August 2022 | Science

[DIYbio] Four questions about reliability of a wet lab action

5:55 AM |

Greeting reader(s):

I posted this question buried in a deeper strand, I think maybe it has to bubble up to get noticed enough to snag opinions, answers; maybe some direct experience 'stories':

Question began 'sort of' to to Sean Sullivan, possibly but really a general question

Here are four questions about creating a gene edit and expression, ex Yeast, even e coli, etc... Some plate and Petri dish wetlab style work. All are about framing expectations and resources, the assumption is the technology is 'best we can get', and/or almost isn't the point, anyway.

Reference Situation:

*) Gene BP count is 3K NT, promoter, tails, introns if any etc known to work generally.

*) Expression system known, like locally managed Yeast or 'whatever'

*) Selection itself is reliable, UV florescence or some other property

Questions:

Q1) How many labor hours is typical, no paperwork time, just wet lab including end game verification ?

Q2) Whats the success rate of a new germ line emerging in percent ?

Q3) How often is a success rate wrong ultimately. not that the Gene did do what is expected, but some 'other' flaw made it not really get expressed ?

Q4) Is sequencing the believed usable final modified colony invariably reliable enough to make Q3 irrelevant ?

Is Q2) Closer to what ? 30% 80 %

For Q1) I mean hands on time with endless revisits to the plates/wells, dishes, tubes, etc. and so on, not including incubation times, centerfuge, etc.

Thanks in advance of anyone who answers. As people like me want to make this technology, not science, this is a big thing hugely.

Thanks!

Daniel B Kolis

my ref: nafl, 31 Aug 2022, diybio

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[DIYbio] Line item success fail rate

8:34 AM |

Question to Sean Sullivan, possibly but really a general question

Anytime the question is asked ? "How often does this succeed ?" The usual answer starts with a long observation; ex: "It depends ...". Ok

Recall from a strict numerical method approach, if success or failure criterea can be decided upon per act, and acts batched into some kind, there is always a number in the noise, and an important one. What is the line item success rate, as usually composed ?

Sure some if;s in the question ! Situation:

*) Gene BP count is 3K NT, promotor tails, introns if any etc well known to work.
*) Expression system known, like locally managed Yeast or 'whatever'
*) Selection itself is reliable, UV florescence or some other property

Q1) How many labor hours is typical, no paperwork time, just wet lab including end game verification ?
Q2) Whats the success rate of a new germ line in percent ?
Q3) How often is a success rate wrong ultimately. not that the Gene did do what is expected, but some 'other' flaw made it not really get expressed ?
Q4) Is sequencing the believed usable final modified colony invariably reliable enough to make Q3 irrelevant ?

Thanks in advance of anyone who answers. As people like me want to make this technology, not science, this is a big thing hugely.

is Q2) Closer to what ? 30% 80 %

For Q1) I mean hands on time with endless revisits to the plates and so on, not includng incubation times, centerfuge, etc.

Thanks!

Daniel B Kolis

my ref: nafl, 30 Aug 2022, diybio

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[DIYbio] NGS Next Gen machine going, LCD Human factors too

7:39 AM |

Hello

https://www.youtube.com/watch?v=tEv_CZqYM2c

This video about an automation sequence apparatus is pretty interesting.

I think the human factors of the LCD screen 6m to 11m for instance, is interesting.

The scripting language would interesting to understand. specifically, a few arms run independently and dont crash into each other. I wonder if the program is messy for that or its solved by the algorithms that read it.

Late in the explanation a user shows a slide noting the staggered ability of the machine to work in time means normally variable times are super close. that is, '90 min' gets to be exactly that without the human factors to do the squirting by hand....

Regs

Dan

my ref: 24 Aug 2022, nafl

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Re: [DIYbio] Multichannel Arm (MCA) trouble on Evoware !

5:47 AM |

Basics first: have you double-checked the worktable definitions? And are you able to share screenshots of your worktable setup (also the one that works, please)?

Cheers,

Rikke

On Mon, Aug 22, 2022, 07:12 Isabelle Simon <is.isabellesimon@gmail.com> wrote:

Hello there,

I'm actually working on a script using the MCA96. It is supposed to use stackable DiTis sites such as in this video : https://www.youtube.com/watch?v=ItH-2kebS_8 . However, on the evoware script, I'm only able to enter site 1 and 2 as DiTis sites, whereas the other values trigger an error "illegal tip length".
Another script using the exact same Labwares, Carriers and line code can make it work, but it does this error whenever we apply this script to my working table

I cannot find any information online, Does someone have any idea ?

Thank you

Isabelle

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[DIYbio] Re: Files for DNA / RNA creation via automation - oh wow

7:59 PM |

Thank you Jake.

My proposal which has morphed into a running IDE ( predictably, a X11 App in Python3 ) seems like it co-exists unusually well with a number of Synthetic Biology software integration projects. Thank you for your message, it was VERY helpful nearly immediately. I had no difficulty in getting accredited for Google group BioProtocols and have been reading the supplied materials over the last 2 hours...

