Fwd: [DIYbio] Re: A hypothetical protocol for DIYBIO DNA synthesis

[Bryan Bishop]

(Don't mind me, I'm just looping in the https://groups.google.com/group/enzymaticsynthesis people.)

---------- Forwarded message ----------
From: Nathan McCorkle <nmz...@gmail.com>
Date: Fri, Jul 15, 2016 at 5:32 PM
Subject: Re: [DIYbio] Re: A hypothetical protocol for DIYBIO DNA synthesis
To: diybio <diy...@googlegroups.com>


On Fri, Jul 15, 2016 at 10:20 AM, CodeWarrior <code.w...@gmail.com> wrote:
> My apologies for perpetually spamming and bumping this thread. One more
> simplifying idea has occurred to me. Suppose the synthesised TdT enzyme had
> a long tail at one terminal which was then covalently bound to the glass
> slide along with the short oligo seeds pre DNA synthesis. No enzyme would
> then need to be deposed in each new cycle the existing TdT could be reused.

I've considered using nanopores, or a matrix of them, like a
strainer... they're cheap and easily orderable too for about $32 each
(they even sent me a free sample), from places like:
http://www.temwindows.com/product_p/sn100-p20q05.htm

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[Bryan Bishop]

---------- Forwarded message ----------
From: CodeWarrior <code.w...@gmail.com>
Date: Sat, Jul 16, 2016 at 12:21 PM
Subject: Re: [DIYbio] Re: A hypothetical protocol for DIYBIO DNA synthesis

To: DIYbio <diy...@googlegroups.com>

lodging it in a pore wouldn’t work, it needs to move about to interact with the fairly short seed oligos you’d start with.

In an effort to improve the method by finding a way not dependent on special reagents like cleanamp dNTPs I’ve been looking at synthesis by sequencing type methods. Consequently I’d like to propose yet another protocol.

1. a oligo sequence we will call A is introduced to TdT and ordinary dNTPs

2. the resulting extended oligos are separated by length

3. the oligos are introduced to micro beads suitable for manipulation with optical tweezers covered with covalently bound (at the 5’ end) oligos of the A sequence and a second sequence we shall call B. By shear chance a small number of the oligos will have the B’ sequence (we use ‘ to indicate the complimentary sequence) and will anneal to a small fraction the beads.

4. beads will be separated into wells and subjected to PCR.

5. both A and B sequence oligos bound to fluorophores at the the 5’ end are introduced and used to mark those beads which are moved into a separate pool and washed clean on the tagging oligos.

6. a super thin layer of metal is evenly deposited on an optically flat side of a prism and A and B sequence oligos are covalently bound to the surface at the 5’ end in spall regular patches.

7. the separated micro beads are deposited on the surface one on each patch and PCR takes place after which the beads are discarded.

8. the surface is washed and placed in a surface resonance imaging frame and a reference reading of the surface is taken.

9. primer A (or B) is added and allowed to anneal, a second reference image is taken.

10. a PCR mix containing only one nucleotide (dA, dT, dC or dG) is added and extension takes place (but no heating occurs)

11. the surface is washed and dried and another reference reading is taken, the difference in the surface plasmon resonance effect between rounds should indicate how many nucleotides have been added.

12. 10 and 11 are repeated with a different nucleotide (dA, dT, dC or dG) in cycles until no more extension takes place.

13. for extra proof reading 10 to 12 can be repeated with the alternate primer (B or A) to read the complimentary sequence.

We now have a metal surface with many patches with bound oligo nucleotides of the sequences metal-5’-ANB’-3’ and metal-5’-BN’A’-3’ and we know what the sequence N is for each patch. if we have enough patches we likely have every short sequence posable. This is a reusable resource. this metal surface can be used to synthesis DNA sequences by transferring the sequence back to micro beads (coated in A and B oligos) placed on it by using PCR. These beads can be moved and brought together in sequence, treated with type type iis restriction endonuclease specific to the A or B sequences to release the bound sequence where necessary or leave only the sense sequence intact for annealing to other sequences for ligation or PCR. By these methods longer sequences could be assembled.

