Re: Bacteria Lava-Lamp
214 views
Skip to first unread message
Mega
Jul 22, 2012, 4:43:27 AM7/22/12
to diy...@googlegroups.com
Hey, I've done something like this...
Took the pVIB plasmid (no need for a specific substrate! just oxygen + glucose) in E.Coli and put them into a marmelade jar containing LB medium (without ampicillin).
In the dark it was glowing much brighter than a petri dish with those bacteria (obviously).
It stopped glowing after a while (I think one week) perhaps because they have consumed all the sugar, or too much endotoxins?)
The best approach however would be to use B.Subtilis. They don't produce Endotoxins afaik. And they make spores, so you can just let them run out of sugar, wait for month, put sugar into it, and they'll grow and glow again!
And, of course, bacteria will lose those plasmids after some time. Best bet for an eternally glowing lamp is that you insert it into their chromosome. (Wanna do this ;) ) And cut out the marker gene (or take a marker that's not a clinical antibiotic; such as heavy metal resistance?), to avoid making resistant germs...
Am Sonntag, 22. Juli 2012 01:15:33 UTC+2 schrieb Chowe:
Took the pVIB plasmid (no need for a specific substrate! just oxygen + glucose) in E.Coli and put them into a marmelade jar containing LB medium (without ampicillin).
In the dark it was glowing much brighter than a petri dish with those bacteria (obviously).
It stopped glowing after a while (I think one week) perhaps because they have consumed all the sugar, or too much endotoxins?)
The best approach however would be to use B.Subtilis. They don't produce Endotoxins afaik. And they make spores, so you can just let them run out of sugar, wait for month, put sugar into it, and they'll grow and glow again!
And, of course, bacteria will lose those plasmids after some time. Best bet for an eternally glowing lamp is that you insert it into their chromosome. (Wanna do this ;) ) And cut out the marker gene (or take a marker that's not a clinical antibiotic; such as heavy metal resistance?), to avoid making resistant germs...
Am Sonntag, 22. Juli 2012 01:15:33 UTC+2 schrieb Chowe:
Hello! Im very new to DIYbio and I am looking to do an exciting first project. I work in a genetics lab so I have experience with most techniques. I have had many ideas for projects, that I happily found out most people have had the same ones too (glowing plants, glowing yogurt) but I thought a lava lamp would be the simplest way. I am wondering how you guys would go about completing this. I saw that cambridge igem team made a lava-lamp, seen here http://www.youtube.com/watch?v=tUFscEVK5Ks I would like to make something just like that. I would prefer if I didn't have to induce it or if I did it would be through oxygen like the cambridge lava-lamp. It would be cool to have in my room to light it up at night. As with the glowing plants and yogurt they seem to be a lot harder and it seems like not many people have been successful so I wanted to choose a project that I would be more likely to succed. Thanks!Corey
Mega
Jul 22, 2012, 4:47:59 AM7/22/12
to diy...@googlegroups.com
>As with the glowing plants and yogurt they seem to be a lot harder and
it seems like not many people have been successful so I >wanted to choose
a project that I would be more likely to succed.
Well, that's because of this: When you have the gene synthesized, it would fit then. But it costs lot of money. Or you take the traditional approach, get a (very good!) viral plant promotor template, RBS template, etc. attach them to the lux genes (also a lot of work), take many steps to make codon adaption (essential for good yields), ..... You see, a lot of work (and chemicals also cost money). Or a lot of money.
Chowe
Jul 22, 2012, 1:10:23 PM7/22/12
to diy...@googlegroups.com
Thanks for the info! I think I will first try out the more simple route and transform pVIB into E.Coli and grow in LB. I am definitely interested in making an eternally glowing light source. That could possibly be a project I work on this coming fall, I will have full time access to my schools laboratory. For that project would you recommend B.Subtilis? Also how exactly would you build a plasmid that would insert part of its genes into the chromosome, any certain plasmids need to be used? Thanks.