Here is the abstract of a primary document I wrote about the centroid of my interests:

The creation of a computer-script language named Nucleotide Assembly Functional Lan-
guage (NAFL) attempts to achieve two objectives: Firstly, improve the scope of FASTA
searches by making them more generalized and include non-Central Dogma atomic
elements correctly. Secondly, host the modelling of many life science programmes in
new Synthetic Biology with less or no custom programming.

The amount of sequence, sequence-like and ancillary information when working through
a specific life science problem is immense. NAFL supports metadata that follows com-
puter queries and modelling as work proceeds automatically. This is particularly relevant
in the frequent case where work sessions span multiple sittings and intermediate re-
sults are shared over time and space with multiple workers. FASTA and sequences of
nucleotide and gene-like sequences are the most prominent organisational elements
in the code's Integrated Design Environment around NAFL. Improving the interac-
tion of searches executed for DNA, RNA and proteins against databases has direct
utility; but this new nomenclature in machine readable form also avoids changing
programs to understand returned sequence matches. The computer dialog focuses on
the user experience and avoids repeating similar steps with slightly different criteria
by representing lists of project components as opposed to working through one at a
time sequentially. The subsidiary tasks for many tasks like protein folding and mRNA
planning is supported with similar syntax human-machine dialog.

NAFL is constructed from first principles to be uniform across computing platforms;
Enabling the execution of stored scripts on personal, cloud and cluster configurations
interchangeably in any common operating system. A goal is to move the user experience
into direct real-time encounters with problems and solutions. In contrast, the usual
creation of endless database flat files and maintenance of notes is seen as an old world
practice. Instead, NAFL substitutes interactivity and automates much note-taking. Since
sizable delays do occur, NAFL specifically implements asynchronous human-machine
interactions; Instead of waiting many works-in-process results are returned out of order
as partial solutions evolve.

Back to your helpful message; ( thank you again ! )

SBOL(3) is useful for some sorts of improvements of sequence BP management to outcomes, I've carefully pulled apart a vast whole cell simulation by a bunch of workers in the shadows of Venter... Maybe 2/3 of the 40 models inside as motifs of simulation are imported as SBOL.

This is that project, its pretty remarkable really:
https://www.cell.com/action/showPdf?pii=S0092-8674%2821%2901488-4

Jake said:
" PAML is a draft standard for a machine-independent protocol specifications.
http://bioprotocols.org/ It currently supports export to Markdown for human execution and to
Autoprotocol, and there are current efforts to support OpenTrons, and to map from SEP055 plans
into PAML protocols. "

Dan continues:

I work on the IDE which barely even addresses the notion I propose. I am so invested in a non-batch processing, real-time s/w for Base Pairs the deeper notion is still barely touched.

I tell people: "Im trying to write a masterpiece"....

If any ideas occur to you about the NAFL language notion, lets poke some chars into this pipeline here !

I toy with the idea of rebuilding this work into a real IDE so Venters DIY-cell could be start, stopped, etc like a program in a debugger. This article points ot more formal papers, etc. This was written 100% ass-of-fire any old way to get it to work for show and tell. That's just wonderful, but its not orderly enough to use too directly. Its rough nature proves the point its possible, a serious milestone achieved.
https://www.cell.com/action/showPdf?pii=S0092-8674%2821%2901488-4

I think on a few computers together it could simulate a single Venter cell at 10 times real time.

Regards,
Dan Kolis

my ref: nafl, blogs, 22 Aug 2022, jake101

On Saturday, August 20, 2022 at 9:30:57 AM UTC-4 jake...@gmail.com wrote:

To the best of my knowledge, what you're looking for doesn't currently exist, but there are two open standards efforts that I'm involved in that are aiming to bring it into being.
1) SBOL best practices proposal for "Representation of Parts and Devices for Build Planning": this is a set of vocabulary and representations to describe a synthesis and assembly plan. https://github.com/SynBioDex/SEPs/blob/master/sep_055.md
2) PAML is a draft standard for a machine-independent protocol specifications. http://bioprotocols.org/ It currently supports export to Markdown for human execution and to Autoprotocol, and there are current efforts to support OpenTrons, and to map from SEP055 plans into PAML protocols.

In short: there's a group trying to put together the capability you're looking for as a free & open source community project, and if you're interested in contributing, more hands are welcome!

Thanks,
-Jake

On Friday, August 19, 2022 at 9:33:49 PM UTC-5 dank...@gmail.com wrote:

Thanks again Ravi Ramana

That was spot on helpful, Thanks 1e6 dude...

The way the entire assembly task is specified in machine readable form is what I wanted. That's is a wonderful negative example really.

Nearly a hundred CSV files mostly two columns of identifiers, then 9 base pair sets "deep in the heap". Starting with duplicating this completed project is months of work to just detangle those relations with iterated smashes of scary wet-lab. Assuming the goal is to vet the equipment, workflow and some science buried in those big ominous details....

Just because somebody else's computer understands a heap of files, does NOT mean any human does, so the transmission of know-how is negligible; ( but not completely absent. Depends on how many months you have ).

My presumption to improve such a thing is also pretty suspect in some regards. But that doesn't keep me from trying ...

As attached, X11 app for doing this with some semblance of rationality...

