Team:Cornell/project/wetlab/chassis

Chassis

Escherichia coli BL21-A1

We are also using BL21 competent strains of E. coli, which are designed for high-efficiency protein expression of any gene under control of the T7 bacteriophage promoter system. T7 RNA Polymerase is regulated by arabinose induction and glucose inhibition of the araBAD promoter in its chromosomal DNA. Proteins under control of T7 promoter driven vectors are then expressed at high levels. The cells also contain the T7 lysozyme gene within the pLysS plasmid, which suppresses T7 RNA polymerase activity caused by leaky basal expression [2].

Cochliobolus heterostrophus

Cochliobolus heterostrophus is an ascomycete fungal species widely studied for its pathogenic traits as well as its ability to uptake recombinant DNA. Typical methods to integrate recombinant DNA into Cochliobolus include flanking the gene of interest with homologous regions to the fungal genome, restriction enzyme mediated integration (REMI), and random insertion. Typically using homologous recombination offers the highest efficiency while random insertion offers the lowest. The fungal strain is transformed by protoplasting followed by PEG transformation [3].

Ganoderma lucidum

Ganoderma lucidum is a wood decaying basidiomycete fungus known for its medicinal properties and pharmacological applications [4, 5]. Inspired by Ecovative Design’s fungal materials, we selected G. lucidum as one of our fungal chassis for expression of our modular genetic circuit for a multitude of reasons [6]. Firstly, G. lucidum displays a trimitic hyphal physiology of generative, skeletal, and binding hyphae, which endow the fungus with a tough consistency ideal for material applications such as Ecovative’s mushroom packaging [4, 6]. Specifically, G. lucidum's binding hyphae justify Ecovative’s manufacturing methodology: the hyphal fibers incorporate agricultural waste in the mycelium as the fungus grows, rendering a sustainable, biodegradable material after the fungus is heat killed [6]. Thus, we designed our constructs to transform into G. lucidum for practical applications in industry, such as the prevention of product contamination during fungal material manufacturing. Secondly, G. lucidum is among the better genetically characterized fungi, which was useful in determining which homologous genetic parts from other fungi, such as promoters, would be suitable for our fungal genetic toolkit [7, 8]. Additionally, we were able to accurately extract, amplify, and incorporate G. lucidum DNA, specifically a native fungal promoter, using the genome sequence to design primers for PCR. This was important in incorporating fungal DNA into our constructs for efficient expression in G. lucidum. Lastly, there are several documented protocols for transforming G. lucidum, which we readily employed when introducing our constructs into G. lucidum. These included the protoplasting of G. lucidum cells and restriction enzyme mediated integration of our constructs for more efficient electroporation [9, 10].

References

1. Dh5-Alpha Competent E. coli. (2013). Accessed from http://www.mclab.com/Dh5-Alpha-Competent-E.-Coli.html

2. Competent Cell BL21(DE3)pLysS. (2013). Accessed from http://www.biodynamics.co.jp/images/prd_ds250/DS260BLysShp.pdf

3. Cochliobolus heterostrophus. (2013). Accessed from http://genome.jgi-psf.org/CocheC5_1/CocheC5_1.home.html

4. Thakur, R. (2013). Studies on Genetic Variability in Ganoderma lucidum (Curtis) P. Karst. for Identification of Elite Strains. Doctor of Philosophy Thesis, CSK Himachal Pradesh Agricultural University, Palampur, Himachal Pradesh, India.

5. Ganoderma spp. (2013). Accessed from http://genome.jgi-psf.org/Gansp1/Gansp1.home.html

6. Mushroom Materials. (2013). Accessed from http://ecovativedesign.com/mushroom-materials/

7. Chen, S. et al (2012). Genome sequence of the model medicinal mushroom Ganoderma lucidum. Nature Communications, 3, 913. doi: 10.1038/ncomms1923

8. Yu, G. et al (2012). Deep Insight into the Ganoderma lucidum by Comprehensive Analysis of Its Transcriptome. PLoS One, 7(8), e44031. doi: 10.1371/journal.pone.0044031

9. Sun, L. et al (2001). Efficient Transformation of the Medicinal Mushroom Ganoderma lucidum. Plant Molecular Biology Reporter, 19, 383a-383j.

10. Kim, S., Song, J., & Choi, H. T. (2004). Genetic transformation and mutant isolation in Ganoderma lucidum by restriction enzyme-mediated integration. FEMS Microbiology Letters, 233(2), 201-204. doi: 10.1016/j.femsle.2004.02.010