Tag: DIYBIO

Micropropagation of wild-type Vaccinium Vitis-idaea

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A few years ago my friend introduced me to a tasty and edible berry while we were on a hike up mount Washington. I had no idea what it was, and much later I learned that they were lingonberries, which are related to a lot of other shrubby berry plants like blueberries.

Recently I learned that plants are very sneaky and do not always come from seeds. In fact, most plant cells seem to be totipotent (capable of creating any other kind of plant cell) and capable of creating undifferentiated cell mass (callus culture) if you feed them the right stuff. I had previously considered a plant (particularly woody plants like trees) to be single monolithic organisms with parts that fall off that are dead/not useful. It seems like it is more the case that any part of a plant can turn into another copy of the plant, under the right conditions. Think about that next time you are looking at cabbages in the supermarket.

It turns out that most ornamental plants, and many commercial plants are grown not from seed, but basically from cuttings that are carefully grown in lab conditions to maximize growth and multiplication of the plants. This is called micropropogation, and its useful for getting (usually) genetically identical copies of plants. This is helpful for preserving desirable mutations.

You can probably see where this is going: tasty plant + new technique = a small project that will likely take many years to complete. I want to grow our local variety of lingonberries!

Media Prep:

these blueberries were growing right on top of the lingonberries!

After weighing my options on media I decided to go with a media that was actually recommended for blueberry bush micropropagation from “Plants From Test Tubes: An Introduction to Micropropagation”. While there was more specific media recommended in some papers on propagation, it was not clear to me (as a neophyte) that “modified ms media” was actually a specific modification of MS media, not MS+2ip. It was possible to have ordered that media, but a lot of items were backordered for months, so I ended up with regular MS media, and 2ip. At this point it was too late to get the specific modified MS, and blueberries and lingonberry plants grow right next to each other in the wild, so it seemed like it might work.

The media I made was not exactly per the book either, since I think my MS mix already had inositol. The final mix for initiation media was:

  • 1L distilled water
  • 2.3 g MS basal media
  • 5ml 2ip @ 1mg/L (roughly .25 umolar)
  • 20 g sucrose (refined white sugar)
  • 6 g agar

The 2ip concentration was in line with what I had read was ideal for initiation from Jakkola et al. 2001, which was encouraging. The media was not checked for pH since I didnt have a meter or strips, but I would have liked to adjust it down to 4.8, which is a reasonable pH for a lingonberry plant.

One nice thing about this media is that it does not seem particularly rich (surprising given the sugars). I intentionally exposed a plate to contaminate it and it took weeks for some mold to show up. With LB, I would have expected it to be disgusting almost immediately.

Explant Collection:

Explants were collected from a population near the alpine garden trail on mount Washington. only a few grams of plants material was collected over about a hundred yards of trail, in order to minimize impact on the environment. Explants were stored in sterilized falcon tubes for transportation.

Explant Preparation and Plating:

Explants were placed under running water in a strainer with a bowl beneath it in order to create an agitated water bath. The plant tissue was washed for 10 minutes under running water to remove dirt. explant material was then sterilized in 70% IPA for 30s, and then washed in 1:10 bleach solution for 25 minutes. a drop of dawn soap was added to the bleach as a surfactant. After sterilization, the plants were washed three times in sterilized water.

Plants were handled inside of a plastic bin that was sprayed regularly with bleach solution. Tools were sterilized/stored in bleach as well in between uses. I would recommend a clear bin for better lighting. It was challenging to cut the plants in a dark box, but after roughly three weeks, there was only one obviously contaminated sample.

Results

I’d like to call this section something more optimistic, like “success”, but I never really got anything to root. However, there was evidence that the media is sufficient to support the plants. In the photos above, the left photo (with new leaves) was grown from the small browned sprig on in the right photo. The right photo was taken on October 5, and the photo on the left was taken on the 19th, two weeks later. New leaves are clearly visible, and it seems like the plant is producing chlorophyll.

I’m not sure if this is strictly useful, but the lack of contamination and the plant being alive is a good first step.

GelIS: The Box

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GelIS yet?

GelIS yet?

HEY, if you are interested in beta testing one of these systems, I am looking for users.  I will supply the box, the transluminator, and 100v variable power supply for $150.  Fill out this form if you are interested.  I expect to ship by mid august.

