Team:ECUST China/Demonstrate
We want to equipped our Paper Transformer with 3 abilities—cellulose degrading ability, cellulose producing ability, and inversion-control ability.
We designed a series of experiments in different dimension to demonstrate the functionality of our Paper Transformer.
Cellulose degrading ability
We equipped our E.coli with cellulase activity to degrade short cellulose at first. Since the cellulase, including endoglucanase, exoglucanase and β-glycosidase, functions as a whole to break down cellulose directly into glucose, yet the glucose can be easily consumed thus not being a suitable intermediate. So we chose to suspend the reaction at the pre-glucose stage, namely accumulating cellobiose as intermediate by introducing only endoglucanase(CenA) and exoglucanase(Cex) but not β-glycosidase(Cep94A). The cellobiose won’t be converted into glucose now because the disruption of β-1,4-glycoside bond is catalyzed by β-glycosidase(cep94A). And since the cellulose were present in the extracellular place, we adopted α-hemolysin secretion system to transport cellulase across the cell membrane and cell wall.
Dimension1: Measurement of cex and cenA activity inside the cell (without hlyA-tag)
We constructed the cex and cenA into a pET-28b plasmid separately at first, and we measured the cellulase (CMCNase) activity by mixing the two crude enzyme extracts together. We mixed two enzyme together to test enzyme activity because the CMCNa test could identified mostly endoglucanase activity and a small margin of exoglucanase activity. At the same time, we set two pH condition and pET28b empty vector as negative control. The cellulase activity were measured about 400mU/mL at pH6 and 450mU/mL at pH7, this low activity might be credited to inclusion body formation.
Figure 1. Enzyme activity of different temperature and pH, measured by CMCNa assay
Dimension2: Verification of secretion system functionality (mRFP)
We constructed mRFP-hlyABD to examine whether the α-hemolysin secretion system could work. Not only that, we optimized the linker between the hlyA-tag and mRFP to eliminate the conformational disturbance of hlyA-tag to mRFP. It could be easily noticed that the 4 types of different linker-transformants were expressing mRFP. We prepared the protein sample of pellet and supernatant, and loaded them on SDS-PAGE. The SDS-PAGE results showed that all the fusion protein were secreted outside the cell even only a small fraction.
Figure 2. SDS-PAGE of supernatant and pellet of types of linker-transformants.
Dimension3: Identification of the secretion of cex and cenA (with hlyA-tag)
After we proved the effectiveness of Cex/CenA activity and secretion system, we constructed a complete module to achieve secretory expression of cex and cenA. We collected the protein sample from pellet and supernatant fraction, and ran them on the SDS-PAGE and Western Blot thanks to a his-tag present in N-terminus of cex and cenA. The WB and SDS-PAGE results denoted that the cex and cenA were both successfully secreted into the media. And latter we tested the enzyme activity with concentrated crude supernatant extracts by CMCNa test. The cellulase activity were measured to be 150mU/mL at pH7, 37℃, supernatant concentrated by 20 times before measurement, and the enzyme activity had been adjusted according to standard IU.
Figure 3. Supernatant enzyme activity of CenA measured by CMC-Na assay (pH=7.0 temp.=37℃)
Dimension4: Identification of the products(cellobiose) of cex and cenA
We ran a TLC test to identify whether and how much the substrate cellulose could be degraded to cellobiose product by loading cellobiose and glucose as positive control. The preliminary results of TLC showed that cellobiose were indeed present in the post-reaction solution as there was a band with the similar track with cellobiose positive band. Given the preliminary results, we further tested the content and complexity of post-reaction solution by TLC. The results showed that our hydrolysis products exhibit spots at the same horizontal position as cellobiose standard solutions, revealing that the reaction products have the component of cellobiose. According to these two results, we proved that cellobiose could indeed be used as our secondary inducer of inverter system.
Cellulose producing ability
Then, we equipped our E.coli with β-glycosidase(cep94A) and cellulose synthase(acsAB) activity to convert cellobiose into glucose and utilize glucose as substrate for BC synthesis, respectively. Besides, we also co-expressed two important accessory protein—acsC and acsD—to facilitate BC fiber formation.
Dimension1: Measurement of cep94A activity inside the cell
Since we chose cellobiose as the stable intermediate to accumulate for latter BC synthesis, the cellobiose must be converted to glucose ahead of being converted to BC. By incubating the crude Cep94A extracted from supernatant fraction of disrupted recombinant cell with pure cellobiose at 50℃ for 30 min. The β-glycosidase activity tested by DNS assay showed as 0.781U/mL.
