Category Archives: Startup

Fraction Collector

I haven’t posted much about my builds- mostly because I’m swamped with work and family. But I can’t resist with this one.

Our flash chromatography system was acting up, and so the saying goes: necessity is the mother of invention. Rather than try to coax the equipment to keep eeking out 48 samples with the occasional hardware failure, I just decided to program my own system that could do exactly what I wanted. More sample collection capacity, more control, and more arduino!

I hijacked my XY plotter (purchased a few years ago to build a microbial biopointilism plotter), and coded a relatively simple XY coordinate grid that was based on a cheap test tube rack (Amazon/Ebay). In doing so, I now have 5 racks of 24 tubes, for a whopping 120 tubes at 50ml each. That’s a grand total of 6 liters of fractionation capacity. Oh yeah.

The plotter already had simple limit switches on the X+/X- and Y+/Y- planes, so I started out writing a simple “homing” sketch to get to (0,0) at the bottom left, then basically just placed the first tube rack under that first coordinate. The collector head will stay over the tube for a defined period of time, which is linearly proportional to the volume that we want to collect. Once that time is up, it moves a specified number of steps to the next tube. The spacing between the row of 8 tubes is constant, but then it needs to move to the next row like a typewriter- same movement between racks. The movement is actually pretty easy, and it only took some minor tweaking to get all of the tube racks aligned with the code.

Next I implemented a 12vdc pinch valve to be able to close the flow while the collector head moves between rows and racks. Tube to tube didn’t suffer any sample losses, so it only turns off the flow while zig-zagging from one row to the next.

I then made a custom 25-pin din cable end to plug into a masterflex digital peristaltic pump, and this allows us to remotely control the solvent flow on vs off. No need to change the flowrate, just keep it at a nice simple isocratic flow. (or one thing I am considering is altering the flowrate based on the column backpressure, so the fraction volume doesn’t tail off as pressure increases).

There is a serial interface with a computer, so the collector head can be steered manually, the valves and the pumps activated, etc.¬†The software/code is arduino-based, and I hosted it over on Github here. Please be kind, I’m a biochemist not a programmer. ūüėČ

Regarding the code… Right now it is only using a delay function to set the time for each sample. I have a draft program coded that uses the system clock to keep track of the fraction time, so the microprocessor can actually think and do other things- like pause, or modify the program while it is running. I’ll upload that when I get it working, but for now the delay is working well. Also I have considered abandoning the laptop and just using a LCD screen or some form of readout, with buttons or some human interface to control the robotic arm and program, but the serial interface is easy and convenient, and it works. Done is better than perfect!

Hardware involved:

  • Makeblock XY plotter kit- with electronics
  • BioChemValve Pinch valve, normally closed, 12vdc
  • A masterflex peristaltic pump and tubing
  • Chromatography column of your liking
  • Luer lock fittings for the pump tubing
  • Zipties to hold the tubing to the cables and the fraction head
  • 5vdc 2x Relay board (or suitable setup to control the pinch valve)
  • 25 pin din, male, solder cup adapter (or as needed for the pump interface)
  • rj11 6 wire data cables to tap into the MakeBlock Orion (Arduino) board
  • A 12vdc 2A power source for the pinch valve
  • And a laptop or computer with Arduino IDE installed on it

I have programmed our Tecan and have done some other small robotics projects in lab, but this is my first project that has become a workhorse of the lab. With my initial success, I’m now trying to hijack a $22 ebay robot kit for fraction collection.

The number of fraction collectors that one needs is always n+1…

-BZ

Crohn’s and Colitis

I recently went to an event hosted by the Crohn’s and Colitis Foundation (CCF)’s Northern California chapter. The event was an educational symposium for patients and families with IBD, and I was impressed with the CCF organization as well as the amazing community of patients and caregivers. I want to thank Kayla Kraich for all of her hard work organizing such a nice and welcoming event.

I went to the CCF event to learn more about IBD, and specifically biologic treatments for IBD, their benefits, as well as their limitations. Our startup is developing a new small-molecule treatment of IBD, so I would like to stay on top of the standard of care and see what else is going on in the field.

