png)
**Figure 3.3** The PCR results of the parts of pIN2

After obtaining the PCR products, we ligated the backbone and target gene using Gibson-Assembly method. Ligated plasmids were transferred into *E.coli* DH5α competent cell, then applied onto the Chl-containing LB plate(pIN1) and Chl-containing LB plate(pIN2) . After incubating the cells at 37℃ for 12 hr, we selected several colonies of each plate and used the test primer to verify whether the fragments linked properly.

![](https://static.igem.org/mediawiki/2019/e/ee/T--ECUST_China--experiment_cPCR.png)
**Figure 3.4** Colony PCR results shown on 1% agarose gel

We chose the positive colonies (pIN1-2 and pIN1-9; pIN2-2 and pIN2-6) which were also sequenced successfully to perform the further characterization experiment.

Detailed circuit measurements were performed in liquid cultures. pIN1 and pIN2 were initially co-transferred to *E.coli* DH5α competent cell. The positive colony was then grown in 5mL M9 at 37℃ for 12 hr, then transferred to 100 mL M9 for enlarge cultivation. After the cells growing to OD600=0.5, we added different concentration of IPTG(0.1μM,1μM, 10μM, 100μM, 1000μM ).

![](https://static.igem.org/mediawiki/2019/9/98/T--ECUST_China--regulator_m9.png)
**Figure 3.5** Liquid M9 media in 15hr of dual-plasmid (pIN1 + pIN2) in different concentration of IPTG

We took samples of each IPTG concentration before and after induction (almost 16hr after induction). The samples were then transferred to a 96-well microplate in which RFP fluorescence (590 nm excitation, 645 nm emission, top 50% cutoff) was measured by using a fluorescence microplate reader. The fluorescence data were normalized against cell densities which were measured by using a microplate reader at 600 nm.

![](https://static.igem.org/mediawiki/2019/7/7d/T--ECUST_China--regulator_inverter.png)
**Figure 3.6** Fluorescence intensity induced by different concentrations of IPTG
Measurements of the circuits in response to varying IPTG levels were summarized in the transfer curve, where each point on the curve represented fluorescence data from three independent cultures of the same circuit under the same induction conditions.

Just as shown in the figure, as the concentration of IPTG rose, the expression of mRFP decreased, indicating that the inverter system worked effectively. The LacI protein was constitutively expressed from *PlacIQ* and repressed the *Plac* promoter.*Plac* transcriptional activity was controlled by modulating the concentration of an externally added inducer, IPTG. The expression of the CI repressor was controlled by Plac. Repressor CI acts on *PλRO12* on pIN2 to repress the transcription of the mRFP gene, the output fluorescence indicator. So without IPTG, the CI level was low and mRFP level was high; adding IPTG increased CI levels and in turn decreased mRFP levels.

### Cellobiose response element

The cellobiose response element is our new biobrick. We chose the cellobiose as the response element ranther than glucose because cellobiose could accumulate in the cytosol, while glucose is easy to metabolize. Only when the cellobiose accumulated to a certain concentration, could the cellobiose operon be active to reverse the inverter.

![](https://static.igem.org/mediawiki/2019/4/41/T--ECUST_China--regulator_circuit-cello.png)
**Figure 3.7** Gene circuit of cellobiose response element

Cellobiose response element is the mutant of *chb* operon in *E.coli*. Wild‐type strains of *Escherichia coli* are normally unable to metabolize cellobiose. The *chb* operon is the inducible genetic system involved in the catabolism of N,N′‐diacetylchitobiose([Plumbridge J et al.,2004](https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2958.2004.03986.x.)). However, single base‐pair changes in the transcriptional regulator *chbR* that translate into single‐amino‐acid substitutions constitute the cellobiose operon which can response cellobiose([Kachroo AH et al.,2007](https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2958.2007.05999.x?tdsourcetag=s_pcqq_aiomsg)). So, ECUST\_China iGEMers performed several types of mutation: chbRN137K, chbRY30C and chbRN238S, aiming to find a efficient cellobiose response element.

