Team:ICJFLS/Engineering
We created some new parts.
In this virus epidemic season, we are lucky to have the opportunity to do some experiments in the laboratory. Although there is a limitation on the number of people in the lab for preventing infection of virus, we cloned some genes involved in the amylose synthesis. For creating parts, they were cloned into the plasmid pSB1C3, hoping they are useful contribution for future iGEMer.
1.BBa_K3867001
This part is ADG1 which serves as the small subunit of ADP glucose pyrophosphorylase (AGPase). The reaction catalyzed by AGPase is as flows:
AGPase catalyzes Glucose-6-phosphate (Glu-6-P) converted to adenosine diphosphate glucose (ADPG). It is a key enzyme in amylose synthesis and catalyzes the first, rate limiting step in amylose biosynthesis. The AGPase gene contains 2 subunits, ADG1 and APL1. ADG1 encodes the small subunit which is the catalytic isoform responsible for AGPase activity. And APL1 encodes the large subunit which is the large subunit playing a regulatory role in the catalytic process.
Fig.1 The sequence information of ADG1.
For construction the new part, ADG1 (1563bp) was inserted into the vector pSB1C3. PCR method was used to identify the new part, showed in Fig.2.
Fig.2 The identification result of ADG1 gene cloning. M: Marker; 1: plasmid of pSB1C3-ADG1; 2: PCR result of ADG1 gene.
When the ADG1 gene was constructed to the expression vector pETDuet-1, ADG1 protein was expressed. His tag was used to purify ADG1 protein. The identification result was showed in Fig.3.
Fig.3 The expression of ADG1 cloned in pETDuet-1-ADG1 in E. coli, M. Marker; 1. Purified ADG1; 2. The whole protein of E. coli with IPTG induction; 3. The whole protein of E. coli without IPTG induction.
2.BBa_K3867005
For construction the new part BBa_K3867005, the APL1 gene (1569bp) serving as the large subunit of AGPase, was inserted to the vector pSB1C3 by restriction enzyme (EcoRI and Pstl). Figure 4 shows the sequence information of APL1.
Fig.4 The sequence information of APL1.
The Fig.5 showed the identification result for construction of pSB1C3-APL1.
Fig.5 The identification result of APL1 gene cloning. M: Marker; 1: plasmid of pSB1C3-APL1; 2: PCR result of APL1 gene.
After the APL1 gene was constructed into the expression vector pETDuet-1-APL1, APL1 protein was expressed. His tag was used to purify APL1 protein. The identification result was showed in Fig.6.
Fig.6 The expression of APL1 cloned in pETDuet-1-APL1 in E. coli, M. Marker; 1. The whole protein of E. coli without IPTG induction; 2. Purified APL1; 3. The whole protein of E. coli with IPTG induction.
3.BBa_K3867007
The part BBa_K3867007 is about Granule-bound starch synthase 1 (GBSS1). The reaction catalyzed by GBSS1 is as follows:
GBSS1(1883bp) is another key enzyme for the amylose synthesis. It belongs to UDP-Glycosyltransferase superfamily protein. GBSS1 is an enzyme that catalyzes the transfer of glucose from to glucose-containing polysaccharides with α1,4-linkages, which is the main gene controlling amylose synthesis.
In order to construct the new part, GBSS1 (1833bp) gene was inserted into the vector pSB1C3 with restriction enzymes EcoRI and PstI.
Fig.7 The sequence information of GBSS1.
Fig.8 The identification result of GBSS1 gene cloning. M: Marker; 1: plasmid of pSB1C3-GBSS1; 2: PCR result of GBSS1 gene.
After the GBSS1 gene was constructed into the expression vector pETDuet-1-GBSS1, GBSS1 protein was expressed. His tag was used to purify GBSS1 protein. The identification result was showed in Fig.9.
Fig.9 The expression of GBSS1 cloned in pETDuet-1-GBSS1 in E. coli, M. Marker; 1. The whole protein of E. coli without IPTG induction; 2. The whole protein of E. coli with IPTG induction; 3. Purified APL1.