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.

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
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.

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.

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.

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Notebook

ECUST_China

EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY

Shanghai, China

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