Building Skills
tyso...@gmail.com
experience. We have ideas of things we would like to accomplish but
little clue as to how. I suggest a small page with a list of
experiments and detailed instructions as to how to accomplish them.
For example, once a person builds or buys a gel electrophoresis
machine how do they use it. A small treatise on analysing the DNA from
ones own cheek cells would prove invaluable. Or maybe an experiment in
which a succesful splice of DNA into another organism has occured. A
list of books which one might read and get help from would also be
invaluable. I feel hopefull about this community and look forward to
seeing it flourish.
Bryan Bishop
> Many people, myself included, have little to no practical lab
> experience. We have ideas of things we would like to accomplish but
> little clue as to how. I suggest a small page with a list of
> experiments and detailed instructions as to how to accomplish them.
Throughout research labs, these are known as "protocols" - sets of
instructions on how to do something, basically. There's a ton of
protocols in highly convoluted terminology available from sites like
protocol-online.org. Another option, more along the lines of what
you're looking for, is to go look at instructables.com, which contains
a "photoblog" method of exploring instructions. ((And at the moment
I'm working on something similar, something more specific, but in the
mean time, instructables is a good bet.))
> For example, once a person builds or buys a gel electrophoresis
> machine how do they use it.
You clip on one of the nodes to one end of the box :-) and the other
to the other end. The chemical stuff is just some mixing (in a
speciifc order) of APS, acrylamide, etc., unless you're doing a
dangerous gel like ethidium bromide. Actually, it's more than just
clipping, since you need a machine to supply the voltage, but the
interface to those machines is pretty simple- I've seen machines with
only one knob, for voltage. :-)
> A small treatise on analysing the DNA from ones own cheek cells would
> prove invaluable.
A good place to start might be 23andme or deCODEme, but that's not
do-it-yourself really. I'm unaware of do-it-yourself genechips at the
moment, actually, but recent projects, like the paper+tape
microfluidics system might be able to provide some backbone to a
project like that.
> Or maybe an experiment in which a succesful splice of DNA into another
> organism has occured.
http://openwetware.org/wiki/Protocols
(click "Show" next to Escherichia coli, search for the word 'plasmid'
for a good place to start)
> A list of books which one might read and get help from would also be invaluable.
There are so many :-). I actually recommend just reading through the
Wikipedia pages at this point. There's even biotech books on
wikibooks:
http://en.wikibooks.org/wiki/Biotechnology
Anyway, there's also--
A very dense collection of information has been available for about a year now-
http://heybryan.org/~bbishop/docs/ellingtonia/biotech/
or see: http://heybryan.org/gitweb.cgi?p=biotech.git;a=tree
This also includes many bookmarks and links to the questions that
you're asking about. However, part of the process is that others need
to comment on the material and do feedback, suggestions, revisions,
and so on, otherwise it's just going to stay the way it is.
> I feel hopefull about this community and look forward to seeing it flourish.
Yep, but we need some active feedback going on here too to keep improving.
Jim H
Give us an example of a "thing" you'd like to accomplish and we can
provide some tips and more specific references.
As far as analyzing DNA from ones own cheek cells, you'll need to
isolate the DNA first. Mac put together a nice, simple protocol for
this already: http://www.instructables.com/id/5_minute_DNA_Extraction_in_a_Shot_Glass/
The steps and reagents are pretty basic. In terms of "analyzing" it,
you'll need to be specific in terms of what you are looking for.
Analyzing for what and/or why? We can point you in the right
direction, some of which cannot be "hacked" at this point in time.
Aaron Hicks
Makezine is a good platform for this sort of thing. Although you'll need to buy the hard copy, here's some past projects:
DIY thermocycler:
http://www.make-digital.com/make/vol07/?pg=75
I think Make volume 4 also had some other experiments.
