Science

On Thu, Jan 31, 2013 at 9:54 PM, Marc Dusseiller <dusjagr@gmail.com> wrote:
> hoi zäme,
>
> there is a lot more info on the hackteria wiki:
> http://hackteria.org/wiki/index.php/DIY_Micro_Dispensing_and_Bio_Printing

marc, how are you dispensing in that project? I see the syringe, but I
don't see something pushing it.

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Is the water level above the top glass slide? If so you're using a ton
more current than needed. The olde-tyme drawings of paper
electrophoresis setups were triangular, with two separate buffer tanks
connected only by the wet paper:
http://www.funsci.com/fun3_en/exper1/exper1_21.gif

On Thu, Jan 31, 2013 at 6:40 PM, Dakota Hamill <dkotes@gmail.com> wrote:
> http://imgur.com/a/t1SEv
>
> Re-doing the experiment but with coffee filter paper, and a real gel box,
> and a real power supply. As you can tell from the first picture, the chem
> filter paper I have allows the dye to disperse way too fast, as it should,
> since it's supposed to let solvents pass through it quickly. Sebastian
> seemed to have decent sounding results using the brown coffee filter paper,
> so we'll see how it works with the dyes.
>
> I tried to reduce pouring turbulence too much when pouring in the buffer
> but, was unavoidable when the solvent front rushed through the slide
> sandwhiches. Running at 120V 100mA or there abouts.
>
> There is already noticeable migration of one of the components of the
> markers to one direction---anyone fancy a guess? You'll win the Marker
> Migration badge of honor.

From what I've been reading today, this might depend on the pH, so
what's your buffer???

:)

>
> I'll try to post a video later if you want to experience the most intense
> marker dye coffee filter electrophoresis you've ever seen. Blow your mind
> if it was in 3D.

Cool!

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hoi zäme,

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I don't even know what thread is what anymore.

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Well I cut 3 strips of filter paper and put them on glass slides.  Soaked one in salt water, one in 1x TAE, one in 10x TAE.

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http://imgur.com/a/t1SEv

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On Tue, Jan 29, 2013 at 4:06 PM, Dan <dgrushkin@gmail.com> wrote:
> Hello DIYers,

Are the survey results released yet?

- Bryan
http://heybryan.org/
1 512 203 0507

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On 01/31/2013 09:46 AM, Dakota Hamill wrote:
> Let's say we have a piece of filter paper 10cm long, 1 cm wide, and 0.1 cm thick.
>
> Is it wrong to think it could handle similar voltages and currents that an agarose gel could?
No, that sounds plausible/possible.

> Say it was running at 100V and 0.050A. Could you then use V=IR to calculate the resistance of the buffer soaked paper would be
> 2000 Ohms?
>
> Or, via P=IV, P = (0.050A) x (100V) = 5W

Yep. That's plausible/possible. A 5Watt rated resistor is only 1 com long and .4 cm diameter
and it can get warm to hot at 5W, but not char to black. A 10 cm long strip 1cm wide might
be barely perceptibly warm in still air while dissipating 5W since its surface areas are so
much greater. Your guess at resistance could be way off, since gel boxes aren't so skinny
as you described. What if The 5 Watt level happened at a current of .005Amp? then the
Volts would need to be 1KV. I think that might be more normal for a 10X long as it is wide
gel or paper/electrolyte.

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On 01/31/2013 09:46 AM, Dakota Hamill wrote:
> What I'm wondering is...WITHOUT trial and error, could you calculate the "perfect" conditions to run an agarose gel under -
> meaning voltage + current assuming a tris buffer?
>
> Likewise, could we calculate the optimal conditions to run an electrophoresis 10,1,.1 cm thick piece of paper with tris buffer?

Probably this should go off list, then come back with results. Short answer, "Sure."

Dakota, how about you reply to me about some actual ohms measurements and I'll help you calculate
and compare. I'm in the middle of a non-bio project and won't make any agarose gel to measure it myself
anytime soon.

John Griessen

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Hi Lawrence,

You can see all the classes we teach on our Meetup page:

http://www.meetup.com/BioCurious/

What classes get taught really depends on who's interested in teaching something (or who's arm we can twist...). We don't have any funding to offer a professional teaching stipend, but we do share part of the income from the classes with the teachers.

Whether or not the teachers come up with a written outline (or videotape the class) and share that online is entirely up to them. We don't really have anything set up to do that in any formal way right now (actually, you may have noticed that our entire web presence needs some serious work ;-)

Patrik


On Thursday, January 31, 2013 1:03:39 PM UTC-8, Lawrence wrote:

I was looking at the Biocurious website. They offer classes, does anyone know what type of classes they teach? Is there any way to access freely the information they teach? The course outline and notes for it? It is great they offer courses but not everyone can get to them.

Are there any other groups that do the same thing? Are these courses available for free and online?

Thanks

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On Thu, Jan 31, 2013 at 1:19 PM, fiaraz <fiaraziqbal@googlemail.com> wrote:
> I was looking into it and spoke to the madlab group in Manchester who
> informed me that it was ok but not as good as the lamp based PCR machine.
> which i am now looking to construct but time is such a rare commodity.

