Chloroplast export peptide

[Mega]

Hi everyone, 

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...

Does anyone know a good database?? 

thx

[Cory Tobin]

> 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

[Andreas Sturm]

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?

[Dakota Hamill]

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!

[Andreas Sturm]

Yeah, we got pGreenII with kanamycin and firefly luciferase. 35S GFP is aslo delivered with it, and must be ligated into. Problem: GFP has EcoRV blunt ends. 

And we just got 1ug, so we need to amplify it. Because on the gel there were two bands for pGreenII, one maybe supercoiled, one unopened. Maybe the enzyme is too weak already, and we didn't really use much of it. 

However, the Genome Compiler GlowingPlant kickstarter project will be started within a few weeks, and then we get a bioluminescence - not just GFP - design!! Thus, there will hardly be time to use pGreen-GFP-Luc because, obviously, bioluminescence is much much better and it's gonna have highest priority!!! 

[Andreas Sturm]

However, the Genome Compiler GlowingPlant kickstarter project will be started within a few weeks, and then we get a bioluminescence - not just GFP - design!! Thus, there will hardly be time to use pGreen-GFP-Luc because, obviously, bioluminescence is much much better and it's gonna have highest priority!!! 

However, we keep trying to restrict and ligate - as an exercise and because I promised Sebastian to send him some ;) 

[Dakota Hamill]

Ah yeah I recall reading the post about the blunt ends and the fear of poor ligation efficiency.  Did you give the ligation a try, then follow it with a transformation?  1ug seems like it'd be enough at 20-50ng per transformation, though I don't know the amount needed per ligation.

Still, all you need are a few colonies that pass the screen test and you can culture them up and mini prep out the plasmid

[Andreas Sturm]

Yeah, as I said, probably the enzym didn't work well.

As for the 1ug we just used 500 ng in the first attempt. And you could hardly see the bands.... So my guess is that the two bands were supercoiled and circular plasmid, and the amount of cut plasmid was too small to be seen.

However, we are doing a midiprep to be able to try it again.

On Fri, Feb 1, 2013 at 3:07 PM, Dakota Hamill <dko...@gmail.com> wrote:

Ah yeah I recall reading the post about the blunt ends and the fear of poor ligation efficiency.  Did you give the ligation a try, then follow it with a transformation?  1ug seems like it'd be enough at 20-50ng per transformation, though I don't know the amount needed per ligation.

Still, all you need are a few colonies that pass the screen test and you can culture them up and mini prep out the plasmid

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[Andreas Sturm]

Thank you Paul.

Well the direction is not an issue, as the casette contains both the promoter, GFP and the terminator.

A lab worker told me about the dephosphorylation enzyme but mentioned we don't have one :D

Adding basepairs just needs free A and free T and ligase, right? Have to dig into this a bit ;)

On Fri, Feb 1, 2013 at 11:39 PM, Paul Sebexen <pau...@gmail.com> wrote:

Just a suggestion regarding ligation - instead of dealing with blunt ends you might have good luck adapting a TA Cloning protocol (adding complementary 3' overhangs to insert and vector), depending on whether you have the necessary enzymes/reagents. This mitigates low efficiency and backbone recircularization as sources of error (the latter may also be addressed with a dephosphorylation step). Keep in mind that the result will still require screening as ~50% of the products will have inserts reading in the wrong direction (I haven;t looked at the expression vector to see if this matters).

Best,

Paul

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[Cathal Garvey]

From memory, TA cloning relies on the fact that some polymerase enzymes
like to add an extra "A" (or was that "T"?) to the end of a copied
sequence as they finish. That means that you can immediately clone the
PCR product in with fairly high efficiency into a vector that's cut to
leave a "T" (or was that "A"? :)) overhang.

There are some commercial vectors specifically designed for T/A cloning,
but if you cut a vector with EcoRV, adding an extra A to the end of your
sequence is likely to reduce blunt-cloning efficiency, not adding anything.

Most error-correcting polymerases don't add a trailing base, so I think
people tend to use mixes of Taq + Proofreading polymerase, or a mutant
proofreader that leaves an overhang. I've never done it myself.

