In order to carry forward the spirit of iGEM, and inherit and spread the value of iGEM, we specially searched the iGEM Biological Parts library for luciferase proteins and picked BBa_K1159001 the Firefly luciferase and the Renilla luciferase. This is a biological part submitted by iGEM13_TU-Munich in 2013 to introduce a new member of the luciferase reporter gene/protein family. Our team carried out a controllable gene expression system for this part in the laboratory, adding data from regulating gene expression in mammalian cells. This information can be a good reference for future iGEM teams working on gene expression regulation.
In addition, through literature research, we found that we can regulate gene expression through the hFoxO1 gene, which is a family of transcription factors.
Besides our contribution to the Parts library, we also have another contribution from the dry lab on the side of business plan drafting for future iGEMers.
Last but not least, we have accumulated rich experience in writing business plans. To exert the value of inheritance, we have specially produced a guide for writing Business Plan Guidance <Structure and Insights> for future teams to refer to.
Optical in vivo imaging is widely used in biotechnology. There are two common luciferase enzymes: Firefly luciferase and Renilla luciferase. Firefly luciferase is the light-emitting enzyme responsible for the bioluminescence of fireflies and click beetles, and its substrate is D-Luciferin. While Renilla luciferase’s substrate is Coelenterazine. Both the enzymes require oxygen and ATP, and they are always used as reporter genes in experiments.
In our project, we choose these two luciferases as our reporter gene to verify the expression of hFoxO1 protein in HepG2 cells.
In order to construct a FoxO1 expression plasmid that can shuttle both in E.coli and HepG2 cells, we designed the DNA sequences of hFoxO1 to be inserted into the XhoI and KpnI sites of the pcDNA3.1 vector (Fig.1), and transfected the HepG2 cells with the recombinant plasmid to set up our experiment platform.
Fig.1 The map of recombinant plasmid pcDNA3.1-hFoxO1
To test if hFoxO1 protein can be used to regulate both Firefly luciferase and the Renilla luciferase expression in HepG2 cells, we cloned the luciferase into two different plasmids and named pGL3-3IRE and pRL-SV40 (Fig.2). We transfected HepG2 cells with two plasmids containing hFoxO1 and luciferase. 12 hours after transfection, we detected the luciferase activity and collected data.
Fig.2 Plasmid profiles (A) pGL3-3IRE plasmid (B) pRL-sv40 plasmid
As shown in Fig.3, “+” represents the presence of the corresponding plasmid (or transcriptional regulation) in the sample, and “-” represents the negative control. The results indicated that luciferase activity was only affected by pcDNA3.1 plasmid and hFoxO1 transcription regulator.
Fig.3 hFoxO1 transcriptional activation platform test results
We not only provided experimental data for both Firefly luciferase and the Renilla luciferase as reporter genes, but also supplied several new parts as follows.
Forkhead box protein (Fox) is a family of transcription factors. The DNA binding region of this family of proteins has a conserved winglike helix structure. There are currently 17 subfamilies in this family, of which the FoxO subfamily is the most well studied. There are four subtypes: FoxO1, FoxO3, FoxO4, and FoxO6 in mammals. FoxO1 has four domains, which are DNA-binding domain, nuclear localization domain, nuclear export sequence, and transcriptional activation domain. It binds with IRE sequence and plays a role in regulating downstream genes. FoxO1 is mainly expressed in insulin-responsive tissues, The main role of FoxO1 is to regulate downstream target genes, such as PEPCK, G6Pase, PGC1-α, and PDK-4 to promote gluconeogenesis and can regulate cell proliferation, gluconeogenesis, and energy metabolism.
pcDNA3.1-backbone is one of the most commonly used mammalian expression vectors, which use a strong CMV promoter to regulate the expression of exogenous genes. The vector is a high-copy-number plasmid. When expressed in the prokaryotic system, the Amp+ resistance can be used to screen the right colony, while in the eukaryotic system, Neo+ resistance can be used for screening. This plasmid backbone can be used to express different proteins in the future.
pGL3pro-backbone is a high-copy-number luciferase reporter system carrier, which uses an SV40 promoter to regulate the expression of luciferase genes, and it can be used for rapid and quantitative assessment of factors that affect the expression of specific genes in mammalian cells. When expressed in the prokaryotic system, the Amp+ resistance can be used to screen the right colony.
These new composite parts are important for both our project and future projects.
We have accumulated rich experience in writing the business plan as the method to present our product and the concept behind the final end product. Therefore, not only does our business plan itself acts as an example for future teams but also we made the business plan writing guidance <Structure and Insights> for reference. This 50-page guidance book includes the structure and key ideas that a business plan should present to the audience. Because we know how our precious experience of writing the business plan can help future iGEM participants, we want our comprehensive business writing key guidance can act as the starter helper to the teams to allow their flow of creativity and imagination.