I grabbed a cup of coffee, sat down, and waited for the talks to start. The people sitting around me all looked like happy and healthy adults leading vibrant lives. The guy in front of me looked about my age, well dressed, clean-cut, and maybe a touch sciencey. I thought, hey, maybe he is another researcher? The first talk focused on biologics for IBD, when they are indicated, pros and cons, etc. At the end of the talk the MD invited up a panel of patients to talk about their experience with biologics. The man in front of me stood up and joined the patient panel. When he shared his story of battling IBD since he was 15, it was eye-opening. I have heard that IBD is one of the most disruptive diseases that can be largely hidden from the outside world.

At the first break, the woman sitting next to me introduced herself. Right after her name, she launched into her history with ulcerative colitis, her multiple surgeries, and how she ultimately tamed her disease with a balance of diet and medications. At the end of her quick life story, she then said, “what about you Brandon?”

I have to admit that I felt like a poser. I don’t have IBD. I haven’t had multiple surgeries. I haven’t seen numerous doctors, received infusions of antibodies, worried about infections from a suppressed immune system. I haven’t struggled to knock down GI flares with steroids, and then dealing with their crazy side effects. Nor have I endured the never-ending visits to the bathroom (a metric for ulcerative colitis activity is the number of daily bowel movements, with the highest tier being anything over 10 times a day).

I told her that I’m just a researcher, but we are working on IBD and have a new drug that we are rapidly advancing to the clinic. She put me at ease with her genuine appreciation for my research, and although I felt insignificant and like I didn’t belong, she welcomed me to the event and wished me the best of luck in our work.

At this point, I may have drank a couple cups of coffee trying to remain alert and sharp during the morning talks, so I made my first trip to the men’s room.

What I encountered in the men’s room was subtle at first, but after recounting the experience and putting it into context with the diseases of the colon, it started to make sense. On my first trip to the men’s room I noticed a number of people coming and going, but the notable interaction was with someone who introduced himself while walking from the urinal to the sinks. The man was very extroverted, and the tone and content of the conversation could have taken place in any setting. Only this time, it was in the men’s room. And that level of forward communication in the men’s room seemed a little odd to me.

Back to the talks, the next speaker gave a nice overview of IBD (the MD and I overlapped as undergrads at UC Davis- go Ags!). After this second talk, my bladder was losing the battle to the coffee and water I was drinking, so I made my second trip to the men’s room. This trip, there was another very genuine introduction and discussion that started in the bathroom. I chatted, and eventually made my way back to the conference room, and didn’t think much of it.

After the talks I headed home, and during the drive I reflected on the great talks and on my interactions with the amazing members of the CCF community. And of course those very genuine interactions that I had in the men’s room…

And then it hit me.

For a patient population that uses the restroom so much, the CCF community is very, very comfortable in the bathroom. Especially at an event filled with fellow patients, doctors, and caregivers, and when it is hard to tell who has what disease, everyone wearing a CCF nametag must seem like an ally. I laughed out loud when I put it together. And then the realization actually felt heavy on my face and my chest. There was a lifelong struggle that was underneath the interactions with the people in the bathroom. I have now attended two CCF events, at which the first item of business was telling everyone the location of the bathrooms. My gastrointestinal health is something I have always taken for granted, but I am now looking at it with a new perspective.

The more that I learn about Crohn’s and Colitis, it just kills me to think of what IBD sufferers are going through. As we are so preoccupied with the pharmacology and interactions of our drug compounds in lab, we don’t often think about of these complications that IBD patients suffer daily. I am extremely grateful for how welcoming and open the CCF community has been with me. It leaves me feeling hungry for our upcoming clinical trials, and it reaffirms that IBD is a worthy cause that I am grateful to be working on.

-BZ

Cannabis sativa

I work on cannabis.

As a PhD biochemist and former cancer researcher, it feels a little dirty to admit. But let me explain…

I love plants. My favorite plant is Sarcodes sanguinea, a native of the high Sierras that is a brilliant red hue because it lost it’s chlorophyll and parasitizes fungi for food:

A fabulous Sarcodes seen while riding my cyclocross bike on the East side of Lake Davis before the Lost and Found Gravel Grinder, June 2017.

I planted lemon, lime, fig, pomegranate, olive, pear, and apple trees in my back yard. I also have a rockin’ garden, and I love teaching my kids where their food comes from:

My main garden in full force, June 2017.