I just took apart an old hot air thermocycler I picked up used for
$40... it has a cheap 500 or 1000W halogen light bulb under a sheet of
aluminum which has been impressed with the PCR tube shape, a cut-out
in the reflector leads to a small squirrel cage fan. The light and fan
are controlled by two solid state relays.

I bet you could replicate something like this using a chinese $8
wal-mart/home-depot flood light, and spray-painted aluminum foil (or
just paint the PCR tube black before cycling)

Here are some pics:
http://nathanmccorkle.com/takeAparts/hybaid/gallery.html

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-Nathan

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http://www.ebay.com/itm/MJ-Research-Programmable-Thermal-Controller-Model-PTC-100-PTC100-Used-Condition-/221168862233?pt=LH_DefaultDomain_0&hash=item337eb10419

Can do 60 samples compared to OpenPCRs 16

OpenPCR costs: $599 not including shipping

This costs: $316 and includes shipping and guaranteed to work or full refund.

Troll eBay for MJ research thermal controllers I found mine for $45 and $35 shipping and it works fine.

Search for "thermal controllers" even though the units are typically called thermal cyclers because people list them as Thermal controllers and no one bids on them.

If you don't believe me check this really nice one that went for <$130 two weeks ago:
http://www.ebay.com/itm/MJ-RESEARCH-PTC-100-PROGRAMMABLE-THERMAL-CONTROLLER-/251212740568?pt=LH_DefaultDomain_0&hash=item3a7d7237d8

If OpenPCR gave back to the community I might be more inclined to tell you to support them but look at their website. No PCR protocols or tutorials. No links to tell you were to buy reagents. Most comments on their forum are not even answered. Seriously, don't support people who are just out to make money off of DiyBio folks.


On Wednesday, January 30, 2013 7:00:02 PM UTC-6, G Lyons wrote:

Hello all-

I was wondering what peoples' experiences have been with the OpenPCR thermal cycler. Is it simple to construct and is it just as effective as the regular ones?

Thanks!

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IIRC you'd need to input the diameter of the tube just under the lip, and the additional mms added by the lip. Honestly I wouldn't recommend an open ended design for pcr tubes though, they are so small that errors in printing due to dodgy skeining or alignment could easily lead to ejection.

Possibly if you make the rim thickness big enough it'll enclose the tubes entirely, I don't recall offhand how closely the cutouts matched the shape of a tube. Play around in openscad I guess!

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Have you attempted a transformation yet?  Didn't you get pGreen II with a kanamyacin cassette?  ...or am I thinking of someone else.  I'm eager to see some glowing plants!

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I havn't heard of any amateurs or DIY'ers who created a fully functioning light-bulb PCR.  Russel Durret had one and I re-created the circuit from it, but he never to my knowledge tested ramp times or ever completed a successful PCR.  I havn't either.  I have a "professional" build light-cycler I got off ebay, the Idaho Technology RapidCycler, and although it is a bit faster than the peltier machines I've tested it against, so far the peltiers seem to beat it out on amplification quality / concentration (only done 2 amplifications...not a great sample size).  Though, this could be because I am not providing ample enough program times to account for the poorer heat transfer between plastic/air vs plastic/aluminum.

Dealing without heated lids kind of sucks because pipetting mineral oil is annoying, and the OpenPCR offers that I believe.  You could get an older peltier based machine and hope it worked off ebay (sometimes it is cheap enough to take the gamble), but for newer PCR machines with heated lids that really work, you may even find the price comparable to OpenPCR.

From observing OpenPCR over the past few years, but never actually using one, I'd say they did a nice job filling the complete void that existed for a relatively easy to assemble plug-n-go machine.  Are there areas where one could improve upon it? Sure; price, ramp time, sample #, size.  But you could argue anything and everything in existence could be improved upon, and that's how progress is made.


 

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What may be feasible (but labour intensive): Make the cell produce the antidote with nuclear localization signal and toxin with chloroplast transit peptide and kanamycin resistance (for cell selection!).

At the same time you'd have to transform the chloroplasts with the genes of interest and the resistance.

On Thu, Jan 31, 2013 at 10:55 PM, Andreas Sturm <masterstorm123@gmail.com> wrote:

Thanks for that Nathan!

Now I have the sequences of biosynthesis pathway for kanamycin and streptomycin  antibiotics ;)

However, both are kind of big. KanSynth ~ 10 kbp ; strepto Synth ~ 18 kbp.


The toxin/antitoxin has just some <1kbp in total!!

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Thanks for that Nathan!

Now I have the sequences of biosynthesis pathway for kanamycin and streptomycin  antibiotics ;)

However, both are kind of big. KanSynth ~ 10 kbp ; strepto Synth ~ 18 kbp.


The toxin/antitoxin has just some <1kbp in total!!

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If the toxin and antitoxin are expressed in the chloroplast, and you export only the toxin, would that not kill the cell? The toxin in question can kill yeast, could probably do it to plants too?

