Overview
We learned the engineering cycle concept advocated by iGEM to implement our technical solution. We divided the main project technical part into two cycles. For the first part, we completed the expression of laccase in E. coli BL21 (DE3), and the second part we embedded the laccase on the UHWMPE membrane to achieve its function.
In the initial stage of the project, our initial design was to directly put bacteria with a suicide mechanism into the bottom of sea. However, through our experimental verification, We found that laccase would remain inside the bacteria after cultivation and not be released until we break the integrity of the E.coli. Therefore, we directly killed the bacteria to make them broken and released the extract with laccase that we needed to use.
For biological concern, we can not directly put the engineered E.coli into the sea. Besides, the flowing seawater is not a suitable environment for the enzyme to function. So we only used E. coli BL21 (DE3) as a tool to produce laccase, and embed the enzyme into the membrane for use.
Cycle #1
Target Enzyme
Laccase (multicopper oxidase family protein)
Gene symbol
AMC88_RS25540
Gene description
multicopper oxidase domain-containing protein
Locus tag
AMC88_RS25540
Gene type
protein coding
Organism
Rhizobium phaseoli (strain: R650, biovar: phaseoli, nat-host: Phaseolus vulgaris)
Lineage
Bacteria; Pseudomonadota; Alphaproteobacteria; Hyphomicrobiales; Rhizobiaceae; Rhizobium/Agrobacterium group; Rhizobium
Chassis：
BL21(DE3)
Plasmid
Suicide module
Using Seawater to kill BL21(DE3): The high osmotic pressure will kill the bacteria not living at the ocean.
Transformation：
Gene transient transfection
Build
We offer Yunzhou Biotechnology Co,Ltd to build our vector and transfer the plasmid into BL21(DE3).
Test
We entrusted Yunzhou Biotechnology Company to conduct sequencing identification and confirmed that the target plasmid we wanted was transferred into BL21 (DE3). However, for some unknown reasons, we detected the activity of laccase, and our Elisa kit could not give the concentration trend of laccase. Fortunately, the subsequent estradiol decomposition experiment verified the existence and effectiveness of the product.
Our seawater sterilization experiment failed, and the sterilization mechanism was ineffective.
Learn
1.We found that laccase basically did not come out of the bacteria, so we need to break the bacteria to release laccase.
2.For biological safety, it is best for us to only use the products of bacteria or the extracts of it after we devitalize the bacteria.
3.We need a container to contain the product to ensure laccase function at the bottom of ocean.
Cycle #2
After communication with Dr. Yuan Shijie from the HKUST(GZ), we learned that there is a high polymer material with special physical structure, invented by Professor Gao Ping, who is his professor. While achieving ultra-high strength, it allow the small molecules to pass through and trap large molecules. The existing research (about to be published) indicate that this membrane can be embedded with proteins. Therefore, we designed a prototype of the hardware combining with the UHWMPE membrane and enzyme.
Design：
The cultivation of engineering E.coli and the induction of laccase will be taken place on the membrane. After culturing for a sufficient time, laccase will deposit on the membrane and be entangled by its structure. Then, we eliminate all the bacteria on the membrane and rinse the membrane with buffer. Then the UHWMPE membrane with embedded laccase is obtained.
Build
Embedded membrane
protocol 'enzyme induction'
Test
The Elisa test of Estradiol degradation rate show the validness of our hardware.
Learn
The prototype of our hardware is proved to be valid. We should consider about appending more functions which satisfies the solution to the problem mentioned in our description.