I also have a soft spot for interesting plant chemistries. While at U.C. Davis I worked with antioxidants produced from broccoli and other cruciferous vegetables. I have worked with the biochemical pathways producing the stevia sweetener, and contrast that with working on the genetics of noxious plants that are toxic to humans.

I am an ¬†endurance athlete, and I like to cycle or trail run for hours. Mostly from the health benefits, but I do enjoy the endorphin rush from an epic workout. I’m generally risk-averse, and I’m an Eagle Scout in most senses of the stereotype.

But I work on cannabis. Or more specifically, cannabinoids that originate from the cannabis plant.

Cannabis sativa. Pot. Weed. Marijuana. Ganja. Reefer. (Insert your favorite slang here). Yeah, that cannabis.

It is weird to me how some people think that I am part of a counter-culture movement full of pot-smoking hippies (or whatever the equivalent is for the millenial generation – pot smoking, avocado-toast-eating, millenials?). In actuality, we are a bunch of over-educated, lab-coat clad nerds, drinking coffee and geeking out about the amazing chemistry of cannabinoids and the corresponding human cannabinoid receptor system.

Nerds!!!

As Alexander Shulgin wrote in PiHKAL, “Among the drugs that are currently illegal, I have chosen not to use marijuana, as I feel the light-headed intoxication, and benign alteration of consciousness does not adequately compensate for an uncomfortable feeling that I am wasting time.”

None of us partake in cannabis for recreational or medicinal uses, and whenever a question on human use comes up- we are forced to consult Google to learn about typical usage and exposures. I personally, and our startup are fully compliant with all state and federal regulations, and we have all necessary approvals to perform our research. When it comes down to it, we are just doing science. Some plant biochemistry, some mammalian receptor biology. But our work could have an impact on inflammatory bowel disorders, colon cancer, and some other potential applications that could be pretty damn cool.

A friend from grad school was concerned that by researching cannabis, I may be a marked scientist- unable to rejoin the ranks of the normal scientists hard at work on cancer, neurobiology, and other respectable areas of study. I completely disagree. What I have found is that the cannabinoid and cannabinoid-receptor research field has been stifled by undue regulatory pressure for so many years, so instead of being a dead-end for science, it is ripe for discovery.

Pharmacological uses of non-psychotropic cannabinoids

The cannabinoid receptor is the most abundant G-protein coupled receptor in the brain, hence, people get really high with THC exposure (an agonist). CBD on the other hand is an inverse-agonist, so it has somewhat of an opposite effect on our receptors and doesn’t get people “high”. It is on the fast-track to be approved for treatment of specific epilepsy subtypes, is in multiple clinical studies for schizophrenia, and has tremendous potential as an anti-inflammatory. So the pharmaceutical applications are real, and have even been vetted by the Food and Drug Administration.

Cannabinoid receptors are also found throughout the body, and to use the “lock and key” analogy for enzymes- we have discovered locked doors throughout the body- now we just need to figure out the keys, and what doors we can open with this new knowledge. The therapeutic ¬†potential for cannabinoids is unmistakable. I look forward to seeing what doors we can open with our work.

-BZ

Startup Quotations

A fun image borrowed from yhponline.com on their UK Startups page

As I approach my fourth year of grinding it out in a startup biotech, I thought I would collect my favorite quotations of startups and productivity. As I read more about business and startups I keep finding new quotes that encapsulate the feeling of a startup, so I hope to add to this list as time goes on.

Move fast and break things. (attributed to Facebook, and humorously depicted on XKCD).

A ‘startup’ is a company confused about: 1. what its product is, 2. who its customers are, 3. how to make money.” – Dave McClure of 500 Startups.

The reality is, Silicon Valley capitalism is very simple:
Investors are people with more money than time.
Employees are people with more time than money.
Entrepreneurs are the seductive go-between.
Marketing is like sex: only losers pay for it.” – Antonio Garcia Martinez in Chaos Monkeys: Obscene Fortune and Random Failure in Silicon Valley.

Done is better than perfect. (described as a Facebook slogan in Chaos Monkeys).

A good idea on Monday is better than a great idea on Friday. (not sure where I heard this one).