Of course this depends on the surface plasmon resonance being sensitive enough to detect a single added nucleotide  Since all SPR really cares about is the mass on the surface having lots of A and B primers initially bound on the metal should give a suitably strong SPR effect. ... I hope. I'm no SPR expert.

On Saturday, July 16, 2016 at 5:11:27 AM UTC+1, Nathan McCorkle wrote:

On Fri, Jul 15, 2016 at 3:47 PM, CodeWarrior <code.w...@gmail.com> wrote:
> I'm curious how you would exploit the nanopore to achieve synthesis?

oh, just a way to retain something like tDt or other enzyme for
repetitious reactions, instead of tethering it. A chunk of rock (the
silicon nitride with holes in it) seems like less variant/dynamic than
a coupling system in use (i.e. we don't have to do any covalent
chemistry)... plus it doesn't go bad sitting on the shelf like
biotin/streptavidin coupling kits might.

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[Bryan Bishop]

---------- Forwarded message ----------
From: Nathan McCorkle <nmz...@gmail.com>
Date: Sat, Jul 16, 2016 at 2:00 PM
Subject: Re: [DIYbio] Re: A hypothetical protocol for DIYBIO DNA synthesis

To: diybio <diy...@googlegroups.com>

On Sat, Jul 16, 2016 at 10:21 AM, CodeWarrior <code.w...@gmail.com> wrote:
> lodging it in a pore wouldn’t work, it needs to move about to interact with
> the fairly short seed oligos you’d start with.

Right, I said nothing about lodging it.

>
> In an effort to improve the method by finding a way not dependent on special
> reagents like cleanamp dNTPs I’ve been looking at synthesis by sequencing
> type methods. Consequently I’d like to propose yet another protocol.
>
> 1. a oligo sequence we will call A is introduced to TdT and ordinary dNTPs
> 2. the resulting extended oligos are separated by length

My idea is essentially looping over those two steps, except you do it
recursively (feed output back into input), and stop at some N-number
of length and reserve it for a pool to do sterically-constrained
gibson synthesis (in a reaction chamber less than the polymer
persistence length, so to prevent secondary and tertiary structure
from screwing with ligation). If you command 10-additions with
sufficiently dilute nucleotide solution (dilute enough to
statistically say there is 1 nucleotide per volume introduced to the
reaction chamber), then just discard any molecules that don't meet
your target length (indicating an addition failed or there were more
nucleotides introduced than expected, during a reaction period). You
don't need sequencing either, since you control the input nucleotide.

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[Bryan Bishop]

---------- Forwarded message ----------
From: CodeWarrior <code.w...@gmail.com>
Date: Sat, Jul 16, 2016 at 2:39 PM
Subject: Re: [DIYbio] Re: A hypothetical protocol for DIYBIO DNA synthesis

To: DIYbio <diy...@googlegroups.com>


Ah that's workable for supper short sequences but the yield will be low and the needed input masive. Imagine you tune your TdT reaction perfectly and get a 30% yield of single nucliotide aditions (which I expect is optimistic). Then let's argue your seperation procedure is 100% lossless (highly unlikely). After 8 rounds you'll have less than 0.007% of the oligos you started with. You've also had to do 8 seperation a probably 8 consecutive PAGE procedures. It's just not efficient to add 8 nucliotides to a sequence. After that you need to jam a primer on the end and do some PCR or you'll be working with a sample diluted out of existence.

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[Bryan Bishop]

---------- Forwarded message ----------
From: CodeWarrior <code.w...@gmail.com>
Date: Sun, Jul 17, 2016 at 7:15 AM
Subject: Re: [DIYbio] Re: A hypothetical protocol for DIYBIO DNA synthesis
To: DIYbio <diy...@googlegroups.com>

sorry just to be clear your going to use a lab on a chip to perform TdT reactions, purify out single nucleotide addition oligos, transfect them into e coli, extract them from e coli and repeat the whole process several times? Ok I won't say this is impossible but to construct such a lab on a chip would be a good deal harder than even doing something as ambitious as building your own surface plasmon resonance imager. Also wouldn't it be far easier to use basic PCR than e coli to amplify the sequence.

actually I did a mathematical analysis based on modelling TdT reactions as a system of ODEs and the theoretical maximum efficiency is 100*e^-1% for one nucleotide additions which is approximately 36.7879%.