![](https://static.igem.org/mediawiki/2019/f/f1/T--ECUST_China--regulator_snapgene.png)
**Figure 3.8** The mutation sites of *chbR*

During the experiment, we used mRFP as the reporter. The sequence of *Pcel* and *chbR* was obtained from *E.coli* K12 MG1655 via PCR .Then we performed inverse PCR to achieve the site-directed mutation of amino acids of *chbR*. According to the literature([Kachroo AH et al.,2007](https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2958.2007.05999.x?tdsourcetag=s_pcqq_aiomsg)), we chose three site-directed mutation : chbRN238S, chbRY30C and chbRN137K.

![](https://static.igem.org/mediawiki/2019/b/bb/T--ECUST_China--regulator_cellomrfp.png)
**Figure 3.9** Gene circuit of cellobiose response element characterization

![](https://static.igem.org/mediawiki/2019/6/67/T--ECUST_China--regulator_pcr-pcelchbr.png)
**Figure 3.10** The PCR results of pCEL backbone and *chbR*

After constructing the mutation plasmids: pIN1-NK and pIN1-YC-NS. Both of the mutant clones and the wild-type strain were incubated in M9 medium containing 0.4% glycerol and 0.4% casamino acids, with or without cellobiose for about 40 hours. All of the samples were transferred to 96-well plate to measure the mRFP fluorescence.

![](https://static.igem.org/mediawiki/parts/3/3c/T--ECUST_China--Cellobiose.jpg)
**Figure 3.11** Fluorescence intensity induced by cellobiose

The results revealed that presence of both chbR mutants resulted in a high basal level of expression. More importantly, the transformant carrying ChbR-YC-NS showed an approximately threefold induction over the basal level in the presence of cellobiose whereas no induction was seen in the presence of wild-type ChbR.

# Linker optimization

Since the cellulose fiber cannot cross the cell membrane, we expected the cellulase, including endoglucanase(CenA) and exoglucanase(Cex), to be secreted through α-hemolysin system.

The hemolysin (type I) secretion system belongs to the ABC transporter family, which recognizes the C-terminal amino acids of hemolysin toxin HlyA for protein secretion without requiring an N-terminal signal peptide.

So we planed to fuse the *cex* and *cenA* gene with *hlyA* tag at the C-terminus. Besides, we also co-expressed the HlyB and HlyD, two inner membrane proteins necessary to recognize and transport fusion protein across the inner and outer membrane directly.

![](https://static.igem.org/mediawiki/2019/5/5a/T--ECUST_China--design_circuit-secre.png)
**Figure 4.1** Gene circuit of cellulase secretion system

But before we constructed our final plasmid, which is load with *cex-hlyA, cenA-hlyA, hlyB, and hlyD*(Figure above), we firstly tried to replace the function gene, namely *cex* and *cenA*, with *mRFP*.

![](https://static.igem.org/mediawiki/2019/5/54/T--ECUST_China--experiment_mrfp.png)
**Figure 4.2** Gene circuit of mRFP secretion system

We used [BBa\_K1166002![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](http://parts.igem.org/Part%3ABBa_K1166002) as the template to acquire the *hlyA-tag, hlyB ,and hlyD* as a whole by PCR. And in the similar way, we cloned the mRFP-containing pIN2 backbone form one of our former constructed plasmid. In order to diminish the conformation effects of the *hlyA* on the target gene(in this case, *mRFP*), we constructed 3 types of linker— flexible, rigid([Xiaoying Chen et al., 2013](https://www.sciencedirect.com/science/article/pii/S0169409X12003006)), and OmpT-cutting linker([R. A. Kramer Ye et al., 2001](https://febs.onlinelibrary.wiley.com/doi/full/10.1046/j.1432-1327.2000.01073.x))—form the original linker which was reported to interfere with the GFP folding when fused together.

