Team:Cornell/project/wetlab/future work

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For practical application to fungal biomaterial manufacturing, our future work will center on successfully transforming Ganoderma lucidum via agrobacterium mediated transformation, a protocol that has been well documented in literature<sup>1</sup>, due to our failure to successfully transform by protoplasting. In addition, we aim to design new Gibson constructs to improve the transformation efficiency of Cochliobolus heterostrophus. These new constructs will depend on homologous recombination for integration into the genome instead of random insertion. Some constructs will combine fluorescence markers as well as resistance markers to aid in selection. Expression stability in the C. heterostrophus will be evaluated using an alternating selection system where transformants are plated on a non-selective plate and subsequently plated on a selective plate; transformants with successfully and stably integrated constructs would survive the resistance selection and also fluoresce. Evaluating the site of construct integration would involve using a fluorescent in situ hybridization<sup>2</sup>.
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While we have already made great strides towards improving the accessibility of fungal genetic engineering to industry, academia, and other iGEM teams, we still strive to expand the utility of our toolkit through additional characterization and troubleshooting. In order to apply our work specifically to biomaterials development, we have constructed numerous vectors and are working to perfect a protocol for <i>Agrobacterium tumefaciens</i>-mediated transformation into the <i>Ganoderma lucidum</i> genome [1].  We are also working to incorporate all of our pre-existing fungal expression constructs into <i>Cochliobolus heterostrophus</i>-specific vectors that allow for homologous recombination into the fungal genome. Expression stability will be tested by alternate plating on selective and non-selective plates and maintained by constant selection. Our goal is to test and compare the expression of each of our constructs in <i>Cochliobolus</i> and <i>Ganoderma</i> to provide insight into the expression compatibility of a wide breadth of fungal chassis.
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We also plan on acquiring superior fluorescence data and quantifying promoter strengths on a rating scale, similar to that of the Anderson promoters available in the iGEM parts registry, with the specific goal of utilizing the pelA promoter as a controlled, inducible activator of the kill switch system. To further biosafety, we will work on constructs to implement the Cre-lox system. The resistances that are successfully cloned into C. heterostrophus and G. lucidum can then be restricted from the genome, preventing horizontal gene transfer between fungi, thus preventing wild fungi from expressing fungal resistances.  
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All of our genetic parts will be integrated into Ecovative’s manufacturing pipeline for the modification and creation of the novel biomaterial, furthering the push to a greener, more sustainable society.
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<b><center>References</center></b>
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<sup>1</sup>Shi, Liang et al. (2012). Development of a simple and efficient transformation system for the basidiomycetous medicinal fungi Ganoderma lucidum. World J Microbiol Biotechnol 28:283-291. doi: 10.1007/s11274-011-0818-z
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<br><sup>2</sup>Salvo-Garrido, H. et al. (2004). The Distribution of Transgene Integration Sites in Barley Determined by Physical and Genetic Mapping. Genetics, 167, 1371-1379. doi: 10.1534/genetics.103.023747
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We also plan on acquiring additional fluorescence data in order to quantify the relative strengths of the constitutive promoters in our library on a rating scale, similar to that of the Anderson promoter collection available in the iGEM parts registry [2]. We also specifically hope to use the glucose-repressible, polygalacturonic acid-induced <i>pelA</i> promoter as a controlled, inducible activator of the <a href = "https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety"> kill-switch and site-specific recombination systems</a>. Furthermore, we are in the process of characterizing a promoter and terminator from Wageningen University’s iGEM team.
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The foundations that we have laid for fungal genetic engineering, and are continually striving to improve, provide a platform for other researchers to apply these tools to a wide variety of sustainable industries.
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1. Shi, Liang et al. (2012). Development of a simple and efficient transformation system for the basidiomycetous medicinal fungi Ganoderma lucidum. <i>World J Microbiol Biotechnol</i> <i>28</i>, 283-291. doi: 10.1007/s11274-011-0818-z
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2. Promoters/Catalog/Anderson. iGEM Registry of Standard Biological Parts. Accessed from <a href = "http://parts.igem.org/Promoters/Catalog/Anderson" target="_blank"> http://parts.igem.org/Promoters/Catalog/Anderson </a>
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Latest revision as of 00:57, 29 October 2013

Cornell University Genetically Engineered Machines

Future Work


While we have already made great strides towards improving the accessibility of fungal genetic engineering to industry, academia, and other iGEM teams, we still strive to expand the utility of our toolkit through additional characterization and troubleshooting. In order to apply our work specifically to biomaterials development, we have constructed numerous vectors and are working to perfect a protocol for Agrobacterium tumefaciens-mediated transformation into the Ganoderma lucidum genome [1]. We are also working to incorporate all of our pre-existing fungal expression constructs into Cochliobolus heterostrophus-specific vectors that allow for homologous recombination into the fungal genome. Expression stability will be tested by alternate plating on selective and non-selective plates and maintained by constant selection. Our goal is to test and compare the expression of each of our constructs in Cochliobolus and Ganoderma to provide insight into the expression compatibility of a wide breadth of fungal chassis.

We also plan on acquiring additional fluorescence data in order to quantify the relative strengths of the constitutive promoters in our library on a rating scale, similar to that of the Anderson promoter collection available in the iGEM parts registry [2]. We also specifically hope to use the glucose-repressible, polygalacturonic acid-induced pelA promoter as a controlled, inducible activator of the kill-switch and site-specific recombination systems. Furthermore, we are in the process of characterizing a promoter and terminator from Wageningen University’s iGEM team.

The foundations that we have laid for fungal genetic engineering, and are continually striving to improve, provide a platform for other researchers to apply these tools to a wide variety of sustainable industries.

References

1. Shi, Liang et al. (2012). Development of a simple and efficient transformation system for the basidiomycetous medicinal fungi Ganoderma lucidum. World J Microbiol Biotechnol 28, 283-291. doi: 10.1007/s11274-011-0818-z

2. Promoters/Catalog/Anderson. iGEM Registry of Standard Biological Parts. Accessed from http://parts.igem.org/Promoters/Catalog/Anderson

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