Reality is not created by gluing together 33K Excel files to make a fancy critter like you.

my ref: 17 Aug 2022, Toronto, NAFL, blog

On Wednesday, August 17, 2022 at 11:24:09 AM UTC-4 Nathan McCorkle wrote:
Just search Google for genbank. You'll find plenty of files.

On Tue, Aug 16, 2022, 3:16 PM Dan Kolis <dank...@gmail.com> wrote:
Greetings,

I asked this on this blog earlier but maybe it god lost in threads of other issues at hand.

Of course its possible what I'm asking about doesn't really quite exist, and/or the question's just been disregarded.

Hmmm.

I'm hoping for a file or two that is used for submission to a machine or hybrid of piece of equipment and people to spec making a B.P. fragment, either/or DNA or RNA.

The file ( ex. example ) maybe a URL for its definition. Im interested somewhat in more then just a string a nucleotides but what sort of metadata is closely coupled to the files design.

Any feedback is appreciated,

Dan Kolis

my ref: 16 Aug 2022, NAFL, blog, bio

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Re: [DIYbio] Purchasing DNA synthesizer

9:15 AM |

Been thinking about part of @AndrewHessel's response earlier in the thread where he said:

"I don't recommend doing it at home/garage/etc if you're planning on working with proteins or short metabolic pathways just because of the economics. Your time and money are better spent on protein/metabolic engineering etc."

Do y'all think he was talking more about the importance of improving/developing automation around constructing and testing different sequences or simply that enough brainpower is going towards the DNA synthesis problem and more should be spent on what we are going to make with cheap DNA (any why)?

@Dank in terms of hiding a lot of the complexity that goes into running a given experiment, many vendors now sell kits that contain all the reagents + consumables + instructions needed to do things like isolate plasmids, generate cDNA libraries, prepare DNA for next-gen sequencing, etc. Even since the start of my PhD, I feel that is has become easier to find 'off-the-shelf' kits for well-established methods.

On Tuesday, August 9, 2022 at 2:52:48 PM UTC-4 Andrew Hessel wrote:

Great that DNA writing is popping up again. It's so foundational yet has received so much less attention than reading DNA. This said, making gene-length fragments is pretty standard and inexpensive these days and is offered by a spectrum of commercial groups. I don't recommend doing it at home/garage/etc if you're planning on working with proteins or short metabolic pathways just because of the economics. Your time and money are better spent on protein/metabolic engineering etc. Also, if you're planning on using older, organic chemistries to make DNA, keep in mind the chemicals required and waste products produced are not great to have around your home or garage. You may want to explore newer enzymatic-based chemistries coming online that are much greener. The idea of a chip-based oligo assembler has been around for a while but to my knowledge no one has been able to get it to work well -- it would be great to have this revisited, but I still would have the oligos pre-synthesized and focus mainly on getting the assembly processes working well. Note that chip-based foundry systems and chip-based test and measurement systems are getting a lot of attention these days. I point people to these two papers to get a sense of where things stand -- Venter's recent review of synbio -- pay particular attention to Figure 4, which describes Avery bio's chip-based DNA synthesis system -- and Roswell's description of their Molecular Electronic chip -- a general purpose, single molecule sensing platform. The problem of making long DNA assemblies necessary for synthetic genomes has not yet been solved. My baseline is E. coli K12 from ATCC, which retails for $400. The genome is about 4.5 megabases. At current synthesis prices, about $0.10 base, K12 would be a $450,000 print job. When it's $450 to print the genome -- and we have base-level control of the entire chromosome -- no one will order the microbe from ATCC again. I look forward to this day.

As Bryan says, onwards and upwards.

Cheers, Andrew

On Tue, Aug 9, 2022 at 5:05 AM Bryan Bishop <kan...@gmail.com> wrote:
Thanks everyone. I am still around (and so is Nathan), and I actually sent an off-list email earlier indicating my interest in funding this project. Onwards and upwards,

- Bryan
https://twitter.com/kanzure

On Tue, Aug 9, 2022, 3:15 AM Brian Degger <brian....@gmail.com> wrote:
https://diyhpl.us/wiki/dna/dna-synthesis.html a the page Bryans on DNA Synth.

Otherwise, search the research papers on microfluidic dna synth.

Cheers,
Brian

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[DIYbio] Multichannel Arm (MCA) trouble on Evoware !

6:13 AM |

Hello there,

I'm actually working on a script using the MCA96. It is supposed to use stackable DiTis sites such as in this video : https://www.youtube.com/watch?v=ItH-2kebS_8 . However, on the evoware script, I'm only able to enter site 1 and 2 as DiTis sites, whereas the other values trigger an error "illegal tip length".

Another script using the exact same Labwares, Carriers and line code can make it work, but it does this error whenever we apply this script to my working table

I cannot find any information online, Does someone have any idea ?

Thank you

Isabelle

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Re: [DIYbio] Purchasing DNA synthesizer

4:33 AM |

+2. I'd like a copy as well. Pls

Sent from my Galaxy

-------- Original message --------

From: "Cory J. Geesaman" <cory@geesaman.com>

Date: 2022-08-09 8:28 a.m. (GMT+03:00)

To: DIYbio <diybio@googlegroups.com>

Subject: Re: [DIYbio] Purchasing DNA synthesizer

Bryan Bishop, that was his name - if he's still around I'd love to get a copy of that research repo if he still has it.