![](https://static.igem.org/mediawiki/parts/0/03/T--ECUST_China--cleavable-linker-SnapGene.png)
**Figure 4.3** 3 types of linker optimization

We modified the linker by introducing desired 5’ primer sequence.Through touch-up PCR using Phanta high fidelity polymerase from Vazyme, we got all the fragments and tested their length through electrophoresis.

We purified the PCR products and detected the concentration of DNA using NanoDrop2000.

![](https://static.igem.org/mediawiki/2019/7/76/T--ECUST_China--experiment_fig4.png)
**Figure 4.4** Touch-up PCR results
O:original linker, F:flexible linker, R:rigid linker, C:OmpT-cut; line1-4: pET+mRFP plasmid backbone(～3500bp); line5-8: hlyA+secretion system(～5200bp); line9: marker III

**Diagram 4.1** Touch-up PCR of hlyA+secretion system
![](https://static.igem.org/mediawiki/2019/d/d1/T--ECUST_China--experiment_pcr1.png)

Using Gibson-Assembly method, we ligated the backbone and target gene. Ligated plasmid were transferred into*E. coli*DH5α competent cell, then applied onto the Kan-containing LB plate and incubated at 37℃ for 12hr([Marc Baaden et al., 2004](https://linkinghub.elsevier.com/retrieve/pii/S0006349504737674)).

**Diagram 4.2** Concentration of purified DNA measured by NanoDrop2000
![](https://static.igem.org/mediawiki/2019/f/f0/T--ECUST_China--experiment_table2.png)

**Diagram 4.3** Gibson Assembly Reaction System
![](https://static.igem.org/mediawiki/2019/d/db/T--ECUST_China--experiment_table3.png)

We selected 8 visible(purple) colonies of each four plates and used 3 pairs of primer to test whether these 2 fragments are connected.

![](https://static.igem.org/mediawiki/2019/8/88/T--ECUST_China--experiment_plates.png)
**Figure 4.5** Plates of DH5α transformants (pIN2 + mRFP + linker + *hlyA + araC + pBAD + hlyB + hlyD*) of 4 types of linker on LB-kan plates and in liquid M9 media.

Three different pairs of primer has been used to test the connection link. and the colony PCR results showed that positive clones were: flexible-linker F12357, rigid-linker R12367, OmpT-cleavable-linker C12357, and original-linker O4568.

So we chose F3, R2, C3, and O8 to be amplified and further tested by sequencing(BGI company). The link sequences were all proved to be in accordance with our designed linker sequence, indicating we’ve successfully constructed 4 types of plasmid with original, flexible, rigid and cleavable linker respectively. These four clones were then use to prepare protein sample to run SDS-PAGE and Western Blot.

![](https://static.igem.org/mediawiki/2019/3/33/T--ECUST_China--experiment_fig6.png)
**Figure 4.6** Colony PCR results shown on 1% agarose gel.

All the colonies proved to be positive by colony PCR and first generation sequencing could be easily spotted red on plates and liquid media, directly indicating that the all the linker+HlyA tags were not interfering the emission of fluorescence of mRFP. Compared with using GFP as the secreted target gene, mRFP is apparently more suitable for the fusion of HlyA to testify the secretion efficiency of hemolysin system.

![](https://static.igem.org/mediawiki/2019/d/d6/T--ECUST_China--experiment_fig7.jpg)
**Figure 4.7** Liquid media of DH5α transformants (pIN2 + mRFP + linker + *hlyA + araC + pBAD + hlyB + hlyD*) of 4 types of linker on LB-kan plates and in liquid M9 media.

Positive transformants with different linker were cultured and induced by arabinose. We collected the same weight of *E.coli* by restricting the value(OD multiple volume) equal to 16.32. And then separate the media and cell by centrifugation. The supernate were concentrated 100 fold and the pellet were resuspended with PBS. Supernate and pellet were both pretreated to prepare protein sample and run SDS-PAGE.