Andreas Sturm
Jul 22, 2012, 2:08:51 PM7/22/12
to diy...@googlegroups.com
> Also how exactly would you build a plasmid that would insert part of its
genes into the chromosome, any certain plasmids need to be used?
http://www.bgsc.org/Catpart4.pdfHere are some plasmids listed. It is written that on request, they may send you a plasmid for free if you use it for non-profit things.
Well, the plasmid needs two flanking sites that fit to a gene in the Bac. Subt. Chromosome. Then in 1 out of 10000 cases, homologous recombination occurs. The plasmid does not replicate, so if the bacillus doesn't die from the antibiotic and forms a colony, it has the gene casette integrated.
In theory, you could also use pUC19, fit in flanking sites of B.Subt. (where to get the template?, can you design the primers?), inside the flanking sites Lux operon + Resistance , amplify it in E.coli, transform B.S.
> I will have full time access to my schools laboratory.
> For that project would you recommend B.Subtilis?
The spores of B.S., when they 'smell' better conditions, will 'wake up' again and in theory, still after 50 years of dryness, etc. you'll get a glowing B.Subt. again.
Chowe
Jul 22, 2012, 4:15:40 PM7/22/12
to diy...@googlegroups.com
That source was very helpful, I had never heard of B.Subtilis, seems like a great bacteria. I think I would definitely choose one of their plasmids. Since their plasmids already have resistance, could I make the construct by just cutting pVIB and my vector of choice with Sal1, ligating together, then transforming into B.Subtilis?
Andreas Sturm
Jul 22, 2012, 4:34:48 PM7/22/12
to diy...@googlegroups.com
Yes, definitely. This way you get the gen into bac. subt. Sal1 has to be in the vector too, but I think one of their vectors will have.
But:
The lux operon is from gram negative bacteria, and b.s. is a gram positive. It may glow weaker (or even not at all. )
But if you have access to the lab, and can use the chemicals for free, I'd try it ;) And the distributor too may give you the plasmid for free.
Those plasmids don't cut out the resistance. So it's not wise to handle them without care so they escape into the wilderness. Although, b.s. is quite the opposite of a pathogen. Just make sure to take the same care you should with each bacterium.
Best, choose a resistance that's very common in natural bacteria. I read an organization saying "Kanamycin resistance is very wide-spread in wild bacteria. So it's the best bet for use in genetically modified crops" (So if you make the bacilli resistant to kanamycin and if one escapes into the wilderness, it's still better than chloramphenicol resistant etc. )
But:
The lux operon is from gram negative bacteria, and b.s. is a gram positive. It may glow weaker (or even not at all. )
But if you have access to the lab, and can use the chemicals for free, I'd try it ;) And the distributor too may give you the plasmid for free.
Those plasmids don't cut out the resistance. So it's not wise to handle them without care so they escape into the wilderness. Although, b.s. is quite the opposite of a pathogen. Just make sure to take the same care you should with each bacterium.
Best, choose a resistance that's very common in natural bacteria. I read an organization saying "Kanamycin resistance is very wide-spread in wild bacteria. So it's the best bet for use in genetically modified crops" (So if you make the bacilli resistant to kanamycin and if one escapes into the wilderness, it's still better than chloramphenicol resistant etc. )
--To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/2SBvM4GIZDwJ.
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To post to this group, send email to diy...@googlegroups.com.
To unsubscribe from this group, send email to diybio+un...@googlegroups.com.
Cathal Garvey
Jul 22, 2012, 5:08:21 PM7/22/12
to diy...@googlegroups.com
Just an additional FYI:
Getting DNA into E.coli works best with supercoiled plasmid DNA, but
getting DNA into B.subtilis works best with linear DNA.
The reason is that B.subtilis uses an active uptake system to absorb DNA
in the environment, which can only absorb DNA if it can find a loose end
to chew on. It's been speculated that most B.subtilis transformants from
plasmid solutions are actually those that found damaged or cut plasmids
and absorbed them, which contributes to B.subtilis' undeserved
reputation for being genetically unstable (that is, the DNA was already
damaged, it's not the bug's fault! :))
The differences in efficiency are pretty dramatic. Especially because
the active uptake system includes chaperone proteins that help trigger
homologous recombination with the chromosome if there are suitably
homologous regions.
