Opentrons OT-2 release

[Koeng]

Hey all, 

Saw OT-2 was released from OpenTrons http://opentrons.com/

The new robot is a lot faster and more polished than the previous version. The 'open' aspect of the software is really useful - you can make scripts to do almost anything! 

Koeng

[Dakota Hamill]

I think OpenTrons people are on this list.  Followed them with interest for quite sometime.

If you're on here, any colony pickers in the works?  Or 8 span heads? 

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[Dakota Hamill]

Well I should have spent more than 5 seconds on the website. 8 spans are on there.  But an open source colony picker would be huge.

[Koeng]

Right now, we are working on a colony picker (FreeGenes project). It's been going slowly since we've been jamming on some database organization stuff. In combination with the Bionet project, we have a system in the works called OpenFoundry which should allow completely automated assembly, transformation, plating, and picking of DNA for MoClo assembly. Everything actually works so far on the bot except the picking. To solve that, we got a the XL transilluminator box from IO rodeo, and cut out some acrylic that should be able to perfectly fit a 96 well plate. Since we do everything on Nunc rectangular plates which are the standard size, we should be able to attach a webcam to the top of the box to get really nice clear and standard pictures of the colonies. Then port that data through OpenCFU, and convert image coordinates to OpenTrons coordinates. Pick with pipette tips.

Anyway, the robots really work well for automated cloning. The multichannel pipettes are also quite nice, and this one is about 2x faster than the sped up versions of the OT-1 (we have one in lab)

Koeng

[Victor Sayous]

Hello, 

I am writing to you because we are currently on the verge of buying on Opentron OT-2. One of the main purposes is making DNA mix for MoClo assembly. I have seen on your message that you are working on adding colonies picking feature to the OT-2. How is the work going? Is it an opensource work? Do you need some help? 

I am a Ph.D. candidate from France in molecular biology.  I have relatives that are very good developers, so maybe we can help a little. 

Looking forward to hearing from you soon,

Victor 

[Koeng]

It is Open Source work! And it actually works now, so we can colony pick with the OT1. The problem is is that the code isn't very generalized yet. We would love to get collaboration going though!

Sending you an email off-list with more information.

Koeng

[Nathan McCorkle]

On Mon, May 14, 2018, 10:27 AM Koeng <koen...@gmail.com> wrote:

It is Open Source work! And it actually works now, so we can colony pick with the OT1. The problem is is that the code isn't very generalized yet. We would love to get collaboration going though!

Throw it on GitHub gist so we can take a look and maybe provide some refactoring or tips on how to do so:

https://gist.github.com

[Dakota Hamill]

I'd be very interested in getting one if it can colony pick accurately from an image.  I can't codw my way out of a paperbox but know a few people that may want to contribute! 

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[Koeng]

I can also just provide the actual git repo of where all development is happening:

https://github.com/EndyLab/bionet-synthesis/blob/master/pipeline/testing/colony_picking/col_picking.py 

feel free to clone and pull request any changes. Or just recommend them here and we can change the code around.

@Dakota if you are careful with your calibrations, the OT1 can definitely colony pick. The OT2 is even more accurate, so I'd be willing to bet you can definitely make it colony pick.

--- Bit of wall text, sorry ---

The entire "bionet-synthesis" repo is actually the OpenFoundry, which is a codebase developed for the FreeGenes project (freegenes.org). Conary Meyer (a lab colleague of mine) and I learned python a few months ago to figure out how to clone the quantity of stuff that was coming through our pipeline. 

In theory, one should be able to deploy an OpenTrons to any lab and immediately be able to clone literally hundreds of plasmids a week with GoldenGate assembly (we've only cloned about 700 plasmids so far, but we recently got about 2500 more and are going to try to clone every single one within a week). No more thinking about DNA cloning! Just have a robot do it. So, in that way, colony picking is only a small part of a bigger idea on how to process DNA (and one that took a shocking short amount of time).

Conary is writing up a paper on the idea which should publish on bioarxiv within a couple of weeks. 

A lot of the code is quite hacked together, as these projects often are, so any help with cleaning would be appreciated. 

https://github.com/EndyLab/bionet-synthesis/tree/master/pipeline is the main location for scripts we actually use to run things. (so the basic stuff of resuspending synthetic DNA -> Building GoldenGate reactions -> Transforming -> Plating -> Picking). Picking happens to be in "testing" because so much is going on with it.

