Synthetic biology is a branch of science that combines biological science and engineering science. It uses standardized biological components and gene circuits to assemble and synthesize new biological systems with specific functions under the guidance of rational design principles. Synthetic biology emphasizes "design" and "redesign", and design-build-construct are the basis of synthetic biology.

Figure 1. Iterations of biological systems.
In the frame work of our project, we first designed the part and system of the waste paper degradation. Then, we inserted them into the microbial chassis, and finall tested the efficiency of the system. To achieve our target, we constantly iterated our system. The figure above shows all the changes we have made and the goals we want to achieve.    

Design

We plan to artificially synthesize cex, cenA and cep94A genes and insert them into E. coli plasmids. The engineering Escherichia coli was cultured in test tube for a period of time to fully produce the corresponding degrading enzymes.
We then put the broken paper in the first test tube, and then added the mixture in the test tube to the ultrasonic crushing system, which allows us to break up the E. coli and release the enzymes associated with degrading cellulose.  Finally, stir thoroughly to allow cellulolytic enzymes to fully degrade the waste paper.

Figure 2.  Illustration of Waste Paper Degradation System.

Build

We used T7 promoter to express cex and cenA genes in pET23b vector. The constructed plasmid was then transferred into Rosetta E. coli for exoglucanase production, which is used for plant cellulose decomposition. Similarly, we synthesized the cep94A gene and inserted it into the pET23b vector. The constructed plasmid was also introduced into Rosetta E. coli to produce cellobiose-phosphorylase for plant cellulose degradation.

Figure 3. Design of the cenA and cex.

Figure 4. Design of the cep94A.

Test

Figure 5. Gel electrophoresis of cex,cenA and cep94A . 

Figure 6. Experimental Results of Bacterial Cellulose Production System. (a, testing of Exoglucanase Cex.b,testing of Endoglucanase CenA.c,d,testing of Cellulose Disaccharide Phosphorylase Cep94A.e,synergistic action of cellulose-degrading enzymes )

As shown in Figures A, B, and C, three plasmids, pET23b-cex, pET23b-cenA, and pT7-cep94A, were constructed and transformed into Escherichia coli. The corresponding enzyme levels were tested, and it was observed that compared to the control group, there was a significant increase in enzyme levels.As shown in the figure 3D, E. coli Rosetta carrying an empty vector, the control group had enzymatic activities of 13.83 U/mg, and the genetic engineered E.coli Rosetta, whose plasmid is recombinant with cep94A gene had enzymatic activities of 255.20 U/mg. The results demonstrate that the enzyme activity of cellulose diphosphorylation enzyme expressed by the cep94A gene is significantly enhanced.Under the same condition, we set up several group to let the engineered E.coli to express the enzyme under different temperature. Quantitatively, our results in  figure 3Eshow that At 50℃, the cellobiose phosphorylase of engineered bacteria is about 255.2 U/mg. By changing the ambient temperature, it is shown that 37℃ is the best reaction temperature, which has the cellobiose phosphorylase about 446.99 U/mg. The results indicate that the enzyme activity of the engineered bacteria's cellulose diphosphorylation enzyme is approximately 255.2 U/mg at 50℃. By altering the environmental temperature, it was found that 37℃ is the optimal reaction temperature for the enzyme.

Learn

Our first system for degrading waste paper has been successfully verified, and our team has decided to focus on researching the application of bacterial cellulose as a filtering material. After several weeks of effort and extensive information gathering, our team has decided to use the cellulose production operon of Xanthomonas campestris pv. campestris to produce bacterial cellulose.

Design

Through scientific literature's studies, we found that bacterial cellulose (BC) produced by microbial cells mainly is an ultrafine network that possesses unique structural, which has a variety of applications in various fields, including water purification. While, among bacteria, Komagataeibacter xylinum are the only primary producer of bacterial cellulose that exist in the nature. Its cellulose synthesis operon has the ability to produce bacteria cellulose. The operen mainly uses enzymes expressed by acsAB, acsC and acsD to conduct BC producing.

Build

The acsAB gene encoding cellulose synthase was synthesized and cloned into pET23b vector, and introduced the constructed plasmid into Escherichia coli Rosetta for bacterial cellulose production.

Figure 7. Design of the acsAB.

Test

We expressed the bacterial cellulose synthase with E.coli Rosetta. We broke them with the ultrasonic wave. Then, we added NaOH into the to dissolves the non-cellulosic components and precipitates the cellulose. After removing the remaining NaOH, we weighed the dried cellulose to determine the cellulose content.

Figure 8.   Gel electrophoresis of acsAB . 

Figure 9.  Cellulose synthesis capacity test of AcsAB.
As the picture shown, in bacterial precipitation samples, engineered E. coli expressing AcsAB produced about 1.71g/L bacterial cellulose in LB medium. But in the culture medium samples, there was almost no detectable presence of cellulose. The results indicate that our acsAB gene's ability to express bacterial cellulose synthase is significantly improved.

Learn

We found that the preparation for the material and food requirement of the cellulose production has a obvious effect on effeciency of cellulose production.

Design

After asking an expert in the field of wastewater purification for his opinion, We decided to add a filtration system before the products from the plant cellulose decomposition system entered the system.

Build

According to the expert's opinion, we used the bacterial cellulose produced in the bacterial cellulose producing system for filtration. Bacterial cellulose can participate in the production of nanocomposites - bacterial cellulose film, which can filter out biological and macromolecular materials. This process is also self-produced and self-sold.

Test

1. Because our research is still going on, we haven't put this idea into practice yet. In the future, we will continue to do the research and try to develop products that can actually be used.

2. Sekar, Ramanan, Hyun-Dong Shin, and Rachel Chen. "Engineering Escherichia coli cells for cellobiose assimilation through a phosphorolytic mechanism." Applied and environmental microbiology 78.5 (2012): 1611-1614.
Lakhundi, Sahreena Saleem. "Synthetic biology approach to cellulose degradation." (2012).
3. Wood, Thomas M., and K. Mahalingeshwara Bhat. "Methods for measuring cellulase activities." Methods in enzymology. Vol. 160. Academic Press, 1988. 87-112.