So, my suggestion is the build the DNA you want in a shuttle plasmid,
with nice big (500bp+) chromosomal flanking regions, in E.coli. Then use
PCR to generate loads of linear copies of the desired flanking regions
and genes, and transform with PCR product. You'll *probably* get better
results that way.
That's only true if you're trying to get chromosomal integration. For
plasmids in B.subtilis, efficiencies are pretty good with lab strains,
so you can ditch E.coli and just work with B.subtilis if you have the
right plasmids and workflow.. an area I'm working on right now myself.
Protip: if you're designing DNA for B.subtilis 168 derivatives, avoid
the XhoI restriction enzyme site, and the TopoI recognition site for
B.subtilis: WCATWTAWWA (W = A/T, acc. to IUPAC DNA notation). You'll get
considerably higher efficiency and DNA stability.
--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
Getting DNA into E.coli works best with supercoiled plasmid DNA, but
getting DNA into B.subtilis works best with linear DNA.
The reason is that B.subtilis uses an active uptake system to absorb DNA
in the environment, which can only absorb DNA if it can find a loose end
to chew on. It's been speculated that most B.subtilis transformants from
plasmid solutions are actually those that found damaged or cut plasmids
and absorbed them, which contributes to B.subtilis' undeserved
reputation for being genetically unstable (that is, the DNA was already
damaged, it's not the bug's fault! :))
The differences in efficiency are pretty dramatic. Especially because
the active uptake system includes chaperone proteins that help trigger
homologous recombination with the chromosome if there are suitably
homologous regions.
So, my suggestion is the build the DNA you want in a shuttle plasmid,
with nice big (500bp+) chromosomal flanking regions, in E.coli. Then use
PCR to generate loads of linear copies of the desired flanking regions
and genes, and transform with PCR product. You'll *probably* get better
results that way.
That's only true if you're trying to get chromosomal integration. For
plasmids in B.subtilis, efficiencies are pretty good with lab strains,
so you can ditch E.coli and just work with B.subtilis if you have the
right plasmids and workflow.. an area I'm working on right now myself.
Protip: if you're designing DNA for B.subtilis 168 derivatives, avoid
the XhoI restriction enzyme site, and the TopoI recognition site for
B.subtilis: WCATWTAWWA (W = A/T, acc. to IUPAC DNA notation). You'll get
considerably higher efficiency and DNA stability.
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
Chowe
Jul 22, 2012, 5:54:49 PM7/22/12
to diy...@googlegroups.com
I have found a site that offers two different plasmids that express the lux operon (one for gram-positive one for gram-negative) http://www.caliperls.com/products/reagents/in-vivo-imaging-reagents/light-producing-cells-and-microorganisms/plasmids/.
Thanks for the info Cathal. Why would you build the plasmid for E.Coli? Since E.coli is gram-negative, couldn't I build my plasmid in B.subtilis then PCR amplify the construct out of the plasmid? Im just thinking that it would save me needing an additional plasmid, since I will already have the B.Subtilis vector to get the flanking regions. I would cut out the gram-positive modified lux operon from the plasmid that I found in the link above. Insert it into a B.Subtilis vector that contains large chromosomal flanking regions for B.subtilis. Then PCR amplify my construct and transform. I could try both ways, plasmid DNA and the linear DNA since I will already have a plasmid constructed.
Cathal Garvey
Jul 22, 2012, 6:16:50 PM7/22/12
to diy...@googlegroups.com
Essentially, the problem is the flanking regions themselves. When
assembling a complex piece of DNA, you want to keep regular "save
points" in the form of transformed cells carrying the intermediate
steps. This is easy in E.coli and in B.subtilis with some basic design
considerations for most projects.
However, two of those design considerations for most species are:
1) Plasmid does not contain regions with strong homology to chromosome.
2) Plasmid does not contain highly repetitive regions.
If you build your integration vector in B.subtilis, you're breaking rule
#1. However, if you build it in E.coli, you're not, as there probably
won't be significant homology between the E.coli chromosome and the
B.subtilis elements you use to target integration.
That's why it's not an issue when you're just building an expression
plasmid without any aim of chromosomal integration; without the
repetitive regions, you don't need to worry about
accidental/premature/incomplete integration.