Also, for the design side, we have synbiolib (https://github.com/EndyLab/synbiolib) which has some generally useful open source synthetic bio tools that can't be found in biopython, like protein optimization or the ability to remove restriction enzymes. (A lot of the tools from the FreeGenes repo https://github.com/EndyLab/FreeGenes will be moving there, such as programs to precision recode sequences, remove restriction enzymes and repeat elements in proteins, and stuff like that). 

Any questions I'd be happy to answer. They're bleeding edge new right now, but we'd eventually like to advertise them to a lot larger crowd.

Koeng

[Dakota Hamill]

Hi Koeng that github is 404.  I brought back up any DIYBio OpenTrons threads because I'm scheduling a demo tomorrow.  Saw someone on reddit that was also trying to develop the OT-2 as a colony picker.  Supposedly it has a camera built in?

Interested to know if anything has progressed on the work.  OpenTrons github doesn't seem to have many open protocols shared but perhaps I'm looking in the wrong place. Wish they had an 8-span P-1000.

Anyone have any experience with sterility inside the plexiglass cabinet it has outside of a laminar flow hood?  I think based on measurements they said the OT-2 rarely fits inside biosafety cabinets.

I don't know if they have a UV light inside or if an aftermarket plus enough ethanol would do the trick.

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[Koeng]

Yea it's been moving around a bit. As I said, under constant development still. 

https://github.com/EndyLab/bionet-synthesis/blob/master/pipeline/colony_picking.py

Got a link to the reddit post? Would love to talk to them as well. The biggest issue in our lab for colony picking is the inconsistent lighting (we have windows) so making colony picking robust is more difficult than it would be to just have a simple box. 

If you take a look in our main pipeline, we have all steps to go from getting synthesis plates in to glycerol stocking clones running on OpenTrons. I just personally ran 960 unique plasmids through this week, and am aiming for ~1500 next week. We had some issues with the colony picking code (the grid layout on our plates is determined by colony density, but variation in our competent cell mixes screwed that. Don't let competent cells settle at the bottom of the trough, kids), so I picked about 700 yesterday. It's honestly not very difficult, and only took about 2 and a half hours. 

Sort of depends on how sterile you actually need it to be. In a rather clean corner of lab, we just sort keep the plates open (on the OT1, which is completely open). The 30 plates I checked out yesterday had absolutely no contamination. Though remember, that is for bacteria. In general, for cloning, it doesn't actually seem like that big of a deal as long as you use the materials quickly and use anitbiotic. I sometimes wipe with ethanol, but most of the time I am just constantly running them. 

Koeng

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[Dakota Hamill]

Thanks for the updated link.

Reddit isn't loading on my computer right now for some reason, probably a bad cookie.

Anyway I believe this is the thread, https://www.reddit.com/r/labrats/comments/8ii734/opentrons_new_robot/

Someone half way down mentioned they were trying to turn it into a colony picker.  I'm sure Ctrl+F will let you find the user.

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[Dakota Hamill]

And now it works when I click via that link....

https://www.reddit.com/user/machinofacture

[John Griessen]

On 06/21/2018 09:39 PM, Koeng wrote:
> If you take a look in our main pipeline, we have all steps to go from getting synthesis plates in to glycerol stocking clones
> running on OpenTrons. I just personally ran 960 unique plasmids through this week, and am aiming for ~1500 next week. We had some
> issues with the colony picking code (the grid layout on our plates is determined by colony density, but variation in our competent
> cell mixes screwed that. Don't let competent cells settle at the bottom of the trough, kids), so I picked about 700 yesterday.
> It's honestly not very difficult, and only took about 2 and a half hours.

Sounds really good. Does your tray need a mini shaker to stay ready longer?

[Nathan McCorkle]

On Thu, Jun 21, 2018 at 7:39 PM, Koeng <koen...@gmail.com> wrote:

Yea it's been moving around a bit. As I said, under constant development still. 

https://github.com/EndyLab/bionet-synthesis/blob/master/pipeline/colony_picking.py

Got a link to the reddit post? Would love to talk to them as well. The biggest issue in our lab for colony picking is the inconsistent lighting (we have windows) so making colony picking robust is more difficult than it would be to just have a simple box. 

