Nights in the lab #1

These posts will highlight progress on various experiments at the Great Lakes Biotech Academy.  One of my key goals with the biotech academy is to reduce the costs associated with performing modern biotechnology research so that more people can participate.  At the heart of biotechnology is the identification and manipulation of functional DNA.  Functional DNA could be DNA that encodes for an enzyme,  a taxonomic marker, or any other sequence that does something.  All living things contain functional DNA -- the challenge is finding that functional DNA and manipulating it to do something useful.  

As highlighted in the April post, fungi are widely used in modern biotechnology.  At the Great Lakes Biotech Academy our focus is principally on white biotechnology and fungal biology.  Like plants and animals, fungi are eukaryotic organisms, however they have much more compact genomes.  A typical fungal genome is approximately 40 megabases and encodes about 10,000 genes.  That's about 100 times smaller than the human genome with only three times fewer genes.  Fungi, especially filamentous ascomycetes, have very little repetitive DNA which makes genome sequencing and assembly relatively easy and straightforward compared to plants and animals.  To identify functional DNA in fungi we need to have access to the DNA from numerous fungal species.  

This is a photo of a stream behind my house where I have collected several samples.  I've isolated ~50-100 species of filamentous fungi from my backyard and the forest behind my house.  

This is a photo of a stream behind my house where I have collected several samples.  I've isolated ~50-100 species of filamentous fungi from my backyard and the forest behind my house.  

I am gathering a large collection of fungal species from the local environment.  I will use these species to isolate diverse fungal DNA for studies at the biotech academy.  All of the isolations are being performed using a minimal medium, Vogel's salts supplemented with corn syrup.  The basic protocol is as follows: (1) homogenize the sample in distilled water with a blender, (2) spread the sample on the minimal medium agar supplemented with three antibiotics to prevent bacterial growth, (3) transfer any unique looking fungal colony to a fresh plate, (4) grow the fungus in liquid culture, and (5) perform PCR to determine what the fungal species is.  See the images below for some examples of how this looks.

DNA analysis in fungi is more challenging than with some bacterial species because fungi have very strong cell walls.  The fungal cell wall is made of chitin, the same tough polymer found in the exoskeletons of insects.  To break the fungal cell wall requires mechanical grinding.  This is typically done by grinding liquid nitrogen cooled fungus with a mortar and pestle or using an expensive piece of lab equipment called a bead-beater.  We have been working to develop a low cost substitute for a bead-beater and have made a lot of progress.  I will highlight the development of the fungal cell homogenizer in another post.  Once I have the thick growth of fungus in a liquid culture, I press out all of the liquid to form a fungal patty (kind of like making a hamburger or meatball, but out of fungus) and store the material at -20C.  Within each of these fungal patties is enough genomic DNA to sequence the whole genome and have plenty left over for thousands of functional DNA isolations using the polymerase chain reaction.  I used our homogenizer to break the fungal cell walls, isolate the DNA, and perform a PCR reaction.  Next step is to get the PCR amplicon sequenced so I can figure out what these species are.  See the pictures below to get an idea of what happens in the process.