On Sunday, July 17, 2016 at 12:01:37 AM UTC+1, Nathan McCorkle wrote:

On Jul 16, 2016 12:39 PM, "CodeWarrior" <code.w...@gmail.com> wrote:
>
> Ah that's workable for supper short >sequences but the yield will be low and >the needed input masive. Imagine you >tune your TdT reaction perfectly and get >a 30% yield of single nucliotide aditions

30% sounds far from perfect... wouldn't perfect be 100%? Also why do you assume 30% when phosphoramidite chemistry is close to 99% already, and we know enzymes work and don't have similar error issues, less stringent  chemistry (enzymes are all water based, phosphoramidite synthesis hates water).

> (which I expect is optimistic). Then let's >argue your seperation procedure is >100% lossless (highly unlikely). After 8 >rounds you'll have less than 0.007% of >the oligos you started with. You've also >had to do 8 seperation a probably 8 >consecutive PAGE procedures.

Oh, i am talking about nanofluidic channels, where you don't need gel or anything more than maybe counter-ion buffers.

>It's just not efficient to add 8 nucliotides >to a sequence. After that you need to >jam a primer on the end and do some >PCR or you'll be working with a sample >diluted out of existence.

Meh, just shove the at-least single molecule into an e.coli with electroporation... 'low' yield 'problem' solved. Seems a boon to me to be able to require at minimum a single molecule of output. Also, I am talking lab-on-a-chip tech, not garage-scale reaction apparatus.

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[Bryan Bishop]

---------- Forwarded message ----------
From: Nathan McCorkle <nmz...@gmail.com>
Date: Tue, Jul 19, 2016 at 3:42 AM
Subject: Re: [DIYbio] Re: A hypothetical protocol for DIYBIO DNA synthesis

To: diybio <diy...@googlegroups.com>

On Sun, Jul 17, 2016 at 5:15 AM, CodeWarrior <code.w...@gmail.com> wrote:
> sorry just to be clear your going to use a lab on a chip to perform TdT
> reactions, purify out single nucleotide addition oligos,

yep, on-chip, using one of the few single-molecule 'is there' analytic
techniques (impedance/stripping voltage, dye like YOYO-1 or gelred,
SERS raman photospec)

> transfect them into

eventually, either doing gibson/SLICE first in-vitro, or possibly
transforming fragments into something like b.subtilis that is known to
chew and re-assemble fragmented DNA during transformation. something
like that (getting to on-chip oligos would be an interesting enough
start for me!!)

> e coli, extract them from e coli and repeat the whole process several times?

I didn't really think of extraction, at the point post-transformation,
just let the cell divide for a few hours then flush into the macro
world to a user in a micro-fuge tube (or maybe there are a few
chambers on the chip that get progressively larger).

> Ok I won't say this is impossible but to construct such a lab on a chip
> would be a good deal harder than even doing something as ambitious as
> building your own surface plasmon resonance imager.

I have to definitely disagree with that.

> Also wouldn't it be far
> easier to use basic PCR than e coli to amplify the sequence.

Well e.coli basically does this kind of thing for you, and I think has
better overall fidelity than a single polymerase (or the more advanced
versions). At least they seem cheaper than in-vitro kits.

>
> actually I did a mathematical analysis based on modelling TdT reactions as a
> system of ODEs and the theoretical maximum efficiency is 100*e^-1% for one
> nucleotide additions which is approximately 36.7879%.