Have you thought of using NIR light? Or another method would be to pulse your light source (any color/spectrum) at a known rate (i.e. something that isn't a harmonic of 60Hz, what artificial lights might be pulsing to) and sync your camera to snap a photo on each ON & OFF and then you can subtract the OFF photo, to normalize the ON data from inconsistent ambient lighting (really you only need an OFF image every once in a while, as fast as the lighting might change... so that could be every other frame, or it could be once or twice a minute).

[Koeng]

Shot him a message a couple days ago. Looks like he didn't pursue the idea to completion because things got in the way. 

>Sounds really good.  Does your tray need a mini shaker to stay ready longer? 

The trays/rectangular plates usually need to dry a LOT to soak up the serial dilutions quickly. For the actual synthesis plates, I just sort of vortex them and then put them in a salad spinner to spin the liquid to the bottom (this works FANTASTIC: Better than what I would ask from any piece of actual lab equipment and it's only like $15).

@Nathan McCorkle I'm more of a biological engineer than an electrical engineer, so I actually would have absolutely no idea how to do that :) If you have any resources I could read up on it that would be appreciated! It seems quite promising for ambient lighting conditions, but more engineering required than just a box. 

Koeng

[John Griessen]

On 06/25/2018 12:46 PM, Koeng wrote:

> On 06/25/2018 11:30 AM, John Griessen wrote:
> >Sounds really good.  Does your tray need a mini shaker to stay ready longer?
> The trays/rectangular plates usually need to dry a LOT to soak up the serial dilutions quickly.

So, that means you leave the plates in the opentrons drying for long periods of time?
If so, it does seems like shaking/circular motion would be good for recovering from your case of
"Don't let competent cells settle at the bottom of the trough"?
Maybe forced dessicating by stirred air is desirable too?
This all starts me thinking of a conveyor for trays and a tray stacker at one end.

> For the actual synthesis plates, I
> just sort of vortex them and then put them in a salad spinner to spin the liquid to the bottom (this works FANTASTIC: Better than
> what I would ask from any piece of actual lab equipment and it's only like $15).

So, does this mean that the salad spinner is a way to get all the liquid out of the trays and all mixed together
in the bottom of the spinner?

[Koeng]

Yep, we leave the plates on the opentrons for drying. 

I think I might have accidentally responded incorrectly: The OpenTrons doesn't aliquot the competent cells. I do that myself in the cold room because we generally don't want to put a robot in there. Also, electronic pipettes make it very easy. However, in that manual pipetting, I didn't mix, which I presume is a major problem. 

For me, the #1 add on to an OpenTrons would be a plate handler + a microplate sealer. If I had that, I could automate nearly everything on the machine. (except making mastermix, because I don't trust the bots to get every last drop of enzyme)

After vortexing, there is a lot of liquid on the microplate seal. When I tear it off, liquid gets everywhere and it sucks because it can cause contamination and majorly reduces the liquid in each well. An improv plate spinner made of a salad spinner fixes all that! Though, now that you mention it, might be a good way to get liquid out of plates after cleaning them.

Koeng

[Dakota Hamill]

Letting the omnitrays dry in a biosafety cabinet or laminar flow does help them to suck up liquid better as Koenig mentioned.  With 96 well spacing I've seen even 1-2uL drops on fresh poured plates find a way to draw into eachother making for a useless bioassay reading from bleeding.

I got the online demo of the OT2 on Friday.  I wish they had an 8 span P1000.   With the list price of the machine plus two 8-span pipettes at ~$6,000 you're looking at a price comparable to some used TECAN EVO's with larger plate decks and a multitude of other head attachments for picking/streaking.

The open source software is a benefit, though I'm code-incompetent so not sure I could make use of it's customizability anyway.

I agree add-ons like hotels, plate sealer, and a small arm/expandable deck would make it even more attractive even at $10k-$15k.

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[Koeng]

Yea, we use about 7.5ul droplets. Also, we just dry them open on the bench :) It works without contamination, which is surprising since we are adjacent to some fungal folks, so I guess Stanford is just pretty clean. 

For the p1000, I'd just pipette 3-4 times with the p300. It's a little bit annoying, but whatever. 

