What is "paper transformer" ?
In order to utilize the less useful short cellulose from wastepaper as the raw material to produce bacterial cellulose(BC) in situ, ECUST iGEMers invented “paper transformer”. It was a dual plasmid system(pCL-pCS) including three devices :
- Cellulose hydrolysis
- Bacterial cellulose(BC) synthesis
- Regulator (including inverter system and cellobiose response element ) .
We choosed E.coli DH5α as the chassis.
Gene circuit of cellulase secretion
cex: Cellulase exoglucanase gene, cenA: Cellulase endoglucanase gene,hlyA, hlyB, hlyD: α-hemolysin system gene
Model of hemolysin secretion
Cellulose hydrolysis device was composed of exoglucanase(cex) and endoglucanase(cen) , both with HlyABD secretory system which allowed the enzyme to be secreted to the extracellular space(ES). The short pulp fibers were degaraded to cellobiose which could be accumulated in extracellular space.
During the experiment, we firstly verified effectiveness of HlyABD secretory system using RFP in liquid supernatant. After that, we substituted RFP for cex and cenA separately to verify the expression effect of the enzyme. Finally, we measured the enzyme activity of exoglucanase and endoglucanase.
Bacterial cellulose(BC) synthesis
Gene circuit of BC synthesis
acsAB, acsC, acsD: Bacterial cellulose synthase gene,cep94A: Cellobiose phosphorylase gene
Model of BC synthesis
BC synthesis device included cellobiose phosphorylase(cep94A) and cellulose synthase(acsABCD), the former degraded cellobiose into glucose(the substrate of BC) while the latter transformed glucose to bacterial cellulose.
During the experiment, we firstly examined the activity of cep94A by DNS test. After that, we tested the BC producing ability by Congo Red Binding assay. Finally, we constructed the cep94A with acsAB, and identified whether and how much cellobiose could be converted into bacterial cellulose.
Regulator consisted of two parts: inverter system and cellobiose response element, which worked synergistically to achieve the purpose of switching the procedure of cellulose hydrolysis and BC synthesis automatically.
Gene circuit of Inverter system
cI: Repressor gene of PλRO12, lacI: Repressor gene of Plac
Inverter system included lactose operon, CⅠprotein and λPRO12. The LacI repressor was constitutively expressed which inhibited transcription from the pLac promoter in the absence of inducer . Thus λPR012 could be active to express cellulase, degrading short fibers to cellobiose. Once the inducer existed, the expression of cellulose synthase was inducible, while the expression of cellulase was turned off because of the exist of c1, hence realizing the reversal of function.
During the experiment, we substituted the functional genes for GFP or RFP to verify the effectiveness of the inverter via observing fluorescence change.
Cellobiose response element
Gene circuit of Cellobiose response element
chbR: Repressor gene of Pcel
For automatic regulation of inverter, we designed cellobiose(intermediate product of our project) response elment (shown in the red box above). This was our new biobrick which could sense when cells were exposed to cellobiose. Only when the cellobiose accumulated enough, could the lacZ gene express, isomerizing lactose to allolactose which worked as the inducer of inverter.
We obtained the sequence of cellobiose operon from E.coli K12 MG1655 via extracting genome and inverse PCR (site-directed mutation of amino acids of chbR). Then we used RFP to verify the effectiveness of cellobiose operon.
"Paper Transformer" Mechanism
“Paper transformer” could achieve the former degradation of short pulp fibers and the latter in situ synthesis of BC under the control of the dual plasmid system (pCL-pCS) which included cellulose hydrolysis device, BC synthesis device, inverter system and cellobiose response element.
Gene circuit of dual plasmid system
The engineered bacteria were firstly cultured in medium containing glucose, lactose and cellulose. The Plac was inhibited in the beginning because of the absence of lacZ gene and high concentration of glucose. While plasmid pCL expressed secretory exoglucanase and endoglucanase under the control of the synthetic λPRO12. In this situation, short pulp fibers were degraded into cellobiose and continuously accumulated. When cellobiose accumulated to a certain extent (at this time, glucose was almost used up), it would activate the cellobiose operon which could express lacZ gene. So that Plac would be active, expressing CⅠ protein, cellobiose phosphorylase, cellulose synthase. CⅠprotein acted on λPRO12. repressing the expression of pCL , cellobiose phosphorylase degraded cellobiose into glucose and cellulose synthase transformed glucose to bacterial cellulose.