On 02/02/13 09:00, Andreas Sturm wrote:
> Thank you Paul.
>
> Well the direction is not an issue, as the casette contains both the
> promoter, GFP and the terminator.
>
> A lab worker told me about the dephosphorylation enzyme but mentioned we
> don't have one :D
>
> Adding basepairs just needs free A and free T and ligase, right? Have to
> dig into this a bit ;)
>
>
>
>
> On Fri, Feb 1, 2013 at 11:39 PM, Paul Sebexen <pau...@gmail.com
> <mailto:pau...@gmail.com>> wrote:
>
> Just a suggestion regarding ligation - instead of dealing with blunt
> ends you might have good luck adapting a TA Cloning protocol (adding
> complementary 3' overhangs to insert and vector), depending on
> whether you have the necessary enzymes/reagents. This mitigates low
> efficiency and backbone recircularization as sources of error (the
> latter may also be addressed with a dephosphorylation step). Keep in
> mind that the result will still require screening as ~50% of the
> products will have inserts reading in the wrong direction (I haven;t
> looked at the expression vector to see if this matters).
>
> Best,
> Paul
>
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[Cathal Garvey]

I thought TDT would just keep adding a splurge of random nucleotides? Do
you use blocked nucleotides to stop it going nuts, or is it less
prolific than I thought?

On 02/02/13 09:46, Paul Sebexen wrote:
> The most common enzymes I've seen used for dephosphorylation are
> Antarctic Phosphatase and Alkaline Phosphatase (CIP).
>
> Generally the A- and T-tailing are done with Terminal deoxynucleotidyl
> Transferase. Taq polymerase can also be used (will add A's
> preferentially even if given a mix of dNTPs), but is a bit less
> efficient at T addition as a 3' overhang (requires only dTTP be added to
> this reaction). After the complementary tails are added, you can proceed
> with ligation as normal; the efficiency should be significantly higher
> than with blunt ends and you can relax the insert:vector ratio a bit.
> There are many variations on this - I've even seen protocols using C-G
> tails.
>
> Not requiring directionality is definitely convenient.

>
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[Paul]

It will keep adding; you can try to work out the proper incubation time so as to not add too many (it's not an overly processive transferase). Blocked nucleotides like ddNTPs will likely interfere with ligation; you would have to use a reversible terminator of some sort.
In any event, the ligation of a single dA to a tail of 3 dTs (for example) is probably still more efficient than blunt ends.
I believe the issue disappears altogether if using taq though.

[Stephanie]

I have begun a web site with the aim of providing crowd funding to medical research projects. This idea was prompted by finding out that a university group had cured type 1 diabetes in mice, successfully completed a phase 1 human clinical trial, and needed funding to complete phase 2. I thought there must be quite a few groups out there who also need funding. What better way would there be than allowing the public to directly have a say in medical research by providing the means for them to find, fund, and follow the research that means the most to them. I am considering also opening this up to the DIY bio community, but I am not quite sure of all the considerations I would need to think about. I have also struggled with whether posting projects should be restricted to verified non-profits, or if I should leave it open. The thinking behind restriction was that I did not want any corporate drug companies on the site. But, by doing so, I may miss a lot of legitimate, innovative, and compassionate for profit groups, especially within the DIY bio communities. How many of you with businesses are non-profit? How many are for profit? What would you think about a site like this?

[Reason]

> -----Original Message-----
> From: diy...@googlegroups.com [mailto:diy...@googlegroups.com] On
> Behalf Of Stephanie
> Sent: Sunday, February 03, 2013 7:46 PM
> To: diy...@googlegroups.com
> Subject: [DIYbio] medical research crowd funding
>
> I have begun a web site with the aim of providing crowd funding to
> medical research projects. This idea was prompted by finding out that a
> university group had cured type 1 diabetes in mice, successfully
> completed a phase 1 human clinical trial, and needed funding to
> complete phase 2. I thought there must be quite a few groups out there
> who also need funding. What better way would there be than allowing the
> public to directly have a say in medical research by providing the
> means for them to find, fund, and follow the research that means the
> most to them

You might look at these items and the links therein to various science
crowdfunding ventures in your search for data points:

http://www.fightaging.org/archives/2012/06/you-cant-just-kickstart-a-science
-project---it-isnt-that-easy.php

http://www.fightaging.org/archives/2012/03/signs-of-progress-in-crowdsourced
-science-funding.php

Reason

[plushzilla]

I guess one of the reasons why crowd funding has worked is that the people who contribute to the cause can get some immediate feedback/return on their 'investment/contribution'. So unless your project produced a deliverable then it would be difficult for people to evaluate/justify why they might want to contribute money to this. 

However, the idea is not without merit, and there has been attempts to get things like this going in the science community. I think the more feasible concept might be for more direct collaborations to take place (outside the formal academic settings and its associated bureaucracy) so that research resources and results can be shared without worrying about things like whether the results can be published or not. I have seen some diy/hacker communities take their projects to a commercial level of maturity, so even if you initially restricted the website to certain types of organizations, you can't necessarily prevent them from veering away from it once they have managed to get the project off the ground.

I'd be interested in specific details, and perhaps others will have different opinions. Basically the best way to test the idea is to get something out their and let the people decide for themselves.

Thanks