Team:Cornell/project/wetlab/future work

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, due to our inability to successfully transform by protoplasting [1]. 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 sites of construct integration would involve using fluorescent in situ hybridization [2].

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 [3]. To further biosafety, we will work on constructs to implement the Cre-lox recombination 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. Furthermore, we are in the process of collaborating with Wageningen University's Genetically Engineered Machines Team, which is sending us a promoter, terminator, and actin-GFP fusion protein for characterization and incorporation into our fungal toolkit.

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.