YIQ Visit

On June 27th a group of students participating in the Young Innovator's Quest visited the biotech academy.  We spent some time talking about functional DNA and the future of biotechnology.  We then performed experiments to determine the genus and species of several non-hazardous mushrooms.  

To determine the species of mushroom, we performed PCR reactions on genomic DNA isolated from each mushroom at the biotech academy.

Lane 1, specimen #1; Lane 2, specimen #2; Lane 3, specimen #3; Lane 4, specimen #4; Lane 5, Ladder; Lane 6, specimen #5; Lane 7, specimen #6.

Lane 1, specimen #1; Lane 2, specimen #2; Lane 3, specimen #3; Lane 4, specimen #4; Lane 5, Ladder; Lane 6, specimen #5; Lane 7, specimen #6.

Participants got to choose some of the parameters in the PCR reaction, including the primer concentration, the genomic DNA template concentration, and the PCR program.  The best looking amplicon for each specimen will be sent for sanger sequencing and used to identify the genus and species later this week.

The three PCR programs used for the mushroom amplification.

The three PCR programs used for the mushroom amplification.

Most of the PCR reactions turned out nicely.  The most intense band for each specimen will be sent for sequencing. 

Making and storing competent cells on a budget

All modern biotechnology labs need to make and test many different functional DNA molecules, but DNA in a test tube is little more than a heterogeneous water soluble polymer.  To unlock the power of functional DNA it needs to be inserted into a living cell.  Once the DNA is inside of a cell the molecular machinery of the cell decodes the information stored in the DNA and remarkable things can happen.  

These are agar plates with a selectable marker called ampicillin.  These plates can be used to identify E. coli cells that have taken up functional DNA.

The process of inserting DNA into a cell is referred to as transformation or transfection.  Most cells can be treated so they uptake some DNA, but only a few organisms, like yeast and E. coli, are especially well suited for DNA uptake.  At the Great Lakes Biotech Academy we are developing efficient ways to assemble and characterize functional DNA at a very low cost.  We have made significant progress on the DNA assembly portion (check for a future article on this topic), but we also need new low cost procedures for preparing and storing cells that can uptake DNA.  

These cells are blue because they were transformed with functional DNA that allows the cells to change color. 

Cells that take up DNA efficiently are called competent cells.  Typically competent cells are evaluated based on a metric called transformation efficiency.  Competent cells produced by commercial vendors have transformation efficiencies in the range of 10^8 or 10^9 colony forming units per microgram of DNA.  Translated to common English, that means if you mix DNA of a mass 1/100th the mass of a grain of salt with these commercial cells you will have approximately 1 billion cells uptake the DNA.  These commercial cells are a tremendous asset for biotech labs, but they are also very expensive.  Each use of competent cells from a typical vendor costs >$20.  The process of making cells competent involves treatments that make the cells very fragile and considerable care must be taken when handling them.  Because the cells are so sensitive they must be stored in special lab freezers that are kept at -80 degrees Celsius or lower (-112 Fahrenheit!).  The least expensive of these -80C freezers costs several thousand dollars.  This is way too expensive.

To fix this problem I performed a few simple experiments.  In the first experiment, I evaluated whether or not commercial vendor competent cells could be stored in a standard freezer at only -20 Celsius.  Unfortunately, the transformation efficiency of the cells was severely compromised by storage at this temperature.  Buying a $10,000 freezer was out of the question, so I next performed an experiment where I measured dry ice sublimation rates inside of a styrofoam cooler inside of a low cost chest freezer.  The chest freezer was only $60 and could easily hold temperature at -20C.  I figured that by keeping the dry ice inside of a cooler in a chest freezer it could be maintained for a much greater time.  I want to point out that dry ice is extremely cold (-78 Celsius) and should not be stored in direct contact with the inside walls of a refrigerator or freezer or inside of a sealed container.  The graph below shows the measured sublimation rate for the dry ice.  Under these conditions the dry ice sublimated at a rate of approximately 1.0-1.2 lbs per day.  Dry ice is available from grocery stores and commercial ice vendors for ~$1.60 per lb in central Indiana.  That works out to ~$50-60 per month to keep competent cells at close to -80C.  That is a significant expense, but dramatically cheaper than the deep freezer.  

Graph showing the dry ice sublimation rate under the conditions described in the post.

Graph showing the dry ice sublimation rate under the conditions described in the post.