Build. Measure. Learn. (repeat).  РEric Ries, The Lean Startup

Startups are an odd creature that I am still working on taming (assuming you can tame it!). The energy and speed are addictive, and despite all of the struggles, I wouldn’t trade it for anything.

-BZ

The Budget Biotech

The bench beaver is always watching you

I have been toying with the idea of starting my own biotech startup in my garage, and want to determine the lowest amount of upfront investment it would take to set up a functional molecular biology lab.

OK, a little background to help the discussion. Straight out of grad school I took a job with a startup biotech developing strains for fermentation of high value small molecules. When I arrived the lab had a few tables and some basic equipment, but being that an electrical engineer did the initial purchasing it wasn’t quite ready for much biochemistry. So my first job was to outfit the lab with the necessary equipment and consumables to get things moving. I quickly realized how expensive laboratory equipment and research reagents were without the generous academic discounts I had grown accustomed to. We pieced together a mixture of new and used equipment, took advantage of lab startup promotional deals, and ultimately assembled a fully functional molecular biology lab on a pretty modest budget.

Taking what I learned from setting up my first biotech lab, I know I can do better. How much better? I think it should be possible to assemble a functional molecular biology lab for under $1,000. But wait you say- that is just enough to buy a decent set of pipetters- how could you set up an entire lab for that?

Precisely. That is the heart of the challenge. To create a budget-minded biotech that is functional, but doesn’t cost an arm and a leg. As I wrote up in a previous article, ‚Äúbiotech shouldn’t be so dang expensive!‚ÄĚ

I think it all depends on how you define the core functionality. To me, the minimum functionality needed is to clone genes and express proteins. I am focusing on the steps from source DNA to expressed recombinant protein. So basically from PCR through cloning in E.coli, to expression of proteins.

Things I am not including in the $1,000: Analytical (in my case HPLC or LCMS), basic infrastructure like tables, stools, computer, etc., and little things like surge protectors or extension cords. And I may take the liberty of culturing my own competent cells to cut down on that cost. Vector backbone will be an open-source design to get around IP conflicts and keep things cheap. We’ll see how far we can stretch $1,000 and go from there.

I am looking mainly at functional but well-loved equipment on ebay and craigslist, and even some homemade or hacked projects capable of carrying out the job at hand. EBay has some amazing deals. So does Amazon (I usually avoid Thermo subsidiaries as much as I avoid Wally World!). And if you are willing to do some soldering, coding, and 3D printing, you can make just about anything in the lab. I acknowledge that reliability will undoubtedly be less than brand new equipment still on warranty, but of the three-legged stool of quality, cost, and time- this exercise is focused primarily on cost.

Here is my initial list of equipment, projected cost, and notes on procurement. Let me know what you think, where you think I can cut the cost down, or if there are any glaring errors in my choices or logic. I plan to revise and post updates to my budget biotech quest, so hopefully this is only the beginning!

-BZ

EQUIPMENT
Item Cost Notes
pcr – thermal cycler $200.00 used… Ebay. Or ghetto fab water bath and servo setup?
gel rig $53.00 kit from iorodeo.com
light box $80.00 mini kit from iorodeo.com or build myself
pipetters $100.00 2 pipettes at 50 each. P20 and P200 to start
water baths $12.99 crock pots with arduino thermostat (DS18B20 and SSR)
-20C freezer / fridge $50.00 total budget – cheap chest – or used on CL
incubator $50.00 Cooler/chest freezer with hairdryer/heatgun heater and extra fan? Or foam insulation box.
autoclave $83.99 pressure cooker- presto on amazon. Ikea?
centrifuge $80.00 used Brinkman on Ebay
flame for plate work $60.00 Ebay flame/regulator kit
Micro scale $25.00 amazon
microwave? $20.00 craigslist
gel power supply $15.00 homebuilt (budget molecular biology power supply)
rotary shaker $140.00 used old NewBrunswick on ebay
pH meter $20.00 Amazon or Ebay
TOTAL EQUIPMENT: $989.98
CONSUMABLES
Item Cost Notes
tips $10.00 Ebay- bulk tips – need upfront tip box
weigh boats $10.00 or papers
DNA stain $68.00 SYBR Safe – cheaper alternatives with blue LED illuminator?
gel buffer TAE homemade
DNA polymerase $39.00 mastermind from geneandcell.com
dNTPs included in mastermind mix
DNA buffer TAE homemade
gel extraction kit NA45 paper? Simple protocol? Kit…
1kb ladder $89.00 geneandcell.com
load dye bromophenol blue
comp cells homemade – but lack of -80 will affect competency
Total Consumables: $216.00
Total Combined: $1,205.98 over budget already! And chemicals not even included yet!