There's also a DNA "kit":
http://blog.makezine.com/archive/2005/05/diy_dna_explore.html
Full article: Fingerprint your own DNA:
http://makezine.com/07/fingerprinting/
Makezine allows for people to add their own projects, complete with images, notes, and text, so it'd be a decent venue for archiving.
http://makezine.com/
-AJ
tyso...@gmail.com
that is bioluminecsent. I am currently in Afghanistan so I can't
perform any experiments outside the realm of research and thought but
here is my plan so far. I found a site that will sell a kit to
containing a plasmid to transmit the Lux genes from P. fischeri to E.
coli, specifically the pVIB plasmid. But here is where I hit a wall,
are plasmids only specific to only one species? Will I have to find/
create a new plasmid to make the transfer? When I accomplish that I
have been looking at how to change the colors, most of the differences
that cause color variation, at least for Jamaican click beetles,
occure on exon 4, how do i cut and paste at that small a level. But
that is getting way far ahead of myself.
Daniel Wexler
however transforming yeast with a bacterial plasmid will not work for
two reasons: the plasmid DNA replication machinery for yeast and
gram-negative bacteria are different and therefore the Lux genes (or GFP
from pGlo if you use that system) would have to be subcloned into a
yeast plasmid such as BAC. In addition, you would have to modify the
Lux DNA sequence by site-specific mutagenesis beforehand to account for
differences in codon bias between yeast and bacteria. This would not be
a trivial project.
Christopher Kelty
what the spooks at NSA are saying about this exchange :)
ck
Mary Wahl
What is the ultimate goal for this bioluminescent yeast? It sounds as
though you'd like to make glow-in-the-dark beer. We're all rooting for
you but there are some considerations to make before you start.
Supposing that you made yeast that expressed all of the lux operon
genes, and that the yeast fluoresced...what then? The yeast would
flocculate at the end of fermentation and would not be present in the
beer. What if you made the yeast secrete all of the lux operon gene
products into the beer, and despite all odds those proteins were
capable of functioning together in the extracellular milieu? Then you
still have the problem that reduced flavin mononucleotide is required
for lux bioluminescence but will not be present in the beer.
I don't mean to read too much into your future plans. Yes, you would
need a different plasmid to transform yeast: the primary reasons are
that the bacterial plasmid would not be replicated by yeast and would
not be properly segregated into daughter cells if replication somehow
did occur. (The origins of replication and the systems for segregating
genomes during cell division are different in bacteria and
eukaryotes.) Plenty of appropriate plasmids complete with eukaryotic
promoters are available in yeast. Daniel is right that this would
still be a difficult project.
I don't understand this bit about exon 4...Photobacterium (aka Vibrio)
fischeri is a bacterium and does not have exons. What is the
bioluminescence system used by the Jamaican click beetle?
Mario Raya
> though you'd like to make glow-in-the-dark beer.
Imagine beer that you can drink in the dark... well i don't drink but
i do believe that the fluorescent cell idea is a great step to build
skills, there is a lot of information around about this proteins and
it is easy to see the positive result. Addgene has plenty of plasmids
with all the information that you may need, they sell each plasmid for
$65, even if they can't ship you the plasmid you will be able to find
something useful there.
Mario
cory....@gmail.com
> I don't understand this bit about exon 4...Photobacterium (aka Vibrio)
> fischeri is a bacterium and does not have exons. What is the
> bioluminescence system used by the Jamaican click beetle?
parts of its body. It uses the enzyme Luciferase which oxidizes
Luciferin to produce the light. To make different colors it uses
different versions of the Luciferase protein (that apparently have
SNPs in the 4th exon) as well as different Luciferin molecules
(Luciferin is a class of molecules).
The project that Tyson is suggesting would most likely be using GFP,
so it would only glow under UV light. To make the yeast use a
Luciferase system would be beyond the reach of any current DIY
project. It would require engineering the complete Luicerin anabolic
pathway into the yeast as well as the Luciferase protein. Tyson, if
you really want to use Luciferase in your yeast and don't want to deal
with engineering a complete anabolic pathway, you could possibly add
the Luciferin to the beer just before consumption, assuming Luciferin
isn't toxic. That could get quite expensive (~$1000 for 100mg) but it
might work and would glow on it's own without the UV light source.