GelIS is a backronym for Gel Integrated System.  As I mentioned before, I want to respond to the needs of the DIY bio community with the design.  The design criteria are:

  • Easy to build: You should be able to put it together in less than 10 minutes, and run a gel.
  • High Quality: This should look professional, and you should be able to use it for a long time
  • Low Cost: Many of the potential users are students (who don’t make much money), schools (which don’t have much money) and folks who are new to biology (and are unsure of spending a lot of money).

Traditionally, the gel box itself has been very expensive.  I believe this is the result of the complexity of the design.  One way to quantify the complexity of a design is to look at how many pieces it has, how many materials there are, and how they are fastened together.  Below are some comparisons of boxes, looking at the “box” part alone, including combs and whatever you need to cast, and ignoring everything else.  Included are the prices new.

Pearl biotech ($200 ish): 13 plastic pieces (one thermoformed), 2 rubber gaskets.  13 acrylic weld joints, 2 glued gaskets, roughly 5 materials (rubber gasket, thick acrylic, thin acrylic, white acrylic, amber acrylic)

Owl Mini Gel Box ($400 ish): 15 plastic pieces (several thermoformed, and two injection molded), about 20 weld joints, 2 glued gaskets, roughly 4 materials (gasket, injection molded plastic, yellow plastic, clear plastic)

“BIOTANG” Gel box ($350): 7 pieces, injection molded.  This one is kinda cool because you can stack gels, and run more than one at once!  two or three materials

GelIS Box ($50):  5 peices, laser cut, 1 weld joint.  Connects to the power system via 3 laser cut pieces, which contain 8 bolted connections.  Pending thermoforming, the parts count can be reduced by two pieces of plastic, and all the bolts.

So as far as complexity goes, the GelIS box has more pieces than the BIOTANG, but less than the other boxes.  It also has a dramatically lower price tag.  This may be because it only has a single weld joint, rather than 13-20 joints.  I intend to weld that joint at the “factory” ensuring that assembly is quick and painless, and that the box is leak free.  Assembly should require no tools other than their hands, to screw a few nuts onto bolts on here and there (screwdriver recommended, but optional).

An Electrophresis System For DIYBIO

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IMG_4627

GelIS Gel Box

HEY, if you are interested in beta testing one of these systems, I am looking for users.  I will supply the box, the transluminator, and 100v variable power supply for $150.  Fill out this form if you are interested.  I expect to ship by mid august.

There have been several attempts to build and sell gel boxes to DIY biologists.  As far as I am concerned, they are all too expensive and too complicated.  Lets look at the two products aimed and produced by diy folks: the Iorodeo kit and the no longer available pearl biotech box.

The IO rodeo Box, image from their website

The iorodeo box is a conventional shaped gel box with two deep buffer wells.  It costs $93  for the kit, plus about $10 (and $5 shipping) for the acrylic glue, coming to a total of 108, or you get have it shipped to you assembled for $123.  It does not come with cables, and the assembly can be tricky if you have never welded acrylic before.

Gel box and illuminator, from ginko bioworks blog

The Pearl box, which I no longer see listed on their website, is also a conventional design, but if I recall correctly, it came assembled and was north of $190.  I do like their box, and I use it in the lab, but it is kind of pricey for someone who just wants to try some electrophoresis!

Neither of these kits provide a power supply, although Iorodeo sells one based on what looks to be a boost converter for $65, unassembled.  $95 will get you an assembled power supply, and for $110 you can even get the 15V power supply to the power supply, which is necessary to make it work.

Both companies sell transluminators, with the Pearl product ringing in at a whopping $299 (read: $300 + s/h) for 64 led array.  To give you an idea of how crazy that is, 64 VERY bright LEDs from digikey come out to $23, or you can go on ebay and get a couple RGB panels of the same size for $5 a pop.  The IORODEO kit is a much more reasonable $80 for the bare-bones no-power supply or assembly kit, or $95 for an assembled LED board, or $112 for assembly and power supply.  This means that if you had bought a pearl box, you would have spent about $500 and still needed a power supply, and if you go with the IORODEO box, you are going to spend $345 for a fully assembled kit, or $285 if you do all the assembly yourself (I included buying the power adapters from iorodeo, but not any assembly options).