Of course, if you only assemble your plasmid in-vitro prior to use, you
don't need to use E.coli.. but your project may be more prone to failure
due to the number of backup-free steps involved in assembly. You could
try using a technique like gibson-assembly to mass compile all the
sub-units of your project together all at once, but I can't offer any
advice on implementation there, having never done it. :)
assembling a complex piece of DNA, you want to keep regular "save
points" in the form of transformed cells carrying the intermediate
steps. This is easy in E.coli and in B.subtilis with some basic design
considerations for most projects.
However, two of those design considerations for most species are:
1) Plasmid does not contain regions with strong homology to chromosome.
2) Plasmid does not contain highly repetitive regions.
If you build your integration vector in B.subtilis, you're breaking rule
#1. However, if you build it in E.coli, you're not, as there probably
won't be significant homology between the E.coli chromosome and the
B.subtilis elements you use to target integration.
That's why it's not an issue when you're just building an expression
plasmid without any aim of chromosomal integration; without the
repetitive regions, you don't need to worry about
accidental/premature/incomplete integration.
Of course, if you only assemble your plasmid in-vitro prior to use, you
don't need to use E.coli.. but your project may be more prone to failure
due to the number of backup-free steps involved in assembly. You could
try using a technique like gibson-assembly to mass compile all the
sub-units of your project together all at once, but I can't offer any
advice on implementation there, having never done it. :)
Chowe
Jul 22, 2012, 6:47:18 PM7/22/12
to diy...@googlegroups.com
That makes sense, it would work a lot better in E.coli.
So I read a little more and saw that the integration vectors for B.subtilis from mega's link have an ori that functions in E.coli and not in gram-positives, so I could use that same vector in E.coli to copy the plasmid.
I myself would love to try gibson-assembly but maybe I should save that for another time.
Andreas Sturm
Jul 23, 2012, 6:45:11 AM7/23/12
to diy...@googlegroups.com
You're link is amazing... Didn't know the lux operon had been adapted to Gram+ bacteria.
Is there a plasmid map? Are there restriction enzymes that cut the lux operon out (for then ligating it into your host of choice ;) )?
If so, everything would be quite easy.
Is there a plasmid map? Are there restriction enzymes that cut the lux operon out (for then ligating it into your host of choice ;) )?
If so, everything would be quite easy.
--
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To post to this group, send email to diy...@googlegroups.com.
To unsubscribe from this group, send email to diybio+un...@googlegroups.com.
To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/j1n3gccCWp4J.
Andreas Sturm
Jul 23, 2012, 7:05:26 AM7/23/12
to diy...@googlegroups.com
If I understood correctly, with that you won't need any cutting and ligating.
It's already a transposon which will integrate as soon as put into the bacteria (?)
That'd be fantastic.
It's already a transposon which will integrate as soon as put into the bacteria (?)
That'd be fantastic.
2012/7/23 Andreas Sturm <masters...@gmail.com>
Chowe
Jul 23, 2012, 2:07:44 PM7/23/12
to diy...@googlegroups.com
It was a relief to find the gram-positive lux plasmid! And I haven't found a plasmid map yet, I am thinking of emailing the company.
Well I was thinking of cutting out the gram-positive lux operon and ligating into the integration vector so it can have specific chromosomal flanking regions, I would prefer to get the construct into the chromosome if possible. But yes I do believe just using the lux plasmid for gram-positive bacteria would work to express lux in the bacteria.
2012/7/23 Andreas Sturm <masters...@gmail.com>
To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com.
Mega
Jul 24, 2012, 5:15:17 AM7/24/12
to diy...@googlegroups.com
> I am thinking of emailing the company.
Yeah, that's always good. Worst case they don't write you back. Lost nothing. There are already flanking sites in it, I think. It's written it is a transposon. Am I wrong??
Chowe
Jul 24, 2012, 7:51:33 PM7/24/12
to diy...@googlegroups.com
You are correct. I have never worked with chromosomal integration before so I didn't know that a transposon could be used for integration. Thank you for pointing that out. I guess that saves many steps!