By keeping the cells on dry ice and using a procedure to make our own competent cells we can evaluate functional DNA at a low cost.  The protocol to make the cells is time consuming, but several hundred aliquots can be made in a single day.  Using these approaches brings our total per transformation cost to less than $1 so long as we are doing several transformations per day.  

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.

     

Fungi, food, and biotechnology.

Fungi are remarkable organisms – ubiquitous in all terrestrial environments, yet often unnoticed.  When most people think of fungi, colorful mushrooms come to mind, but mushrooms are only a fraction of the species diversity in the fungal kingdom.  Hidden yeasts, molds, and endophytic fungi all play essential roles in many ecosystems.   For instance, the decomposition of wood and leaf litter is largely performed by microscopic filamentous fungi.  Most plants harbor endophytic and mycorrhizal fungi that can enhance nutrient uptake and plant health.  Fungi are also a big part of human society and the biotechnology industry.

One of the most common uses of fungi by humans is in the production of food and beverages.  The beer, wine, and spirits industry all use the microscopic yeast, Saccharomyces cerevisiae, for the production of alcohol.  Wine has been produced by humans for several thousand years; however it was not until the 19th century when French scientist Louis Pasteur conclusively linked the production of alcohol to the Yeast.   Yeast is also used around the world as a leavening agent in the production of bread.  Molds, or filamentous fungi, are also heavily used in food production.  Foods like soy sauce, blue cheese, and tempeh are produced using filamentous fungi – in the cases of blue cheese and tempeh you actually eat the mold!  Finally, the most widely recognizable form of fungi, the mushroom, is itself a fine delicacy and available for purchase in all grocery stores and pizza parlors.

Outside of the food industry, fungi are also a central component of the modern pharmaceutical and biotechnology industries.  Human health was dramatically improved as a result of the discovery that fungi produce antibiotics.  Antibiotics have conservatively saved nearly 100 million lives.  The biggest pharmaceutical blockbuster of all time, the cholesterol lowering statins, was discovered in the ascomycete mold aspergillus.  On the basic research front, in the 1940s the bread mold Neurospora crassa was used to establish the Nobel Prize winning one-gene one-enzyme hypothesis. Saccharomyces cerevisiae was the first eukaryotic organism to have its genome sequenced in 1996.  Since then yeast has been used as a model organism for studying fundamental processes in molecular biology and for the development of new tools in the biotechnology industry.  Recent breakthroughs using yeast as a tool for biotechnology include the low cost production of the anti-malaria drug artemisinin and the production of the first fully synthetic genome.

Fungi are also great organisms to use for teaching genomic biology.  Even though fungi are multicellular eukaryotes with diverse appearances, their genomes are relatively small compared to animals and plants.  A typical fungal genome is approximately 100 times smaller than the human genome -- fungi have very little "junk" DNA!  Large efforts, like the 1000 fungal genomes project at the US Joint Genome Institute (JGI), are using genome sequencing to further our understanding of fungal diversity.

At The Great Lakes Biotech Academy we have chosen to focus predominantly on the biology of fungi and biotechnology applications involving fungi.  This is because some of the most important organisms used in synthetic biology and industrial biotech are fungi.  Fungi are also excellent organisms for teaching complex topics in molecular biology and physiology because they span the microscopic and macroscopic world.   Using fungi, we will be able to explore exciting areas of biology related to multicellular growth, sexual reproduction, taxonomy, and the effects of genetic mutation.  Have a look at some pictures of fungi taken recently at the biotech academy.

The Biotech Career Path

There are no Steve Jobs, Bill Gates, or Mark Zuckerbergs in the biotechnology industry – why?  Somehow, without even a college degree, these iconic technology founders started companies that changed the world.  Unlike the computer hardware and software industry, practically all biotech entrepreneurs have Ph.D.s and go through a version of the “traditional biotech education path” summarized below. 


THE TRADITIONAL PATH
(HS= high school, UG= undergraduate, G= graduate school, PD= postdoctoral studies)

STAGE 1: Fundamental STEM skills acquisition (HS 1-4, UG 1-2)

Students gain a baseline understanding in the areas of mathematics, chemistry, biology, and physics.  Courses on these topics are a prerequisite for most science and engineering degrees and the classes are filled with students with diverse interests (e.g. pharmacy, medicine, environmental engineering). 

STAGE 2: Core biotechnology skills acquisition – (UG 3-4, G 1)

Students begin to take biotechnology-specific coursework.  Courses in analytical chemistry, molecular biology, bioinformatics, and biochemistry are commonly taken at this stage.  High achieving students may begin to participate in closely supervised investigative research projects.