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Well, the idea is:  when inserting the lux operon into chloroplasts, adding the antitoxin (stays in chloroplast)  and the toxin (secreted into the cytoplasm)

The toxin shall then invade other chloroplasts and kill the non-transformed ones.



It may be good like this: A chloroplast export peptide, which is cut of then, and downstream of it there is a chloroplast import signal which then makes it enter chloroplasts again.


Or would it work even without signaling peptides? Would some of the toxin make it out of the chloroplast (it has a double membrane around!) into the cytosol, just to enter another chloroplast?

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I was looking into it and spoke to the madlab group in Manchester who informed me that it was ok but not as good as the lamp based PCR machine.  which i am now looking to construct but time is such a rare commodity.

On Thursday, January 31, 2013 1:00:02 AM UTC, G Lyons wrote:

Hello all-

I was wondering what peoples' experiences have been with the OpenPCR thermal cycler. Is it simple to construct and is it just as effective as the regular ones?

Thanks!

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On Thu, Jan 31, 2013 at 12:30 PM, Larry James <wulfdesign@gmail.com> wrote:
> oh,
> thats smart,
>
> duh.
>
> thought would have to modify & print up a custom one for different sizes.
>
> sometimes I wonder ;)

Well being that I don't have any tubes now, it is probably cheaper to
just modify the design and print different sized heads.

Cathal, would I just have to change the diameter of the hole?

--
-Nathan

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On Thu, Jan 31, 2013 at 8:53 AM, Cathal Garvey
<cathalgarvey@cathalgarvey.me> wrote:
> Not necessarily; does the toxin enter other cells? Does it get degraded
> quickly by extracellular proteases?
>
> Toxin/antitoxin systems more often appear to be selfish plasmid
> strategies to force individual cells to keep the plasmid. Antibiotics,
> on the other hand, are usually a cellular strategy (which might or might
> not be plasmid-encoded) to kill off the competition.

Well looking through some papers, it seems that plastid transformation
is often selected with spectinomycin resistance genes, where the
spectinomycin is applied externally. So it must be around the main
cell before getting to the plastid for degradation.

It seems theoretically possible that you could just use this 18kb
spectinomycin operon, which is biosynthesis and resistance
http://www.ncbi.nlm.nih.gov/nuccore/EU255259

Found via
The Gene Cluster for Spectinomycin Biosynthesis and the
Aminoglycoside-Resistance Function of spcM in Streptomyces spectabilis
Kyoung-Rok Kim, Tae-Jong Kim, Joo-Won Suh
http://home.kookmin.ac.kr/~bior/Curr.%20Microbiol%2057-371.pdf

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-Nathan

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I was looking at the Biocurious website. They offer classes, does anyone know what type of classes they teach? Is there any way to access freely the information they teach? The course outline and notes for it? It is great they offer courses but not everyone can get to them.

Are there any other groups that do the same thing? Are these courses available for free and online?

Thanks

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> I am looking for a signal peptide that tells proteins to leave the
> chloroplasts.
>
> Google shows a load of import protein, nbut I don't want cytoplasm ->
> chloroplast but the other way round...

I could be wrong, but I've never heard of that happening. I know the
chloroplast can communicate with the nucleus using abscisic acid,
sucrose, H2O2, O3, xanthoxin, tetrapyrrole, etc. But I've never seen
a report of a protein being exported from the chloroplast to the
cytoplasm or nucleus.

Search for "chloroplast retrograde signaling".

Why not encode your gene in the nucleus and have it produced by
ribosomes in the cytoplasm?

-cory

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oh,
thats smart,

duh.

thought would have to modify & print up a custom one for different sizes.

sometimes I wonder ;)

L

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On Thu, Jan 31, 2013 at 12:04 PM, Cathal Garvey (Phone)
<cathalgarvey@cathalgarvey.me> wrote:
> If the toxin and antitoxin are expressed in the chloroplast, and you export
> only the toxin, would that not kill the cell? The toxin in question can kill
> yeast, could probably do it to plants too?
>

Hmm, you could say the same thing about added antibiotic too then, I
think... if the chloroplast is resistant, but the main cell isn't.
Maybe there are some toxins/antibiotics that only affect
bacteria/chloroplast? Or maybe if a plant cell's chloroplast died, it
would simply die as a result? If plant cells live in defined media in
dark conditions, that might tell us if killing/halting just the
chloroplast might be enough.

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On Wednesday, January 30, 2013 11:36:14 PM UTC-5, Patrik D'haeseleer wrote:

Cool! First response is up, and it's about the idea of using amphibian or reptilian cell lines on the BioPrinter at BioCurious (for the record, we're not working with *any* animal cells right now):

You beat me to it!  Was going to post later today.  Had a formatting bug to work out but now resolved.  The second post should go up tomorrow and i'll post it to the mailing list too.

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If the toxin and antitoxin are expressed in the chloroplast, and you export only the toxin, would that not kill the cell? The toxin in question can kill yeast, could probably do it to plants too?

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