I'm strongly against just going with used lab robots. I have a CAS1200 right next to me, which is a fantastic little bot for what it does, but is rapidly getting outdated and I would not invest time in. Tecan I've had horrible experience with. Last lab we had an evo that I wanted to get a PCR protocol on, and they quoted us 10k to have a specialist come out and install it. Sure, you get picking and streaking, but you also get nailed down to a single software source that basically can just screw you whenever they want. Which is what happened back in last lab. Even Tecan's service has been subpar for me. Sure, they respond very quickly, and kindly, but there isn't substance I get out of those conversations ("We have the manual for your model plate reader, let me send it right over!" with no response afterwards even with multiple followups, or "the software for your plate reader can be downloaded for $3000, or you can use our free version, which has basically no features and it only works on windows xp"). And even that has been better than my experience with other companies, which when I mention I'm working at home simply ghost me. 

Another lab next to us works with RNA. Basically, they had to reimplement the tecan API from scratch to get it to do what they want, which has taken hundreds of dev hours. With the OT, since we had their actual source code, we've actually tweaked it itself to do what we needed. Saved tons of headaches and hours. 

Those attachments aren't going to be cheap. The software isn't either. Meanwhile, within a week or two we can get colony picking working on an OT. There's hidden costs in investing in robots, because you also invest in the ecosystem. I bought this CAS1200 for $350 off ebay, and it has all those nice little features like liquid detection, nice metal blocks to keep enzymes cold, and a UV lamp on it's top for sterility. I still use the OT1 far more often, which in almost any metric of hardware is an inferior robot. However, it can seamlessly integrate with the rest of our software stack, and that is HUGELY beneficial for productivity, and we can chat with their actual development team instead of salespeople. 

It's pretty easy to code in python. You can probably pick it up in a couple of days. In addition, your productivity only accelerates once you get used to the platform, in comparison to other more gui-based platforms, that don't scale quite as easily.

TL;DR: Raw price and hardware is not a good metric to determine which robot to use.

Koeng

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[Michael Crone]

Out of interest has anyone tried to use Antha developed by Synthace? We have a Felix in my lab and I'd like to get it up and running because it is easier to justify using that than buying the cheaper opentrons. Would be interesting as well if Antha will also support opentrons.

I agree most of the GUIs developed by these companies are terrible so anything that is command line based would add to flexibility.

[John Griessen]

On 06/27/2018 02:42 AM, Michael Crone wrote:
> We have a Felix in my lab and I'd like to get it up and running because it is easier to justify using that than buying the cheaper opentrons.

What makes it easier to justify? After Koeng's email on how wonderful programmability without vendor lock-in is, I don't get you.

[Michael Crone]

Antha removes the vendor "lock-in" that you are talking about and allows for cross-platform workflows (which becomes useful when you are working in a DNA foundry environment). And when you already have a $30 000 robot it's difficult to justify to your PI to buy a $10 000 one... especially when that robot is already fully equipped with all of the accessories.

I completely agree though that the opentrons is great for every day work in the lab and its programmability makes it very attractive.

[John Griessen]

On 06/28/2018 01:30 AM, Michael Crone wrote:
> Antha removes the vendor "lock-in" that you are talking about and allows for cross-platform workflows

OK. I'll read about antha.
Maybe it will trigger me to develop an inexpensive microplate conveyor system and "forklift arm" microplate handler.

--
John Griessen
industromatic.com Austin TX building lab gear for biologists

[cathal...@cathalgarvey.me]

Antha is nice. I haven't even dabbled, but I got the tour once from the developers (who are great people, by the way). Last time I saw it, it was very similar to a declarative DSL of Go, and could be extended easily enough using Go. This means that for coders who want to add device support they can use low-level manual memory management, but for coders who want to add features for defining experiments etc., they don't need to worry about memory management as Go handles that using a GC. The Go syntax is relatively easy to learn, also; it's not Python, but it's minimal enough to make up for it.

The overarching idea with Antha was not only to be a driver software suite for hardware, but to be an experimental definition language. So you could say "assuming a device to do operations X,Y,Z, conduct an experiment with an 80:20 split of tests to controls that does X then Y then Z".. and in theory you wouldn't even need to define specific volumes or plate formats at time-of-writing, you'd define what you had available for each device at runtime and Antha would figure out how to string that together.

The developers, Synthace, use it in production and last time I heard they were doing great. So Antha ought to be well-supported for a long time hence, and it's GPL licensed so it can outlive them in the worst case.