On Monday, August 8, 2022 at 9:06:21 PM UTC-4 djwr...@gmail.com wrote:

I would also check with Nathan McCorkle and Bryan Bishop.

Dan Wright

Sent from my iPhone

On Aug 8, 2022, at 10:03 AM, jerry maxwell <jerrymax...@gmail.com> wrote:

Contact Sierra Biosystems (Certified Scientific) at 209-288-2445. They build the new SB "Shasta" as well as several variations of K&A machines for synthesis of oligos.

Sent from my iPhone

On Aug 8, 2022, at 8:24 AM, Abizar Lakdawalla <abi...@gmail.com> wrote:

How are you doing the oligos synthesis, cyanoethyl protection?

On Mon, Aug 8, 2022, 8:09 AM Koeng <koen...@gmail.com> wrote:
Hi all,

I am working on creating an open source chip for oligo synthesis. In order to test it out, I'm in need of an oligo synthesizer (that has a flow cell I can hack to do what I need). Any advice on where to buy a good used one? Or even better, does anyone have an oligo synthesizer on hand they'd be willing to sell?

Thanks,

Keoni

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Re: [DIYbio] Purchasing DNA synthesizer - Question about BP build out

12:44 PM |

Hi

With a good practice of Arduino/Raspberry pi, with a 3d printer and some knowledge of synthetic biology (and a huge motivation of course) would it be possible, for educational purposes, to build a machine that would synthesize a very small molecule of DNA ?

I had seen some prototypes on the IGEM site :
https://2015.igem.org/Team:Cooper_Union/Loomino_Description
https://2021.igem.org/Team:Aachen/Description
(they use enzymatic method)

DNA electrowetter below
https://forum.arduino.cc/t/dna-electrowetter-device/956082

(and assuming the reagents would be accessible...)

Cheers

Karim

Le 20/08/2022 à 16:03, Koeng a écrit :

> Independent of the hardware, how do you propose to append promoters, markers, tails, etc into the gene(s) desired ? Getting them expressed may be non-trivial...

GoldenGate. Expression is outside of outside of scope, though it's kinda easy in my experience.

> Is there a Q.C. step that's understandable for intermediate results ?

Sequencing.

> Being specific to answer this may benefit you... Answering a question to others often clarifies it for yourself too.

I've cloned thousands of genes myself and was part of designing of many genetic toolkits (led https://stanford.freegenes.org/ for 3 years). Also a main contributor of Poly (https://github.com/timothystiles/poly), and founded a company doing full automation for synbio (in particular, the DNA assembly space). I've built functioning systems for this task before, which is honestly quite a bit more illuminating than talking about building them.

On Friday, August 19, 2022 at 7:33:49 PM UTC-7 dank...@gmail.com wrote:

Hello 'k-101',

Independent of the hardware, how do you propose to append promoters, markers, tails, etc into the gene(s) desired ? Getting them expressed may be non-trivial...

Is there a Q.C. step that's understandable for intermediate results ?

Being specific to answer this may benefit you... Answering a question to others often clarifies it for yourself too.

ex: a Fat jupyter notebook full of files and notes, or what ?

Big projects are fun projects, you named yourself one.

Good luck !

my ref: 19 Aug 2022, DIYbio, NAFL, koeng101, Question about BP build out

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Re: [DIYbio] Purchasing DNA synthesizer - Question about BP build out

7:03 AM |

> Independent of the hardware, how do you propose to append promoters, markers, tails, etc into the gene(s) desired ? Getting them expressed may be non-trivial...

GoldenGate. Expression is outside of outside of scope, though it's kinda easy in my experience.

> Is there a Q.C. step that's understandable for intermediate results ?

Sequencing.

> Being specific to answer this may benefit you... Answering a question to others often clarifies it for yourself too.

I've cloned thousands of genes myself and was part of designing of many genetic toolkits (led https://stanford.freegenes.org/ for 3 years). Also a main contributor of Poly (https://github.com/timothystiles/poly), and founded a company doing full automation for synbio (in particular, the DNA assembly space). I've built functioning systems for this task before, which is honestly quite a bit more illuminating than talking about building them.

On Friday, August 19, 2022 at 7:33:49 PM UTC-7 dank...@gmail.com wrote:

Hello 'k-101',

Independent of the hardware, how do you propose to append promoters, markers, tails, etc into the gene(s) desired ? Getting them expressed may be non-trivial...

Is there a Q.C. step that's understandable for intermediate results ?

Being specific to answer this may benefit you... Answering a question to others often clarifies it for yourself too.

ex: a Fat jupyter notebook full of files and notes, or what ?

Big projects are fun projects, you named yourself one.

Good luck !

my ref: 19 Aug 2022, DIYbio, NAFL, koeng101, Question about BP build out

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Re: [DIYbio] Re: Off-grid Algae Storage?

4:37 AM |

Even getting 1 kW for 9.5 h per 24 for a m^3 of 'footage' is a hard sell for human created stuff.

Ravi said:
> Conversion to Matter/Materials/Chemicals is what Life is good at.
> Algae are good at it.