Mega
Jul 25, 2012, 4:59:37 AM7/25/12
to diy...@googlegroups.com
Did you ask them about that?
I read transposon and was quite sure about that. But I've too never done chromosomal integration yet (unfortunately), so I can just assume.
On their webpage, many Gram+ bacteria are listed which were engineered by this. It seems, that many bacteria have the recognition sites for this transposon?
EDIT: They don't need any recognition sites, they just jump around and integrate! Strange ;)
I read transposon and was quite sure about that. But I've too never done chromosomal integration yet (unfortunately), so I can just assume.
On their webpage, many Gram+ bacteria are listed which were engineered by this. It seems, that many bacteria have the recognition sites for this transposon?
EDIT: They don't need any recognition sites, they just jump around and integrate! Strange ;)
Chowe
Jul 26, 2012, 1:21:52 AM7/26/12
to diy...@googlegroups.com
I sent them an email about a plasmid map, but still have no reply. Hopefully soon. I will post in here when I get a reply.
Mega
Jul 26, 2012, 4:46:26 AM7/26/12
to diy...@googlegroups.com
Yeah cool.
What would really be awesome if one day you could cut out the erythromycin resistance. Then it would be a totally harmless strain of b.subtillis that just glows in the dark.
It could be a very effective model strain for beginner amateur biologists, because
a) it glows when active thus it's very easy to see if it feels good or if it's dying.
b) B.S. is harmless, if no resistance is added, it stays harmless.
c) the glowing consumes a fair amount of energy, so it makes it even less pathogenic (lower grow rates)
d) You can't kill it by drying or not feeding it. It makes spores and you will still after 50 years be able to strike them on new plates and they will grow again.
What would really be awesome if one day you could cut out the erythromycin resistance. Then it would be a totally harmless strain of b.subtillis that just glows in the dark.
It could be a very effective model strain for beginner amateur biologists, because
a) it glows when active thus it's very easy to see if it feels good or if it's dying.
b) B.S. is harmless, if no resistance is added, it stays harmless.
c) the glowing consumes a fair amount of energy, so it makes it even less pathogenic (lower grow rates)
d) You can't kill it by drying or not feeding it. It makes spores and you will still after 50 years be able to strike them on new plates and they will grow again.
Mega
Jul 30, 2012, 11:18:48 AM7/30/12
to diy...@googlegroups.com
Just wanted to tell,
I plated the glowing colis (from my bio fridge) on new plates, and they were much brighter than the original ones. Maybe because of less satellite colonies because of fresh amp ?
They were even so bright that you could see their glowing in a semi-dark room (10 o clock PM, streetlights illuminating the room weakly) without adapting your eyes to it.
I plated the glowing colis (from my bio fridge) on new plates, and they were much brighter than the original ones. Maybe because of less satellite colonies because of fresh amp ?
They were even so bright that you could see their glowing in a semi-dark room (10 o clock PM, streetlights illuminating the room weakly) without adapting your eyes to it.
Chowe
Jul 31, 2012, 11:19:26 PM7/31/12
to diy...@googlegroups.com
That is great to hear! I have also plated some glowing cells on amp plates (my first at home experiment) and I finally got them to glow. It was a great feeling when I first saw them. Now If I were to put these plates in the freezer to keep for a while, how long do you think I could save them for later use?
Also, I got an email back from the supplier of the gram-positive glowing plasmid and they said that there is information on the plasmid map on the site, he wasn't too clear about it. He also added that it is about $561 US for the plasmid, not very cheap.
Nathan McCorkle
Aug 1, 2012, 5:22:19 AM8/1/12
to diy...@googlegroups.com
Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D,
and E) in Saccharomyces cerevisiae.
http://www.ncbi.nlm.nih.gov/pubmed/14654435
(I can access the full article, so I think it's open-access)
--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
and E) in Saccharomyces cerevisiae.
http://www.ncbi.nlm.nih.gov/pubmed/14654435
(I can access the full article, so I think it's open-access)
> --
> You received this message because you are subscribed to the Google Groups
> "DIYbio" group.
> To post to this group, send email to diy...@googlegroups.com.