![](https://static.igem.org/mediawiki/2019/b/bf/T--ECUST_China--arabinose_induced.jpeg)
**Figure 4.8** Liquid LB media containing positive colonies with(Y) or without(N) arabinose induction after 16 hours. Left three media are pIN2+mRFP, and pIN2(empty vector) as negative control.

The SDS-PAGE shows that, the fusion protein—mRFP-HlyA(in the red frame, about 32.6kD)—were all expressed inside the cell and successfully secreted to the media.And in the OmpT-cleavable linker case, the mRFP and HlyA-tag were also successfully cleaved, since the cleaved mRFP(in the rgreen frame, about 26.0kD) clearly surpassed the fusion protein on the gel. Although the mRFP-HlyA fusion protein and mRFP were both slightly smaller than expected size, we highly suspected that’s because, the pI of the mRFP-HlyA and mRFP are estimated to be 5.12 and 5.07, both were lower than pH7, which might dramatically affect the mobility ratio of these protein in the SDS-PAGE buffer and shows at a lower molecular weight on the gel.

![](https://static.igem.org/mediawiki/2019/d/d7/T--ECUST_China--experiment_fig9.png)
**Figure 4.9** SDS-PAGE of supernatant and pellet of types of linker-transformants.

So we’ve demonstrated the OmpT-cleavable linker to be functional, and the flexible and rigid linker were also proved to be not hindering the secretion of fusion protein while offering several alternative options for perspective users who want to express their protein in an secretory form.

#### ECUST\_China

EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY

Shanghai, China

[![](https://static.igem.org/mediawiki/2019/2/27/T--ECUST_China--ecust_school-w.png)](https://www.ecust.edu.cn/)
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#### GET IN TOUCH

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# Team:ECUST China/Results

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* 01
  [Cellulose degradation](#main1)
* 02
  [Bacterial cellulose synthesis](#main2)
* 03
  [Regulator](#main3)

# Overview

Wastepaper recycling industry is confronted with a serious problem: the inevitable keratinization during the recycling process, which results in the shortening of the cellulose fiber length and subsequently generating paper with lower quality. To utilize the short cellulose from waste pulp more rationally, ECUST\_China iGEMers invented “paper transformer” which could realize the former degradation of short pulp fibers and the latter in situ synthesis of bacterial cellulose.

After the hard work in the laboratory during the whole summer, ECUST\_China iGEMers have overcame many difficulties and accomplished almost all the work needed for this project.

# Cellulose degradation

In this modul, we successfully equipped our *E.coli* with cellulase activity to degrade short cellulose. In order to use cellobiose (the intermediate product of short cellulose) which could be accumulated in the cytosol as the regulated element, we chose endoglucanase (cenA) and exoglucanase (cex), excluding β-glycosidase (whose product is glucose) to degrade short fibers to cellobiose. And since the short cellulose existed in the extracellular space, both endoglucanase and exoglucanase were linked with hemolysin system which allowed the enzyme to be secreted to the extracellular space(ES).

**Key acheivements:**

* Constructed the plasmid: *cex-hlyA* and *cenA-hlyA* to verify the expression and enzyme activity of endoglucanase (cenA) and exoglucanase (cex).
* Constructed the plasmid: *cex-hlyA-hlyBhlyD* and cenA-hlyA-hlyBhlyD to verify the secretion efficiency of Cex-HlyA/CenA-HlyA.
* Optimized the linker between cellulase and hemolysin system to improve the performance of fusion protein.
* Performed TLC/HPLC and confirmed that the product of endoglucanase (cenA) and exoglucanase (cex) was mostly cellobiose which was useful for the latter steps.

# Bacterial cellulose synthesis

Since the substrate of BC synthesis is UDP-Glc, cellobiose should be degraded to glucose or glucose-phosphate firstly by cellulose phosphorylase (Cep94A) and then be transformed to bacterial cellulose via acsABCD. We have accomplished the above two steps.