Both of these options are pricey, and while the iorodeo kits are encouraging, I think assembly stands in the way of people really doing biology.  Not only do you spend a lot of money on the kit, but there is the daunting task of assembly before you can do what you wanted to do when you spent all that money: run a gel.

IMG_4613

My goal is to develop a gel box, transluminator, and power supply that people can buy for $150, assembled.  You buy it, it gets shipped to you for $5 in a flat rate box, and then you open it and do some ikea assembly (put some nuts on some screws) and you are done.  Thats it.  Now you can run a gel.  Today I finished the first major piece of that system- the box.

DIY Vacuum Manifold

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I have been working on several low cost tools for biology, and yesterday it occurred to me to try to make a vacuum manifold.  a vacuum manifold is used to facilitate large volume DNA preparations by applying vacuum to the bottom of the filter column.  They are quite expensive, but simple devices; they are just a gang valve that hooks up to a vacuum pump, generally via luer lock.  As a note, you still have to centrifuge the tubes after you filter them (even in commercial models), but this might be helpful if you have a tiny centrifuge and a lot of preps to do.

My version is simple and cheap, and it works!  There are two main components: the vacuum pump and the manifold.

delrin_fold

The first manifold was manufactured from POM, aka Delrin.  A few notes on delrin: it is incredibly tough, and smells like fish when machined.  The manifold was basically three  holes drilled perpendicular to the axis of a 3″ piece of delrin.  The axis was drilled to a depth of about 2.75, and a hose barb was connected to the end, where I applied the vacuum.  This iteration was a big fail, because the seals on the tubes were not soft or, the right size (I just had them lying around).  This meant that there was a lot of leakage, and that made it not work, despite looking nice.

manifold

While I was testing various parts of the system to find the leak, I found out that 3/16″ tubing, or “standard airline tubing” for fish tanks is the perfect fit for the little nipple on the bottom of silica prep columns!  This led me to a less mechanically stable, but totally working connection.

I happened to have a 5-way gang valve (teal thing) from an aquarium in the lab, from the algae bioreactor project.  I rigged up four tubes to fit hypothetical silica columns, and one that went to a water intake.  This is used to help purge air bubbles from the system.

This setup worked surprisingly well!  If I had a vise big enough to clamp down the gang valve, it would be a totally functional manifold.  Unfortunately, it was just a hair too big for the vise and the pump was not ergonomic enough to prevent the pumping from moving the manifold around.

vac

Here is the vaccum pump.  Nothing fancy- it is just two aquarium check valves, some aquarium tubing, a T connector, and a syringe (for feeding babies formula/medicine) from CVS.  The tubing just slips on the tip!

I have considered rebuilding this by replacing the gang valve with a bunch of T connectors, or by upgrading the delrin part, but there are two problems.  First, you would have to re-plumb the whole system if you wanted to do a different number of preps than last time.  Two, the first prep to drain through the system is going to start to suck a bunch of the air into the system.  Much like a hole with a straw in it, or two unequal resistors in parallel, most of flow will be coming through the path of least resistance, reducing the draw on the other samples.  With such a sucky (haha, couldn’t help myself) vacuum system, this is going to make the device useless.

This vacuum setup is good for anyone who is interested in DIYBIO and cannot get access to an autoclave, of a large centrifuge.  This can be used to filter sterilize media, and bleach can do the sterilizing on the other end.  As you can see here, it can take some of the load off the centrifuge.  I don’t know if small centrifuges are significantly slower at filtering than large ones, but I do know that they can’t hold as many samples, so something like this could speed that process up!

Nice Pictures of Electrophoresis Gels

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Have you ever struggled to take a picture of a gel?  Probably not if you dropped a couple k on a gel documentation system.  But for those of us who to take photos of gels and keep our money, you might look like this:

Image

In this photo, I am trying to hold my gel illumination (a couple of LEDS) in one hand, and hold my amber filter, focus, and take a picture with my other hand.  In low light, my phone camera can’t focus either!  Thankfully, my camera can take voice commands, so I did manage to take a picture.  I have a Samsung galaxy note II, (at the time of writing, this is a recently released, top of the line phone) which has a fixed aperture and TINY sensor.  Here is an example of the quality it produces:

20130715_193243

This is barely usable, but maybe ok for just a quick note.  however, you really have to know where the bands are to see them.  For reference, that is 200ng of DNA.  Notice the greenish tint around the bands, and the lack of definition in the gel wells.