Dimension2: Verification of the BC producing ability of acsAB
Accordingly to Imperial London 2014 iGEM’s conclusion, the acsAB would be enough to synthesize bacterial cellulose. So we constructed acsAB without acsCD, the secretory machinery, on the pIN21 plasmid for the ease of our construction work. We adopted the Congo Red Binding assay to detect minimal BC produced inside the cell by disrupting recombinant cell. The light absorbance at 490nm was decreased about 22%, denoting the existence of free BC binding to Congo Red dye.
Figure 4. The congo red binding assay of acsAB
Dimension3: Identification of acsCD expression
We constructed the acsC and acsD without acsAB, since we have tried to constructe them together for about 2 months yet still couldn’t get the desired plasmid because it’s too big. So we constructed the acsC and acsD on the pIN1. Through SDS-PAGE with IPTG induced and un-induced, we could notice the band sized 138.8kDa, indicating the acsC was successfully expressed. While the acsD estimated to be about 17.4kDa was not conspicuous, we suspected that’s because the limit of marker and buffer condition.
Figure 5. SDS-PAGE results of acsC and acsD
Dimension4: Identification of the products(cellobiose) of cex and cenA
We ran a TLC test to identify whether and how much the substrate cellulose could be degraded to cellobiose product by loading cellobiose and glucose as positive control. The preliminary results of TLC showed that cellobiose were indeed present in the post-reaction solution as there was a band with the similar track with cellobiose positive band. Given the preliminary results, we further tested the content and complexity of post-reaction solution by TLC. The results showed that our hydrolysis products exhibit spots at the same horizontal position as cellobiose standard solutions, revealing that the reaction products have the component of cellobiose. According to these two results, we proved that cellobiose could indeed be used as our secondary inducer of inverter system.
Figure 6. 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
Inversion-control ability
Necessarily, we also equipped our E.coli with regulatory ability. This regulator module consists of two components, inverter system and cellobiose responsive element, which worked synergistically to switching bacterial cellulose producing event from paper cellulose degrading event, thus circumvented the futile cycle.
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1.The inverter system is a combination of two pairs of repressive elements, herein lacI-Plac and cI-Pλ. The inversion was triggered by allolacctose, which was catalyzed by lacZ. If the allolactose is absent, the lacI-Plac element won’t be turned on, indicating the absence of cI protein. The absence of cI will turn the cI-Pλ element on, and the expression of cenA, cex and hlyABD starts, thus indicating the degradation of cellulose in the first stage.
With the cellobiose accumulating gradually, the pulp cellulose degradation should be off and bacterial cellulose synthesis should be on. How to trigger this inversion? That’s why we devised the second component of the regulator module—cellobiose responsive element.
2.The cellobiose responsive element is a new biobrick we developed. It consists of a repressor protein coding sequence(chbR) and its downstream promoter(Pcel), which function as a unit to sense cellobiose concentration and respond accordingly. We inserted this unit and a lacZ gene to the cI-Pλ regulated downstream region, so the repressor(ChbR) would be constitutively expressed and bound to Pcel. Until the cellobiose concentration reached a threshold, denoting the majority of cellulose were degraded, the Pcel would be on because of cellobiose’s binding to ChbR. As a result, the lacZ expression was activated, thus triggering the inversion—cI would be expressed to inhibit cellulose degradation and acsABCD and cep94A would be expressed to perform BC synthesis.
Dimension1: Verification of cellobiose responsive element’s functionality
We constructed the chbR coding sequence and its downstream Pcel promoter together onto the pIN2 plasmid. The chbR coding sequence was point mutated to satisfy the demand of cellobiose sensing capacity. At first, we replaced the lacZ with mRFP reporter gene to prove the functionality of this element by detecting the fluorescence. The results revealed that presence of both chbR mutants resulted in a high basal level of expression. More importantly, the transformant carrying ChbRYC-NS showed an approximately threefold induction, denoting the chbR sensed the cellobiose as expected.
Figure 9. Fluorescence intensity induced by cellobiose
Dimension2: Verification of inverter system’s effectiveness
We constructed two plasmids—pIN1 and pIN2, to achieve timely inversion. By replacing the function genes with mRFP, we measured the mRFP fluorescence curve under different concentration of inducer(IPTG). The results showed that the cI expression could be successfully induced by IPTG and the cI could indeed bind to Plambda thus inhibited downstream mRFP expression.
Figure 10. 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.
Dimension3: Verification of the lacZ activity under cellobiose control
We wanted to construct the lacZ downstream the cellobiose responsive element to trigger inversion with the natural and nontoxic inducer lactose. We haven’t constructed this device yet because the construction problems we met during the initial stage. But we might finish this construction before the Giant Jamboree.
Dimension4: Verification of the regulator as a whole entity.
This is the functional combination of the inverter system and cellobiose responsive element. We are working hard on constructing and characterizing this device. We will try our best to accomplish it before Giant Jamboree.
ECUST_China
EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY
Shanghai, China
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