ABSTRACT
In order to build an "Internet +" innovation and entrepreneurship education practice platform, 3,752 undergraduates from 5 medical colleges and universities were investigated by questionnaire. The results showed that there was a gap between the expectation of the students and the setting of entrepreneurship and innovation courses, project guidance and so on. In view of the contradiction between the supply of educational resources and the needs of students in medical colleges and universities, we have developed the "Internet +" innovation and entrepreneurship education practice platform, that including five main functions and three layers of framework based on cloud computing and open source technology. The platform integrates various resources of medical colleges and universities to make useful exploration for cultivating students' innovation and entrepreneurship ability, accelerating the implementation of projects, and promoting the connotation development of innovation.
ABSTRACT
We constructed inducible and constitutive heterotrophic expression vectors of Dunaliella salina (D. salina) and identified heterotrophic transformants. A gene encoding a glucose transporter (Glut1) was cloned from human placenta tissues by RT-PCR and sequenced. Inducible heterotrophic expression vector pMDDGN-Bar of D. salina, which included a duplicated carbonic anhydrase (DCA) promotor and a Bar selectable marker that could drive expression of the Glut1 gene in D. salina, was constructed by molecular biology methods. In addition, we constructed another vector G5Glut1-Bar that contained a constitutive ubiquitin promotor, Glut1 and Bar box. The two expression vectors were introduced into D. salina by electroporation method, and then screened the transformants with phosphinothricin (PPT). Total RNA of the transformants extracted was used to analyze the integration of the target gene (Glut1) by RT-PCR. The cloned Glut1 sequence was 1479 bp and encoded 493 amino acids. The results of all enzymes digesting showed that two expression vectors were successfully constructed. After screening by PPT for several weeks, the transfomants grew well whereas wild-type cells died completely. The result of RT-PCR indicated that two transformants both had an about 250 bp specific band and the sequence homology was 100% compared with the human Glut1 sequence by Blast analysis. Taken altogether, inducible and constitutive heterotrophic expression vectors of D. salina was constructed successfully and the Glut1 gene was integrated into the genome of D. salina. Expression vectors above-mentioned may be used for the expression of the foreign Glut1 gene in D. salina.
Subject(s)
Base Sequence , Chlorophyta , Genetics , Metabolism , Cloning, Molecular , Electroporation , Genetic Vectors , Genetics , Glucose Transporter Type 1 , Genetics , Industrial Microbiology , Molecular Sequence Data , Transformation, GeneticABSTRACT
To optimize the electroporation system in Dunaliella salina (D. salina), we studied the effects of growth phase of cells, electric parameters, electroporation buffer and concentration of plasmid on transformation efficiency. The results showed that a transformation efficiency of 1.81 per thousand was achieved in D. salina cells at mid-log growth phase electroporated with plasmid (DCA-bar) 10 microg/mL, voltage 0.8 kV and capacitance 25 microF. However, when glycerol was added to electroporation buffer at a final concentration of 0.4 mol/L, the transformation efficiency was increased up to 2.03 per thousand. Additionally, transformation was done with plasmids DCA-bar, NR-bar, pUomega-bar respectively, under above optimum conditions, and similar transformation efficiencies were obtained. The findings indicate that an efficient and stable system of electroporation in D. salina has been developed, providing a powerful tool for the transgenic research of D. salina.