What you really need is a camera with manual focus, and a small piece of plastic.  You want manual focus, because no matter what you do, there won’t be a lot of light coming through the lens.  This makes it difficult for the camera to autofocus, which can be frustrating.  Manual focus lets you use your superior eyes and brain to focus.  Here is the device:

Gel Photography Device

Gel Photography Device

So there ya go.  Thats a UV (clear) filter with a piece of amber plastic in it.  I only bought the filter because I wanted it to be convenient to screw on and off.  Any plastic that is this color will work for blue light- there is no mystery there.  Here is what the world looks like through this lens:

Woah man, it's like instagram

Woah man, it’s like Instagram

Now lets take another look at that gel.  In fact, I will put both here so you can swap between them:

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Which one looks better?  Definitely the one with the filter.  It has a few things going for it:

  • No confusing green tint around the illuminated area.  This is confusing, since the bands are also green
  • No high-ISO noise in the non illuminated area.  More even illumination.
  • Slightly better focused image (Ok, I am to blame, I did not focus very well)

It was also WAY easier to take the picture.  Just point, focus, and click!  If I had a better illuminator, I wouldn’t even have to hold the LEDS!  There is also a difference in pixel count, but that is mostly irrelevant, for the quality of image.  Commercial gel docs use sensors that are much under 2 MP.  By pixels, my phone camera should be 4x better!

What really matters more is the size of the sensor.  As a rule, larger sensors are better, but I don’t need to explain that- you can read Ken Rockwell’s excellent posts on the megapixel myth and look at comparisons at high isos here.  Use the drop downs to look at side-by side comparisons of different cameras at high-ISO (what happens in low light, like with a gel).  Check out the below, which compares the canon rebel xs (10 MP, 419 square mm square APS-C sensor, 185 on ebay, WITH lens), the canon 510 HS (12.1 MP, 28 square mm sensor, on ebay, 12.1 MP) and the Sony CyberShot DSC HX20V (18.2 MP, 28 square mm sensor, 260 on ebay, 18.2 MP).  These are buy it now prices.

Winner is the XS 1000, as you can see the difference in the dark colors!

Winner is the XS 1000, as you can see the difference in the dark colors!

This is a picture of a test swatch, with very similar colors next to each other.  As you can see, the cheapest camera, with the smallest number of megapixels, but the largest sensor has the best color differentiation, and low noise at a relatively high ISO 1600 (for reference, 400 ISO is the most common speed for “general use”).  The XS 1000 also has manual focus!  My conclusion is that if you want to take a picture of a gel, use an older, cheaper APS-C sensor camera, and get a filter for it.  You will thank yourself for it later when you have nice pictures.

DIYBIO – FBI Outreach Conference, San Fransisco

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Actually, we were in Walnut Creek, but it is close enough to call it San Fransisco!

As it turns out, the FBI and other defense organizations (Hello, DTRA) are pretty interested in DIYBIO.  Coming away from the conference, it seems like the FBI is  interested in exactly what you would expect: preventing bad guys (nefarious actors!) from doing Bad Things.  The Defense Threat Reduction Agency on the other hand, is interested in buying technology from people who start in “garages”, or DIY environments, to use for defense work.

The room was pretty full! I didn’t know there were this many diybiologists!  (some of these people are FBI agents/wmd coordinators.  Hard to tell the difference in this photo)

The main focus of the conference was on the interaction between law enforcement and DIY biologists.  It seems to be that the FBI is not concerned with DIY biologists, and that the FBI certainly does not view the DIYBIO “movement” as a threat.  The position of the Bureau is that local DIYBIO folks should get a hold of their local WMD coordinator,  It was also reassuring to know that the FBI hires PHD biologists and a lot of scientists to work in their WMD department- it would be nice if policy makers were just as well informed.