As an existent energy source Methane is misrepresented substantially. First combusted any old way it still benefits pollution abatement with 1/2 the burden of higher C-Chain fuels.

A Black box to take mostly Methane from any source and yield energy + C composited to a solid not a gas makes Methane much more amenable to a lean-in as a energy yield system without any excuses.

A haemoglobin like subsystem in built life con-existent with fuel cell ... whatever to nail down carbon would be worth a substantial energy allocation. H2 whether as 'city gas' from Anthracite or made is usable with considerable difficulties of all sorts. CH4 is easy to use, store, societies have considerable experience handling nat gas safely too..

The usual notion when C and O2 are in a system, someplace CO2 comes out. Ok, but managing Oxygen carefully is something life does well. Again: CH4 in, C-anything solid + usable energy. Combustion generally burdens such a process incredibly with unwanted high temperature products. No life science system works with effluents as hot as burning ... anything. Even if a processor had layer after layer of a microbial life to get C2 isolated its advantage procedurally compared to H2 is may be a game changer. Graphene-ish out I suppose is best. Diamond is a reference mtl in the notion too.

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[DIYbio] Re: Files for DNA / RNA creation via automation - oh wow

4:14 AM |

To the best of my knowledge, what you're looking for doesn't currently exist, but there are two open standards efforts that I'm involved in that are aiming to bring it into being.

1) SBOL best practices proposal for "Representation of Parts and Devices for Build Planning": this is a set of vocabulary and representations to describe a synthesis and assembly plan. https://github.com/SynBioDex/SEPs/blob/master/sep_055.md

2) PAML is a draft standard for a machine-independent protocol specifications. http://bioprotocols.org/ It currently supports export to Markdown for human execution and to Autoprotocol, and there are current efforts to support OpenTrons, and to map from SEP055 plans into PAML protocols.

In short: there's a group trying to put together the capability you're looking for as a free & open source community project, and if you're interested in contributing, more hands are welcome!

Thanks,

-Jake

On Friday, August 19, 2022 at 9:33:49 PM UTC-5 dank...@gmail.com wrote:

Thanks again Ravi Ramana

That was spot on helpful, Thanks 1e6 dude...

The way the entire assembly task is specified in machine readable form is what I wanted. That's is a wonderful negative example really.

Nearly a hundred CSV files mostly two columns of identifiers, then 9 base pair sets "deep in the heap". Starting with duplicating this completed project is months of work to just detangle those relations with iterated smashes of scary wet-lab. Assuming the goal is to vet the equipment, workflow and some science buried in those big ominous details....

Just because somebody else's computer understands a heap of files, does NOT mean any human does, so the transmission of know-how is negligible; ( but not completely absent. Depends on how many months you have ).

My presumption to improve such a thing is also pretty suspect in some regards. But that doesn't keep me from trying ...

As attached, X11 app for doing this with some semblance of rationality...

Reality is not created by gluing together 33K Excel files to make a fancy critter like you.

my ref: 17 Aug 2022, Toronto, NAFL, blog

On Wednesday, August 17, 2022 at 11:24:09 AM UTC-4 Nathan McCorkle wrote:
Just search Google for genbank. You'll find plenty of files.

On Tue, Aug 16, 2022, 3:16 PM Dan Kolis <dank...@gmail.com> wrote:
Greetings,

I asked this on this blog earlier but maybe it god lost in threads of other issues at hand.

Of course its possible what I'm asking about doesn't really quite exist, and/or the question's just been disregarded.

Hmmm.

I'm hoping for a file or two that is used for submission to a machine or hybrid of piece of equipment and people to spec making a B.P. fragment, either/or DNA or RNA.

The file ( ex. example ) maybe a URL for its definition. Im interested somewhat in more then just a string a nucleotides but what sort of metadata is closely coupled to the files design.

Any feedback is appreciated,

Dan Kolis

my ref: 16 Aug 2022, NAFL, blog, bio

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Re: [DIYbio] Purchasing DNA synthesizer

1:16 PM |

Human nature foments to invariably underestimate cost and time, this is no exception. I saw a recipe for a mRNA experiment and the reagents and one-of juices, slides, trays and whatnot was many dense pages. All sorts of different vendors. How many new accounts to open can a P.A. do in a workday ? Maybe 12 ? Some stuff over borders, safety paperwork, on and on it goes.

Like the standard notion: "You see the top poking out, the volume of the glacier below the waterline is 90% of what you see".

But if Synthetic biology wants to take off, these easy no brainer issues could be addressed. But, It's no fun. Nobody gets a Nobel prize for standardising ordering chemicals. Stuff like emulating retail generally and using barcodes, precise minimums and maximums for cell sizes, is possible. There is some chance standardization like this is still now premature.

Imagine the laughter at NIH to read you want to write a machine learning program ( OH ! Its A.I. ! ) to get the right jars of goop in at the lowest price quickly.... To preprint a foot of stickers for experiments instead of hand write them, etc.

All the constraints like temperature, 'best before'. I know a cancer researcher who spends maybe 1/2 her workday in a BSL-3 lab trying to keep some stupid fussy cells alive.

The details !