STAGE 3: Supervised investigative biotechnology research – (G 2-5)

Students have completed all formal coursework and work almost exclusively on investigative research projects guided by their thesis committee under the mentorship of their research advisor.  The quality of the education provided at this stage is highly variable in the United States and is heavily dependent on the engagement of the student’s research advisor and the academic institution.  

STAGE 4: Independent investigative biotechnology research – (PD 1-5)

Students have received their Ph.D. and are now post-doctoral associates (postdocs).  Postdocs are typically given considerable freedom to develop and perform investigative research projects in academic research groups headed by tenured professors.  Postdocs are the core engine of biotechnology research in the United States.  Many new startup biotechnology companies are spun-off from academic research performed by postdocs. 


Origins of the traditional path

The traditional path has remained basically unchanged for the last 100 years and was not developed to create a biotechnology workforce or entrepreneurs like Gates and Zuckerberg.  It is tightly associated with the career path to a tenured professor position in academic research.  A tenured professor must be a master of their discipline, in addition to being an educator, as well as (especially in more modern times) a professional marketer and grant writer.  It is a very celebrated and sought after position – and once tenured, a professor is vested with a lifetime appointment.  Tenured professors are exceptional people, but the training required to be a professor has no direct connection to the training required to be a biotech practitioner or entrepreneur.  The main reason that essentially all biotech entrepreneurs have a Ph.D. is because most investigative biotechnology research happens in universities.  I believe the unintended consequences of intertwining the career paths of a biotechnology entrepreneur and a tenured professor is severely hampering innovation in the field.

The fundamental problem

Being an entrepreneur in any field is challenging.  There are multiple risks: the idea may not work, the economic conditions may change, a competitor may come up with a better product, and the list goes on.  However, the biotech industry has even steeper risks.  To get to the point where you ostensibly have the skills to start a new biotech company, you have to spend 10+ years in school.  A new biotech company also requires specialized equipment and employees with Ph.D.s, which are very expensive.  To top it all off, most people exit the traditional path in their early 30s – which is the same time many consider starting families or buying their first home.  Finally, many of the top people exiting the traditional path end up as assistant professors and their time is consumed by the pressure to receive tenure.  All these factors synergistically discourage and overwhelm many people, including those with great ideas, preventing them from becoming biotech entrepreneurs.

How the Great Lakes Biotech Academy will address the problem

At the Great Lakes Biotech Academy we are developing a new and holistic educational program, outside the traditional path, to provide young people with the skills they need to prototype their great ideas.  Our programs are not a substitute for an advanced degree; they are a launching platform for talented young people to participate in the biotech industry.

There are three main pillars of our approach:

1.  Core skills training in biotechnology via The Fellows Program

2.  Apprenticeship style mentoring for Fellows Program graduates

3.  Dramatically lowering the costs to perform basic biotech research

The Fellows Program will provide the hands-on skills needed to perform biotechnology R&D.  The history, theory, and rigorous derivation of how each technique works will be relegated to optional homework assignments and replaced with practical tips and tricks that practicing scientists use to get work done on a daily basis.  We will rapidly move through topics in microbiology, genetics, and molecular biology – directly integrating the topics we cover in the classroom with hands-on experimental work to reinforce the key points.  A person writing a smartphone app doesn’t need to understand the history and theory behind how a compiler converts c++ code into machine language; they just need to know the one line text command to compile the program.  Why should it be any different with biotechnology?   

Once a student graduates from the Fellows Program they will be invited to participate in ongoing academic research projects at the Academy.  Fellows will get to practice their newly acquired skills and receive mentorship from practicing scientists and older Fellows Program graduates.  We anticipate that as Fellows gain confidence in their capabilities they will begin to make creative contributions to the research programs.  This should create a self-reinforcing positive feedback loop and a fertile environment for new discoveries.

The third pillar of our program is to find creative ways to lower the costs of performing modern biotechnology research.  To realize these lower costs we have made strategic decisions about how we produce, store, and modify DNA that differs from common practice.  We have also elected to not seek or utilize expensive laboratory equipment.  Instead, our focus will be to find consumer grade products to replace many of the essential pieces of laboratory equipment.  Consumer grade products are sold in competitive mass markets at low cost.  A simple example of a consumer product substitute is a Sous-vide immersion circulator in place of a research grade temperature regulated water bath.  When a consumer product substitute isn’t viable, we will use a combination of 3D printing and low-cost parts to “MacGyver” it.  Keep an eye on our news section for more posts dedicated to this topic.