There was also a good discussion about the media- it turns out that both the FBI and DIYBIO folks both tend to kind of dislike the media, because as one attendee put it “They overestimate our abilities, and underestimate our ethics”.  There were some good talks given on how to engage the press in a a way that cannot be misconstrued, and how to do due diligence when someone wants to cover your space.  Rachel had an anecdote from when the BBC approached them to do a piece on the DIYBIO activities at MADLAB/MCR:

The approach that we got. we are interested in debate, is't that lovely, PCR machines, exclamation points. This is what we read: we're going to do a piece on bioterror and flu virus research. And we knew that, we knew that we were going to be portrayed as extreme. We're the only group that can kind of say these things, we weren't the right people, but we were going to be their people anyway, and it was. This is what showed up in the BBC website.. "growing concern about DIYbio.. FBI, oh there you are". Biological threat, all in the same sentence.
( quote from transcript typed by Bryan Bishop )

I thought it was very useful that we had Dan Grushkin, Rachel Turner, and Sascha Karburg -who have both done quite a bit of journalism- to tell us how the journalism works.  It is important to have both sides of the story to really understand what is going on, so DIYers can engage the press more tactfully.

Speaking of Sascha, we got to enjoy his documentary on DIYBIO at the end of the first day.  After a few years in the making, it looked pretty awesome!  I didn’t understand what they were saying most of the time, as it was in German, but the images definitely told a story.

I think that the highlight of the conference was finally seeing who was out there, and what they were up to.  If you want, you can read transcripts here, courtesey of Brian Bishop.

countries from left to right:
USA, The Netherlands (behind the benches), Finland, Denmark, Germany, Turkey

The last day we all went down to Biocurious to play with some DNA.  Biocurious walked everyone through the basic procedure for a chemical transformation, but the real highlight here was working with people from other places, and actually building a plasmid with the Genomikon kit.

Overall it was fun to meet everyone, and exciting to see what the rest of the diybio folks are up to.  I think finally meeting the European counterparts helped bring the community together.  And it was certainly good to learn that the FBI won’t be knocking on our door any time soon.

FBI-DIYBIO Outreach Workshop

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I have been invited to California to the FBI-DIYBIO outreach workshop.  Day one is tomorrow.  As I sit here slurping at the last of of my large java-chip frappacino (with whipped cream) at Bryant and Mariposa, I have to say that I am pretty psyched to see Biocurious, and meet all the other DIYBIO folks to compare notes.  I will be posting my notes here on what happens!

How Do I Get Started In DIYBIO?

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A lot of people have been asking “How do I get started in DIYBIO?”.  The answer is not easy.  Biology is a broad field, ranging from studying entire ecosystems, to the chemicals that allow life to continue.  But I have done my fair share of DIY molecular biology, and I have begun to write up protocols and reviews of equiptment- which I will begin to share on this site on the DIYBIO protocols page, and on BOSSLAB.

Enjoy!  More posts on how to get started to come.

Genomes, Environments and Traits Confrence!

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Jason Bobe moderates a discussion with George Church and Geraldine Hamilton about personalized medicine microfluidic devices

Today I attended the Genomes Environments and Traits Conference.  It was awesome!  There were talks on all manner of technical breakthroughs from faster and cheaper sequencing, to single-cell sequencing (WITH 3D protein/DNA localization on the intracellular level!!!), to hackable drug delivery kits for 3rd world countries.

The more exciting part for me personally was running into all kinds of DIYBiologists.  It was awesome to finally meet them in person!  Ellen Jorgensen from GeneSpace was there, as well as Joseph Jackson from BioCurious, and (obviously) Jason Bobe from the Personal Genome Project.  There were also some people from the BOSSLAB group there (woo! not sure if they want to be mentioned by name).  I even found somebody from Olins’ neighbor college, Wellesley, and I spotted at least one Babson Jacket in the crowd.

Anyways, this conference got me more excited about biology and science, and this summer at BOSSLAB.

Mini-Maker Faire @ Cambridge Science Festival!

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Really bad photo. I apologize…

I was at the mini-maker faire today representing DIYBIO Boston, and all I got was this really bad photo…

Just kidding!  I also talked to a bunch of AWESOME MAKERS and excited participants.  I even got to help Gui and Molly of Artisans Asylum lift a giant motorized barbers chair onto a truck, and see a bunch of <6 year olds run dyes from M&Ms in agarose gels.

If you are looking for my bio work because you met me at the festival, click here to see the things I have done with biology.