I'm interested in more details... I don't really understand what you
mean in this sense. In my arrangement, if tDt didn't add, I'd just
tell it to try again (or wait longer between cycles, or reduce chamber
volume to increase relative concentration), and it it added somehow
too many, either you could waste the molecule... or have an
alternative reaction loop that is some sort of really slow exonuclease
that only works on the growing end. Am I missing something about
efficiency? Regardless of assembly, etc, downstream?

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[Bryan Bishop]

---------- Forwarded message ----------
From: CodeWarrior <code.w...@gmail.com>
Date: Tue, Jul 19, 2016 at 9:36 AM
Subject: Re: [DIYbio] Re: A hypothetical protocol for DIYBIO DNA synthesis

To: DIYbio <diy...@googlegroups.com>

sadly I’m a bit busy but I’ll try to work through the maths quickly.

so we have 4 populations start oligos f0, oligos with one nucleotide additions f1 and oligos larger than that fm we also have a finite supply of nucleotides n. our system of ODEs is

dn/dt=-a n f0-a n f1-a n fm

df0/dt=-a n f0

df1/dt=a n f0-a n f1

dfm/dt=a n f1

the terms in turn are, a n f0 reactions where a nucleotide is consumed changing a start oligo to an oligo with one nucleotide, a n f1 reactions where the desired one nucleotide addition oligos are turned into longer many nucleotide added oligos. a n fm reactions where many nucleotide oligos grow still larger. Notice all reactions are equally likely as TdT doesn’t care about oligo length once you get past oligos a few bases long. The non linear system can be simplified by noticing that df0/dt+df1/dt+dfm/dt=0 implies f0+f1+fm=I the initial number of start oligos. this means we can solve for n and substitute getting a linear system. if you solve this for initial condition then find the time t-max where f1 reaches a maximum and then find f1(t-max) you get I/e. all the other variables cancel out.

adding an exonuclease would be an interesting alteration. in vivo TdT works against exonuclease activity to produce short oligo additions maybe you can get past the 36% yield limit that way.

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[Kent Kemmish]

Hey Nathan,

I've already built such a prototype (what you call "a strainer" I call "a demonpore array") if you want to talk about it? PM me.

It took a year and half and involves a lot of stuff that's not obvious starting out. For boundary pushers I'd like to share it.

-Kent

[Bryan Bishop]

On Tue, Jan 10, 2017 at 8:00 AM, CodeWarrior <code.w...@gmail.com> wrote:

The TdT thus bound is still only non covalently bound to the DNA though. Even if an extreem PH solvent capable of removing it can't be found a sufficiently hot solvent will denature the protien alowing it to unbind but leaving the DNA intact.

Similar methods were described earlier in the thread (such as from Nathan):
https://groups.google.com/d/msg/diybio/FXaT-MjJQYI/tdHZwXqeCgAJ

DNA synthesis chemistry using TdT:
http://2014.igem.org/Team:Cooper_Union/TdT_project
http://2015.igem.org/Team:Cooper_Union/DeNovoSynthesis

There was some discussion about that team's use of TdT over here:
https://groups.google.com/d/msg/enzymaticsynthesis/DApMjXx8gS4/CmKEK9-vJlwJ

There was also an igem team that did some characterization work,
http://2014.igem.org/Team:Rutgers

even earlier mumblings about TdT from Nathan can be found here:
http://gnusha.org/logs/2013-07-13.log
http://gnusha.org/logs/2014-12-26.log

e.g. from a search he asked me to do a few years ago, http://diyhpl.us/~bryan/irc/nmz787-nucleotide.log.txt

I believe Cathal also had some ideas for TdT:
http://diyhpl.us/wiki/dna/synthesis/tdt/

TdT is certainly a useful critter to work with. A bunch of TdT chimeras and mutants would be really useful to investigate. By the way, I think that another area worth considering is DNA assembly; we can somewhat reliably get 100 bp oligos, and constructing longer stretches is a pain in the butt--- if we could somehow make that process simpler (such as using yeast homologous recombination) then we wouldn't need long DNA synthesis methods in the first place.

[Алексей Киселёв]

Is there any news? Somebody was conducting new experiments with TdT?