Team:Cornell/project/wetlab/fungal toolkit/regulatory elements

In designing our genetic circuit, we identified several desired properties: 1) industrialized production of biomolecules (for the augmentation of fungal characteristics), 2) continuous expression in fungi, and 3) modularity. Thus, regulatory elements were determined to be an essential component for the controlled expression of genes we transformed into fungi. As part of our fungal toolkit, we developed a standardized registry of promoters that could interchangeably be used in our genetic circuit.

The T7 promoter was identified as an ideal promoter to meet goal 1 due to its high affinity for T7 RNA polymerase (RNAP) and thus high levels of expression of downstream genes. However, the T7 RNAP consequently consumes resources heavily (i.e rNTPs), which can even stop growth in E. coli [1]. Therefore, we designed our genetic circuit to regulate the expression of T7 RNAP in our fungal chassis. Constitutive fungal promoters were selected to allow for continuous but regulated expression of T7 polymerase, thus meeting goals 1 and 2.

Our genetic circuit is comprised of two parts. The first consists of an interchangeable native fungal promoter upstream of the T7 polymerase gene; the second consists of the T7 promoter upstream of an interchangeable gene we would like to express in fungi. Thus, T7 polymerase is produced in a regulated manner for the strong expression of various genes under the control of the T7 promoter.

To make our genetic circuit modular, we built our parts registry so that our promoters could be cloned into the promoter slot and our genes (i.e. resistances) could be cloned into the gene slot by using the standardized BioBrick restriction system. Thus, if a gene needs to be expressed in fungi for a desired characteristic, it can easily be incorporated and the promoter tailored for a specific chassis, meeting goal 3.

Since the use of homologous promoters can enhance expression levels, we selected two homologous glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoters for expression in Cochliobolus, specifically the Aspergillus nidulans gpdA promoter and the Ganoderma lucidum gpdA promoter. The gpdA promoters have been previously characterized in producing industrial enzymes so we predicted they could be effectively utilized in our promoter library and in several regulatory constructs. The A. nidulans trpC promoter (PtrpC) and trpC terminator (TtrpC) were also utilized for strong homologous and heterologous gene expression [2]. The Anderson promoter (Pc) and the A. nidulans pelA promoter (PpelA) were selected for characterization of certain constructs, which is currently still in progress. Pc is a highly constitutive promoter and, in place of a fungal promoter, could allow for construct expression and characterization in bacteria [3]. PpelA is induced by polygalacturonic acid and repressed in the presence of preferred carbon sources, such as glucose, which makes it appropriate for utilization in our kill switch constructs (designed to be inducible and repressible for practical use during manufacturing processes) [4].

A multitude of laboratory techniques were employed to generate our constructs, including PCR, DNA digestion, gel extraction, DNA ligation, and electroporation.