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Wet Lab

Overview

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Chassis

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Fungal Toolkit

(http://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit)

Regulatory

Elements

(http://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/regulatory_elements)

Selectable

Markers

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Characterization

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Homologous

Constructs

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Biosafety

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Protoplasting

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Carotenoids

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Limonene

(http://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/limonene)

Antifungals

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BioBricks

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Animation

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Future Work

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Wetlab Overview

We are working with the fungus Ganoderma lucidum, a model organism for the class of wood-

rot fungi that is used in Ecovative’s products. We have assembled a number of constructs for

the production of carotenoid pigmentation, fluorescent proteins, and antibiotic resistances that

we are seeking to transform by both random insertion and homologous recombination into the

organism’s genome. These proof-of-concept tools are necessary to optimize our process so

that we can then obtain rigorous, reliable characterization data for the antifungal gene we are

seeking to introduce, a protein that specifically targets Aspergillus niger.

However, as Ganoderma does not have a well-standardized transformation protocol and takes

several days to grow to an appropriate density, we are also conducting much of our basic fungal

characterization work in Cochliobolus heterostrophus, a simpler fungus. In addition, we are

seeking to introduce a novel T7 viral regulation system into fungi. Similar systems have been

used successfully in mammalian cells, and this system would greatly expand the accessibility

of fungal genetic engineering beyond experienced mycologists. This system allows us to

conduct preliminary characterization within E. coli BL21-A1, a much simpler system for

acquiring quantitative data.

We are also implementing a number of biosafety measures within our toolkit, including a

recombination system that has previously been demonstrated to work in simpler fungi, which

will allow us to remove antibiotic resistance genes and targeted antifungal compounds before

the end of the production process, in order to prevent them from spreading into the

environment. The strain we are seeking to develop would be a huge boon to the economic

viability of Ecovative’s sustainable biomaterials, as it would allow them to more easily maintain

the high levels of quality control that are necessary in scaled-up production.

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