Team:ECUST China/Results
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).
- 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 efficiency of protein secretion.
- Performed TLC and confirmed that the product of endoglucanase (cenA) and exoglucanase (cex) was mostly cellobiose which was useful for the latter steps.
Key acheivements:
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.
- 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.
Key acheivements:
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.
- Constructed inverter system into a dual-plasmid (pIN1-pIN2), both of pIN1 and pIN2 were characterized respectively (for more data, please visit:BBa_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.
Key acheivements:
Future plans for this part:
Combined the inverter system and cellobiose responsive element to verify the functionality of the whole regulator.
For more details, visit the 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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+86 021-64253306
ecust_igem_2019@163.com