On Friday, August 19, 2022 at 3:13:10 PM UTC-4 Koeng wrote:

Length, which is a function of reliability

> is it because of business model choices of the companies

Absolutely.

> Some more light on this will be helpful.

I wrote a couple essays on this topic here - https://keonigandall.com/posts/affordable_dna_2.html

> My understanding is synthesis needs to get way more cheaper as per the "data storage" drivers. Wondering if it's just hype or realistically possible.

The reason DNA is expensive right now, IMO, is that companies spend a lot of money on development of technology and the cloning process, and don't have enough users to aggregate the development costs against. You can always just solve the first bit with shittier/older technology, but reduce the requirement of cost aggregation, and therefore get a much more affordable end product.

On Friday, August 19, 2022 at 9:56:24 AM UTC-7 Ravi Ramana wrote:
By Enzymatic synthesis about 5X better you mean in which factor - speed or efficiency or length ?

And oligos being 100x-1000x expensive than what current tech could do - is it because of business model choices of the companies
or
you mean the current tech base is amenable for that kinda improvement. Some more light on this will be helpful.

My understanding is synthesis needs to get way more cheaper as per the "data storage" drivers. Wondering if it's just hype or realistically possible.

On Friday, August 19, 2022 at 7:30:21 PM UTC+5:30 Koeng wrote:
Oligos have to be stitched together into genes. Enzymatic synthesis is about 5x, last I heard, better than chemical, but also much more expensive and complicated to run.

> This barrier shd be broken immediately, if so.

It is very difficult, but not impossible.

On Thursday, August 18, 2022 at 10:17:05 PM UTC-7 Ravi Ramana wrote:
Any specific reasons why the differentials are so high? other than the long writes between gene & oligos?
Also, you saying currently we could do 0.001 to 0.01 per bp already and its business model choices its' 100-1000X higher presently? That's tall.
Enzymatic synthesis offerings even don't get those price points as of today. Curious to look at any pointers. This barrier shd be broken immediately, if so.

On Wednesday, August 10, 2022 at 12:43:36 AM UTC+5:30 Koeng wrote:
@Abizar Yep

@Dan awesome! I'll contact you about that.

@Bryan let's definitely talk!

> making gene-length fragments is pretty standard and inexpensive these days and is offered by a spectrum of commercial groups

Over 200x price difference between genes and oligos. Oligos are about 100-1000x more expensive than they could be with current technology. I am aware of current commercial groups - I used to run the FreeGenes Project and oversaw a few million base pairs of synthesis from Twist.

> The idea of a chip-based oligo assembler has been around for a while but to my knowledge no one has been able to get it to work well

Genscript uses it in production. Already have reached out to Drew Hall + some of the Avery folks, since they're jumping on since the patent is expiring from custom array (just like me)

On Tuesday, August 9, 2022 at 11:52:48 AM UTC-7 Andrew Hessel wrote:
Great that DNA writing is popping up again. It's so foundational yet has received so much less attention than reading DNA. This said, making gene-length fragments is pretty standard and inexpensive these days and is offered by a spectrum of commercial groups. I don't recommend doing it at home/garage/etc if you're planning on working with proteins or short metabolic pathways just because of the economics. Your time and money are better spent on protein/metabolic engineering etc. Also, if you're planning on using older, organic chemistries to make DNA, keep in mind the chemicals required and waste products produced are not great to have around your home or garage. You may want to explore newer enzymatic-based chemistries coming online that are much greener. The idea of a chip-based oligo assembler has been around for a while but to my knowledge no one has been able to get it to work well -- it would be great to have this revisited, but I still would have the oligos pre-synthesized and focus mainly on getting the assembly processes working well. Note that chip-based foundry systems and chip-based test and measurement systems are getting a lot of attention these days. I point people to these two papers to get a sense of where things stand -- Venter's recent review of synbio -- pay particular attention to Figure 4, which describes Avery bio's chip-based DNA synthesis system -- and Roswell's description of their Molecular Electronic chip -- a general purpose, single molecule sensing platform. The problem of making long DNA assemblies necessary for synthetic genomes has not yet been solved. My baseline is E. coli K12 from ATCC, which retails for $400. The genome is about 4.5 megabases. At current synthesis prices, about $0.10 base, K12 would be a $450,000 print job. When it's $450 to print the genome -- and we have base-level control of the entire chromosome -- no one will order the microbe from ATCC again. I look forward to this day.

As Bryan says, onwards and upwards.

Cheers, Andrew

On Tue, Aug 9, 2022 at 5:05 AM Bryan Bishop <kan...@gmail.com> wrote:
Thanks everyone. I am still around (and so is Nathan), and I actually sent an off-list email earlier indicating my interest in funding this project. Onwards and upwards,

- Bryan
https://twitter.com/kanzure

On Tue, Aug 9, 2022, 3:15 AM Brian Degger <brian....@gmail.com> wrote:
https://diyhpl.us/wiki/dna/dna-synthesis.html a the page Bryans on DNA Synth.

Otherwise, search the research papers on microfluidic dna synth.

Cheers,
Brian

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Re: [DIYbio] Purchasing DNA synthesizer

12:50 PM |

Sorry for interrupting the conversation.

