Engineering

Design 1

After studying the existing technology, we found that the technology used in the market today has problems such as pollution and low efficiency. We eventually found that using transgenic technology could be a possible way. Basically, chitosanase genes bind to the genes of E. coli to form a new plasmid and are expressed inside E. coli cells.

Build 1

In the first attempt to use transgenic technology. After we obtained a plasmid with a chitosanase gene, we put it into E. coli Dh5alpha and multiplied it, and finally successfully demonstrated chitosanase in cells.

Test 1

Although we were able to successfully express chitosanase. In terms of the yield of chitosan oligosaccharides, we found that chitosan oligosaccharide preparation efficiency was extremely inefficient.

Learn 1 Learning and Optimization

Based on the results of the first test of our design, we realized that there could be a more productive way to produce chitosan oligosaccharides. In the first round of experiments, we transferred the chitosanase gene into E. coli and found that the protein expressed by it could only be in the cell, and this makes the transmembrane transport of chitosan necessary to break down chitosan, which greatly reduced the production efficiency of the enzyme. We realized that the problem was with the transmembrane transport of chitosan, and perhaps we could find a way to break down chitosan without crossing the cell membrane. In the second cycle of the experiment, following the professor's suggestion, we introduced the N-terminal gene of the ice crystal nucleoprotein, and improved our experiment to achieve this purpose.

Design 2

After receiving expert advice, we conducted a second trial. The chitosanase gene was extracted, but the difference is that we fused the chitosanase gene with the N-terminal gene of the INP (Ice Nucleation Protein) in advance to become our target gene. We synthesize the gene of interest with the gene of E. coli to form a new plasmid and re-insert it into E. coli Dh5alpha, at which time the protein expressed by the N-terminal gene of the INP will be anchored in the cell membrane for fixation. In addition, chitosanase is expressed on the cell surface, which successfully eliminates the tedious step of chitosan transmembrane into the cell.

Build 2

In order to make chitosanase work better, we display chitosanase on the cell surface through cell surface display technology. The INP gene fragment in the newly synthesized plasmid after successful expression allows chitosanase to express and function outside the cell membrane. Furthermore, breaking down the exposed chitosan into chitosan oligosaccharides.

Test 2

First, we compared the crude chitosanase and detected the effect of cell surface presentation technology on maintaining the stability of the enzyme, as shown in Figure A, after 40 days of placement, the engineered bacteria still maintained 80% of the initial enzyme activity, while the crude chitosan only maintained 31% of the initial enzyme activity after 40 days at room temperature. Therefore, it can be seen that the cell surface display technology can maintain and enhance the activity of chitosanase.

Furthermore, we further compare the amount of chitosan produced by engineered bacteria and crude chitosan enzymes with substrate hydrolysis. As shown in Figure B, it can be seen that within day 1-7, the hydrolysis capacity of engineered bacteria maintains a good stability, but the crude enzyme solution is almost inactivated after a day of interaction with the substrate and cannot continue to hydrolyze the substrate. It can be seen that engineering bacteria can work more stably.

Learn 2

Using cell surface presentation technology, the efficiency of our preparation of chitosan oligosaccharides has been greatly improved. This contributes to our next goal of industrializing the project. While the technology has been improved, we are also thinking about how to further improve the efficiency of production and control the yield constant. For example, we may make modifications to existing engineered bacteria to make them more tolerant of different conditions and make them more usable.