> To unsubscribe from this group, send email to
> diybio+un...@googlegroups.com.
> You received this message because you are subscribed to the Google Groups
> "DIYbio" group.
> To post to this group, send email to diy...@googlegroups.com.
> To unsubscribe from this group, send email to
> To view this discussion on the web visit
> https://groups.google.com/d/msg/diybio/-/mUCT3sRy0xcJ.
--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Nathan McCorkle
Aug 1, 2012, 5:26:18 AM8/1/12
to diy...@googlegroups.com
A synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria
http://nar.oxfordjournals.org/content/35/6/e46.abstract
http://nar.oxfordjournals.org/content/35/6/e46.abstract
Nathan McCorkle
Aug 1, 2012, 5:30:10 AM8/1/12
to diy...@googlegroups.com
On Wed, Aug 1, 2012 at 5:26 AM, Nathan McCorkle <nmz...@gmail.com> wrote:
> A synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria
> http://nar.oxfordjournals.org/content/35/6/e46.abstract
See the supplementary doc on that one for sequences that I think are
> A synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria
> http://nar.oxfordjournals.org/content/35/6/e46.abstract
used in a Gibson-assembly--like fashion
Nathan McCorkle
Aug 1, 2012, 5:32:14 AM8/1/12
to diy...@googlegroups.com
On Sun, Jul 22, 2012 at 4:43 AM, Mega <masters...@gmail.com> wrote:
> Hey, I've done something like this...
>
> Took the pVIB plasmid (no need for a specific substrate! just oxygen +
> glucose) in E.Coli and put them into a marmelade jar containing LB medium
> (without ampicillin).
>
> In the dark it was glowing much brighter than a petri dish with those
> bacteria (obviously).
> It stopped glowing after a while (I think one week) perhaps because they
> have consumed all the sugar, or too much endotoxins?)
>
> The best approach however would be to use B.Subtilis. They don't produce
> Endotoxins afaik. And they make spores, so you can just let them run out of
> sugar, wait for month, put sugar into it, and they'll grow and glow again!
> And, of course, bacteria will lose those plasmids after some time. Best bet
> for an eternally glowing lamp is that you insert it into their chromosome.
> (Wanna do this ;) ) And cut out the marker gene (or take a marker that's
> not a clinical antibiotic; such as heavy metal resistance?), to avoid making
> resistant germs...
>
>
A suite of recombinant luminescent bacterial strains for the
> Hey, I've done something like this...
>
> Took the pVIB plasmid (no need for a specific substrate! just oxygen +
> glucose) in E.Coli and put them into a marmelade jar containing LB medium
> (without ampicillin).
>
> In the dark it was glowing much brighter than a petri dish with those
> bacteria (obviously).
> It stopped glowing after a while (I think one week) perhaps because they
> have consumed all the sugar, or too much endotoxins?)
>
> The best approach however would be to use B.Subtilis. They don't produce
> Endotoxins afaik. And they make spores, so you can just let them run out of
> sugar, wait for month, put sugar into it, and they'll grow and glow again!
> And, of course, bacteria will lose those plasmids after some time. Best bet
> for an eternally glowing lamp is that you insert it into their chromosome.
> (Wanna do this ;) ) And cut out the marker gene (or take a marker that's
> not a clinical antibiotic; such as heavy metal resistance?), to avoid making
> resistant germs...
>
>
quantification of bioavailable heavy metals and toxicity testing
Angela Ivask*, Taisia Rõlova and Anne Kahru
http://www.biomedcentral.com/content/pdf/1472-6750-9-41.pdf
Andreas Sturm
Aug 1, 2012, 9:35:33 AM8/1/12
to diy...@googlegroups.com
> Now If I were to put these plates in the freezer to keep for a while, how long do you think I could save them for later use?
I was told you can't put the LB agar into the freezer because it makes crystals.
I got the glowing bacteria from a plate in the fridge and it has been there I estimate a few month, 4 months or so. There were still living cells on it, else the new plate wouldn't have glowed. I put it back into the freezer immediately, so IDK if they would still glow qunatitatively.
Reply all
Reply to author
Forward
0 new messages