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).

    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 ).
  • 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 ).

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 )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


    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

    GET IN TOUCH

    +86 021-64253306

    ecust_igem_2019@163.com