YES, they need technology like de novo DNA synthesis just for seconds. For example, DNA synthesis by dNTPs on virtual matrix (maybe "optical trap") and filling gaps by polymerase-free autocatalysis.

> companies spend a lot of money on development of technology

пт, 19 авг. 2022 г. в 23:13, Koeng <koeng101@gmail.com>:

Length, which is a function of reliability

> is it because of business model choices of the companies

Absolutely.

> Some more light on this will be helpful.

I wrote a couple essays on this topic here - https://keonigandall.com/posts/affordable_dna_2.html

> My understanding is synthesis needs to get way more cheaper as per the "data storage" drivers. Wondering if it's just hype or realistically possible.

The reason DNA is expensive right now, IMO, is that companies spend a lot of money on development of technology and the cloning process, and don't have enough users to aggregate the development costs against. You can always just solve the first bit with shittier/older technology, but reduce the requirement of cost aggregation, and therefore get a much more affordable end product.

On Friday, August 19, 2022 at 9:56:24 AM UTC-7 Ravi Ramana wrote:
By Enzymatic synthesis about 5X better you mean in which factor - speed or efficiency or length ?

And oligos being 100x-1000x expensive than what current tech could do - is it because of business model choices of the companies
or
you mean the current tech base is amenable for that kinda improvement. Some more light on this will be helpful.

My understanding is synthesis needs to get way more cheaper as per the "data storage" drivers. Wondering if it's just hype or realistically possible.

On Friday, August 19, 2022 at 7:30:21 PM UTC+5:30 Koeng wrote:
Oligos have to be stitched together into genes. Enzymatic synthesis is about 5x, last I heard, better than chemical, but also much more expensive and complicated to run.

> This barrier shd be broken immediately, if so.

It is very difficult, but not impossible.

On Thursday, August 18, 2022 at 10:17:05 PM UTC-7 Ravi Ramana wrote:
Any specific reasons why the differentials are so high? other than the long writes between gene & oligos?
Also, you saying currently we could do 0.001 to 0.01 per bp already and its business model choices its' 100-1000X higher presently? That's tall.
Enzymatic synthesis offerings even don't get those price points as of today. Curious to look at any pointers. This barrier shd be broken immediately, if so.

On Wednesday, August 10, 2022 at 12:43:36 AM UTC+5:30 Koeng wrote:
@Abizar Yep

@Dan awesome! I'll contact you about that.

@Bryan let's definitely talk!

> making gene-length fragments is pretty standard and inexpensive these days and is offered by a spectrum of commercial groups

Over 200x price difference between genes and oligos. Oligos are about 100-1000x more expensive than they could be with current technology. I am aware of current commercial groups - I used to run the FreeGenes Project and oversaw a few million base pairs of synthesis from Twist.

> The idea of a chip-based oligo assembler has been around for a while but to my knowledge no one has been able to get it to work well

Genscript uses it in production. Already have reached out to Drew Hall + some of the Avery folks, since they're jumping on since the patent is expiring from custom array (just like me)

On Tuesday, August 9, 2022 at 11:52:48 AM UTC-7 Andrew Hessel wrote:
Great that DNA writing is popping up again. It's so foundational yet has received so much less attention than reading DNA. This said, making gene-length fragments is pretty standard and inexpensive these days and is offered by a spectrum of commercial groups. I don't recommend doing it at home/garage/etc if you're planning on working with proteins or short metabolic pathways just because of the economics. Your time and money are better spent on protein/metabolic engineering etc. Also, if you're planning on using older, organic chemistries to make DNA, keep in mind the chemicals required and waste products produced are not great to have around your home or garage. You may want to explore newer enzymatic-based chemistries coming online that are much greener. The idea of a chip-based oligo assembler has been around for a while but to my knowledge no one has been able to get it to work well -- it would be great to have this revisited, but I still would have the oligos pre-synthesized and focus mainly on getting the assembly processes working well. Note that chip-based foundry systems and chip-based test and measurement systems are getting a lot of attention these days. I point people to these two papers to get a sense of where things stand -- Venter's recent review of synbio -- pay particular attention to Figure 4, which describes Avery bio's chip-based DNA synthesis system -- and Roswell's description of their Molecular Electronic chip -- a general purpose, single molecule sensing platform. The problem of making long DNA assemblies necessary for synthetic genomes has not yet been solved. My baseline is E. coli K12 from ATCC, which retails for $400. The genome is about 4.5 megabases. At current synthesis prices, about $0.10 base, K12 would be a $450,000 print job. When it's $450 to print the genome -- and we have base-level control of the entire chromosome -- no one will order the microbe from ATCC again. I look forward to this day.

As Bryan says, onwards and upwards.

Cheers, Andrew

On Tue, Aug 9, 2022 at 5:05 AM Bryan Bishop <kan...@gmail.com> wrote:
Thanks everyone. I am still around (and so is Nathan), and I actually sent an off-list email earlier indicating my interest in funding this project. Onwards and upwards,

- Bryan
https://twitter.com/kanzure

On Tue, Aug 9, 2022, 3:15 AM Brian Degger <brian....@gmail.com> wrote:
https://diyhpl.us/wiki/dna/dna-synthesis.html a the page Bryans on DNA Synth.

