Team:ICJFLS/Proof Of Concept
The plastic straw is widely used in food industry, which causes seriously environmental pollution, even threatens animal life in the world. Please see our project description:
Under the plastic restriction ban in China, and with the increasing awareness of environmental protection, people begin to use paper straw and PLA straw instead of plastic straw. However, both of them have some defects. After we performed social survey and searched some literature, we know that the disposable products made of amylose and cellulose have higher social value, but most of them come from food crops. Obviously, it's not an economic way to produce billions of amylose straw using food crops, because some countries and regions in the world are still in hungry and poverty.
1. Our project aimed to synthesize amylose in E. coli for the disposable amylose straw production
At present, the research reports of amylose mainly focus on increasing the percentage of amylose in crops and the extraction methods from different crops, as well as some application research. Based on our knowledge, there is no report that amylose is synthesized in E. coli.
Our project aimed to clone the genes related to the amylose synthesis in plant, and transform them to the engineered E. coli, which fermentation conditions are optimized, preservation is convenient, and the industrial production is facilitated to be realized. Hoping the synthesized amylose in E. coli could be used to make billions of straws.
2. We have cloned 3 genes successfully from Arabidopsis thaliana, and transformed them into the E. coli
In order to synthesize amylose in E. coli, we searched some literature to understand the synthesis pathway of amylose in plants. Based on the synthesis pathway of model organism Arabidopsis thaliana, we identified two key synthases, AGPase and GBSS1. AGPase contains two subunits, ADG1 and APL1, which are synthesized respectively and constructed into a bi-functional expression vector pETDuet. Another synthase, GBSS1 gene, was constructed to another expression vector pET-28 (+). The two expression vectors were co-transformed into E. coli. Restriction enzyme digestion and PCR method were used to identify the construction of recombinant plasmids. SDS-PAGE electrophoresis showed that ADG1, APL1, and GBSS1 were successfully expressed in E. coli. The synthesis pathway of amylose was constructed successfully in E. coli.
To avoid repetition, please refer to our results:
3. The E. coli transformed with genes related to the synthesis of amylose works successfully
After completion gene cloning work, the transformed E. coli were cultured in the medium for optimizing conditions, which are used for yielding amylose. With the help of math model work, we got the best culture conditions of transformed E. coli. They were cultured for 22h at 37°C, with pH7.6 and 14% inoculums size, and glucose serving as the carbon source. Then the amylose synthesized in E. coli was harvested. The result is showed in the following figure.
Glu, Glucose; Man, Mannitol; Suc, Sucrose; Fru, Fructose; GluA, glucosamine; 2-DOG, 2-deoxyglucose; 3-OMG, 3-O-methylglucose.
4. Summary:
Our experimental results showed that the amylose was synthesized successfully in the transformed E. coli, indicating that the parts transferred to the E. coli worked well. The results also showed that the production yield of amylose is not high, suggesting that the culture conditions still need optimization.