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[Koeng]

Antha is also pretty pricey per year, I think 4 times an OT cost?

That said, I met with some developers of Antha and I really like it. Their GUI is really great for management and wonderful for multifactorial optimization. Their data from optimizing GoldenGates was very impressive. It's a piece of proprietary software I would consider purchasing, if I had the money to, since mainly what you're paying for is support and the user interface supporting the bottom-level code. Also, yea it's not really vendor lock-in because it works across many platforms - but python is pretty hard to beat when working with libraries.

Here are some videos of our OT1s doing work with our codebase. When in "build-mode" we run about 3 at a time, cloning about 300 plasmids a day. 

OT doing GoldenGates

https://www.youtube.com/watch?v=-_tEMNG0I3U

OT doing transformations

https://www.youtube.com/watch?v=IQA4v-9h0W8

OT doing plating (from above)

https://www.youtube.com/watch?v=FxHn1avxR-4

OT doing plating (from back)

https://youtu.be/xXuF4v3vZ78?t=11s

(At the end of this video, you can see how we're doing the serial dilutions. Extremely important to note: you want to slowly dispense the liquid above the plate, creating a small droplet that sticks to the sides of the tip, and THEN move down to drop the droplets while stabbing the agar a little. If you do it when you're already down in the agar, you're gonna have a bad time)

Koeng

[Koeng]

(actually, if I remember correctly, you have to purchase actual Antha for support of running on the bots? It seemed like it was good for labs doing high throughput work but didn't want to have to think about running robots and developing code)

[Cathal Garvey]

It appears that Antha itself is still FLOSS and gratis: https://github.com/antha-lang/antha

However, perhaps this GUI you mention is not FLOSS, and it's possible that they are not releasing device drivers. Because they are the copyright holders of Antha, they can do that even though for someone else it would be a GPL violation. :/

I'd still expect the OpenTrons Antha drivers to be open though, because it's OpenTrons.

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[Koeng]

Last time I talked to one of the devs, they would like to get an OT port once they're more widespread. It's not here yet. 

And yep, that sounds about right for copyright situation. I'd rather write my own software and own it under MIT.

Koeng

[Michael Crone]

We're looking at getting the OT-2. I was wondering if anyone could please give me an idea of the most useful pipettes and modules for starting out. We're a synthetic biology lab (we have an Echo and Felix, but nobody uses the Felix because it is such a pain). I was thinking that the multichannel 50-300 would be good (mainly minipreps and other wash steps for RNA purification etc), along with probably all the single channel pipettes. And then the magdeck initially (for minipreps and other purification). Is the tempdeck useful? What kind of applications would you use it for? Scale transformations?

I was hoping to get someone's experience just to inform our own decision!

Thank you!

Michael

[Dakota Hamill]

Is there a repository of Open Tron protocols outside of the main websites extremely small library?  Just got ours up and running and it looks like I'm going to need to learn to do some coding.  Have a deep well 24-well plate and there's nothing in the database for that, and no simple UI to adjust plate height.

The software also doesn't allow using an 8 span to go from 24-well spacing to 96 well spacing.  Just planning on removing every other pipette tip and doing it that way, but again looks like something that must be custom coded. 

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[Koeng]

I have a personal repository of opentrons protocols, but they probably wouldn't be very useful to you (I'm also moving towards using all json protocols since they're easy to generate and track with an actual database). In essence, no not really.

Have you used their protocol designer software? That might be able to do what you want. Also, I'm not really sure if the pipette itself is physically able to go from 24 to 96 well plates.. do you actually have one right now?

For complicated stuff like 24-to-96, you'll probably have to roll out your own little piece of software, which shouldn't be too hard given their API. 

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[Dakota Hamill]

Anyone have any dimensions on the OpenTrons tube tack holders or TipOne box holder?  

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[Koeng]

I know that the tube rack holder at least is open source, you can probably just email the OpenTrons folks to get the file

[Dakota Hamill]

We just hired a computer science intern for a few weeks to develop some protocols for us since I'm code-illiterate.  The OpenTrons protocol library is pretty empty.   @Koeng do you still have that file of plating onto omni-tray surfaces from 96-well format?  If not I'm sure we can figure it out soon enough.  Thanks. 

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[Koeng]

I think I have it somewhere on our lab computer. Will try to get you a link once I get back to lab