Otherwise, search the research papers on microfluidic dna synth.

Cheers,
Brian

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Re: [DIYbio] Purchasing DNA synthesizer

12:13 PM |

Length, which is a function of reliability

> is it because of business model choices of the companies

Absolutely.

> Some more light on this will be helpful.

I wrote a couple essays on this topic here - https://keonigandall.com/posts/affordable_dna_2.html

> My understanding is synthesis needs to get way more cheaper as per the "data storage" drivers. Wondering if it's just hype or realistically possible.

The reason DNA is expensive right now, IMO, is that companies spend a lot of money on development of technology and the cloning process, and don't have enough users to aggregate the development costs against. You can always just solve the first bit with shittier/older technology, but reduce the requirement of cost aggregation, and therefore get a much more affordable end product.

On Friday, August 19, 2022 at 9:56:24 AM UTC-7 Ravi Ramana wrote:

By Enzymatic synthesis about 5X better you mean in which factor - speed or efficiency or length ?

And oligos being 100x-1000x expensive than what current tech could do - is it because of business model choices of the companies
or
you mean the current tech base is amenable for that kinda improvement. Some more light on this will be helpful.

My understanding is synthesis needs to get way more cheaper as per the "data storage" drivers. Wondering if it's just hype or realistically possible.

On Friday, August 19, 2022 at 7:30:21 PM UTC+5:30 Koeng wrote:
Oligos have to be stitched together into genes. Enzymatic synthesis is about 5x, last I heard, better than chemical, but also much more expensive and complicated to run.

> This barrier shd be broken immediately, if so.

It is very difficult, but not impossible.

On Thursday, August 18, 2022 at 10:17:05 PM UTC-7 Ravi Ramana wrote:
Any specific reasons why the differentials are so high? other than the long writes between gene & oligos?
Also, you saying currently we could do 0.001 to 0.01 per bp already and its business model choices its' 100-1000X higher presently? That's tall.
Enzymatic synthesis offerings even don't get those price points as of today. Curious to look at any pointers. This barrier shd be broken immediately, if so.

On Wednesday, August 10, 2022 at 12:43:36 AM UTC+5:30 Koeng wrote:
@Abizar Yep

@Dan awesome! I'll contact you about that.

@Bryan let's definitely talk!

> making gene-length fragments is pretty standard and inexpensive these days and is offered by a spectrum of commercial groups

Over 200x price difference between genes and oligos. Oligos are about 100-1000x more expensive than they could be with current technology. I am aware of current commercial groups - I used to run the FreeGenes Project and oversaw a few million base pairs of synthesis from Twist.

> The idea of a chip-based oligo assembler has been around for a while but to my knowledge no one has been able to get it to work well

Genscript uses it in production. Already have reached out to Drew Hall + some of the Avery folks, since they're jumping on since the patent is expiring from custom array (just like me)

On Tuesday, August 9, 2022 at 11:52:48 AM UTC-7 Andrew Hessel wrote:
Great that DNA writing is popping up again. It's so foundational yet has received so much less attention than reading DNA. This said, making gene-length fragments is pretty standard and inexpensive these days and is offered by a spectrum of commercial groups. I don't recommend doing it at home/garage/etc if you're planning on working with proteins or short metabolic pathways just because of the economics. Your time and money are better spent on protein/metabolic engineering etc. Also, if you're planning on using older, organic chemistries to make DNA, keep in mind the chemicals required and waste products produced are not great to have around your home or garage. You may want to explore newer enzymatic-based chemistries coming online that are much greener. The idea of a chip-based oligo assembler has been around for a while but to my knowledge no one has been able to get it to work well -- it would be great to have this revisited, but I still would have the oligos pre-synthesized and focus mainly on getting the assembly processes working well. Note that chip-based foundry systems and chip-based test and measurement systems are getting a lot of attention these days. I point people to these two papers to get a sense of where things stand -- Venter's recent review of synbio -- pay particular attention to Figure 4, which describes Avery bio's chip-based DNA synthesis system -- and Roswell's description of their Molecular Electronic chip -- a general purpose, single molecule sensing platform. The problem of making long DNA assemblies necessary for synthetic genomes has not yet been solved. My baseline is E. coli K12 from ATCC, which retails for $400. The genome is about 4.5 megabases. At current synthesis prices, about $0.10 base, K12 would be a $450,000 print job. When it's $450 to print the genome -- and we have base-level control of the entire chromosome -- no one will order the microbe from ATCC again. I look forward to this day.

As Bryan says, onwards and upwards.

Cheers, Andrew

On Tue, Aug 9, 2022 at 5:05 AM Bryan Bishop <kan...@gmail.com> wrote:
Thanks everyone. I am still around (and so is Nathan), and I actually sent an off-list email earlier indicating my interest in funding this project. Onwards and upwards,

- Bryan
https://twitter.com/kanzure

On Tue, Aug 9, 2022, 3:15 AM Brian Degger <brian....@gmail.com> wrote:
https://diyhpl.us/wiki/dna/dna-synthesis.html a the page Bryans on DNA Synth.

Otherwise, search the research papers on microfluidic dna synth.

Cheers,
Brian

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