Budget Molecular Biology Power Supply

For my first blog post I have chosen to write up a project I did very early in my startup¬†days. The theme of this post is that¬†biotech shouldn’t be so dang expensive.

While setting up a molecular biology lab for a startup biotech, I was shocked at the industry prices on equipment and reagents, something I took for granted while still in academia. Nowadays I am better at vetting Ebay sales for used equipment, but at the time I thought there were two options: new or homebrewed. So I made a simple 120v DC power supply to run SDS-PAGE gels for about $20 in parts. I think you could probably get the parts down to about $10 per power supply.

Disclaimer: I am a biochemist, and not an electrical engineer or someone who designs electronics for a living. Electricity hurts. This post is meant to be informative, but not fully instructional. Use this information with extreme caution, and don’t blame me if you shock yourself or catch something on fire!

So it all started when I needed another power supply to run my SDS-PAGE protein gel system. I didn’t want to buy an expensive power supply because I just couldn’t justify spending hundreds of dollars for something I wasn’t going to use much. So in the early days I used alligator clips to tap into the leads from my¬†BulldogBio DNA agarose gel box. This was pretty sketchy, so after a few runs I decided to open up the power supply and see what was inside. I saw only two switches, a fuse, and an electrical component¬†that was new to me. After googling the writing on the module I learned that it was a Bridge Rectifier- basically a big rectifying diode setup¬†that can convert the AC into a choppy DC supply. The Bulldog power supply obviously worked well for running gels, so I didn’t think that the rough DC signal was a problem. Inside the BulldogBio unit there was also a switch to switch the negative/ground to the electrophoresis rig between the true AC ground and the (-) leg of the bridge rectifier (I think this is how it switches from +60vDC to 120vDC), but I didn’t implement that in my power supply (yet). So I pieced together the parts, built the power supply and it worked!

IMAG1280
Link to fullsize image
IMAG1279
Link to fullsize image

Bill of materials:
Electronics enclosure of your choosing
120vAC Power Inlet (and a cord- or just run the cord into the box)
Double Pole Single Throw (DPDT) switch rated to >5A AC (I used a DPDT switch I had laying around)
Fuse holder and quick burn 1A fuse (you can get one built into the power inlet also)
Bridge Rectifier (I bought an overpriced one from Fry’s)
Banana Plug Terminal
A scrap piece of proto board that I had to drill out to fit the big terminals of the bridge rectifier
Some scrap 20ga wire and blade fittings
Solder and iron to connect to the fuse holder and power terminals
Heat shrink tubing to cover the naked power terminals (I didn’t, but I would next time)

Notes:
I definitely would not use this for sensitive electronic equipment, as the DC output is probably pretty rough. It would require some beefy capacitors and voltage regulators to fine tune the output, but that is way beyond my knowledge.¬†The ability to switch between 60vDC and 120vDC would add on a couple of dollars, and would give me the ability to run a gel a little slower. This would likely only require a single pole-double throw switch to switch the gel negative pole between the true AC ground and the (-) leg of the bridge rectifier. But I haven’t gone that far, for what it’s worth. ¬†Also, I haven’t really tested how much heat is generated by the bridge rectifier after an hour of use. The overall box stays cool, and all components look fine (read: no meltdowns yet). If you are running multiple gels or need to push more current through the rectifier, consider getting one with a heatsink, or at least use a fan to move some air through the box. Also, I haven’t really tested how much heat is generated by the bridge rectifier after an hour of use. The overall box stays cool, and all components look fine (read: no meltdowns yet). If you are running multiple gels or need to push a higher amperage, then definitely keep an eye on the box and component temperatures.

I’ll try to sketch out a schematic when I have a moment- or maybe a Fritzing diagram if I’m feeling saucy.

-BZ