**Key acheivements:**

* Constructed the plasmid: Cep94A to verify the expression of cellulose phosphorylase (Cep94A) via SDS-PAGE.
* Used cellobiose as the substrate and performed DNS assay to measure the enzyme activity of cellulose phosphorylase (Cep94A).
* Constructed the plasmid: *acsAB* and *acsC, acsD* to verify the expression of cellulose synthase (acsAB and acsC, acsD).
* Performed congo red binding assay to verify the BC synthesis functionality of acsAB.

**Future plans for this part:**

Constructing the cep94A, acsAB and acsCD to the same plasmid. Incubating the cell which can express cep94A and cellulose synthase (acsABCD) in the medium containing cellobiose as the sole carbon source. We hope that bacterial cellulose could be synthesized.

# Reguator

In order to achieve the purpose of switching the procedure of cellulose hydrolysis and bacterial cellulose synthesis automatically. A novel regulator has been accomplished, containing inverter system and cellobiose response element. Inverter worked as the switch of the two functional genes while cellobiose realized the automatic regulation of this switch.

**Key acheivements:**

* Constructed inverter system into a dual-plasmid (pIN1-pIN2), both of pIN1 and pIN2 were characterized respectively (for more data, please visit:[BBa\_K3093100![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](http://parts.igem.org/Part%3ABBa_K3093100) ).
* Co-expressed the dual-plasmid (pIN1-pIN2) system and verified the functionality of pIN1-pIN2 via observing the changes of mRFP (the reporter) fluorescence.
* Obtained the sequence of *Pcel* and *chbR* from *E.coli* K12 MG1655 via PCR and performed inverse PCR to achieve the site-directed mutation of amino acids of *chbR* to make it sense cellobiose.
* Constructed the mutation plasmids: pIN1-NK and pIN1-YC-NS. And confirmed that the mutation strain could response to cellobiose.(for more data, please visit:[BBa\_K3093002![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](http://parts.igem.org/Part%3ABBa_K3093002) ).

**Future plans for this part:**

Combined the inverter system and cellobiose responsive element to verify the functionality of the whole regulator.

Since the inducibility of the mutant chbR seemed to be poorer than our expectation. Quite a few reasons gave rise to this phenomenon:

- Problem 1: The modified repressor chbR YC-NS was not sensitive enough to cellobiose.

Improvement methods: Struggling to find a highly cellobiose-specific chbR repressor protein by determinate evolution, however, the workload could be extremely heavy so that other methods have to be found.

- Problem 2: The endogenous chbR of E. coli which might compete with the mutant chbR for DNA binding sites disturbed the response of cellobiose operon.

Improvement methods: The gene of endogenous ChbR protein can be knocked out to eliminate its effect on cellobiose.

- Problem 3: The chbR gene was downstream from a strong constitutive promoter BBa\_J23108 and a strong RBS BBa\_B0034, which might result in an excessive amount of repressor protein, making chbR difficult to induce by cellobiose.

Improvement methods: The expression level of chbR can be adjusted by using different types of promoters and RBS, thereby adjusting the threshold for the cellobiose response element.

- Problem 4: The -35 region of Pcel was TACTAT, rather than the typical TTGACA, leading to the lower efficiency of RNA polymerase.

Improvement methods: The literature ( [Joseph AM et al, 2017](https://link.springer.com/article/10.1007/s00203-016-1299-0#CR11) )documented that a mutation in the -35 region of Pcel could improve the chbR’s response of cellobiose. Therefore, it is possible to adopt a method of changing the -35 region to increase the transcriptional efficiency and thereby increase the induction ratio.

For more details, visit the [EXPERIMENTS![](https://static.igem.org/mediawiki/2019/b/bd/T--ECUST_China--icon_cursor.png)](https://2019.igem.org/Team%3AECUST_China/Experiments)

In summary, ECUST\_China iGEMers have basically achieved the overall goal. If time permits, we really hope that our “paper transformer” could be actually applied to the waste paper recycling industry to “make paper paper again” !

#### ECUST\_China

EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY

Shanghai, China

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