Off topic...
Bryan wrote:
> You clip on one of the nodes to one end of the box :-) and the other
> to the other end. The chemical stuff is just some mixing (in a
> speciifc order) of APS, acrylamide, etc., unless you're doing a
> dangerous gel like ethidium bromide.
when using polyacrylamide gels. Don't get any on your skin. I would
recommend that anyone here who is not confident in their lab skills to
stick to agarose gels. So safe you can eat it. Although, you are
right, ethidium bromide is potentially dangerous.
Cory
tyso...@gmail.com
deployment
tyso...@gmail.com
1. The goal of my yeast project is to create a biological light source
that excretes a useful and safe byproduct. Unfortunately due to
luciferase's dependence on oxygen I am more likely to create vinegar
instead of alcohol because that is the biproduct of aerobic
fermentation. Not that that will stop me from trying ;)
2. I hope that someone can suggest some experiments or protocols which
will give me a better grasp of what is going on and what I need to
learn.
3.I obviously need to have a much deeper understanding of basic
genetics. is there a genetics for dummies book? Registering to go back
to college.
4. Im sorry Mr. Tobin but I must refute the idea that something
cannot be done, it is simply a dearth of knoweledge and supplies I
lack. Fortunately I can gain both. But I do deeply appreciate your
input and hope that you will be willing to talk with me more on this
subject. You seem to have quite a depth of understanding of the
subjects I am frightfully deficient in.
5. I thank you all for bearing with me. I recently downloaded
protocols for making yeast competent and am trying to understand that.
I hope you will all continue to bear with me.
Jim H
If you are looking for bio-luminescence, you might want to use more
complex fungi instead of simple yeast.
There are several reports of natural bio-luminescence with molds and
basidiomycetes. I can't find ant specific references, but I'll try to
locate something. http://www.mushroomexpert.com/taxonomy.html
Cory Tobin
> cannot be done, it is simply a dearth of knoweledge and supplies I
> lack. Fortunately I can gain both.
I'm not saying you can't engineer a complete Luciferin anabolic
pathway into yeast. It can be done and probably will be done (maybe
by you?). Amyris was able to engineer a complete butanol metabolic
pathway into bacteria; E. coli I think. It took a team of expert
metabolic engineers a couple of years to accomplish that feat. So it
may take a good amount of time and resources but it can be done.
> But I do deeply appreciate your
> input and hope that you will be willing to talk with me more on this
> subject.
Surely.
-Cory
Cory Tobin
> Okay, Where to start. Ok why do i need to engineer the entire anabolic
> pathway? Cant a plasmid just inject the genes I need and it glows,
> keep in mind I know this is a simplistic approach. I assume there is
> an extant anabolic pathway in some organism, how do I find it? Is the
> main body of the problem dealing with codon bias? Lets say I engineer
> the pathway, can a plasmid then transfer the neccessary data or will i
> need something like a lamda phage?
> And last but not least, are you interested in working on this with me?
>
> Sincerely,
> Ty
> Okay, Where to start. Ok why do i need to engineer the entire anabolic
> pathway? Cant a plasmid just inject the genes I need and it glows,
> keep in mind I know this is a simplistic approach.
There are different ways to make things glow. One is fluorescence and
another is luminescence. Fluorescence is where you excite a protein
with one wavelength and it emits a completely different wavelength.
For example Green Fluorecent Protein (GFP) is excited at 395nm and
then emits at 509nm. You could simply add a plasmid carrying the GFP
gene to the yeast. When you shine 395nm light on it, it will emit
509nm light.
Luminescence is when chemical energy is converted into light, usually
by a protein. In the case of fireflies and the beetle you mentioned,
they have a protein called Luciferase. This protein oxidizes a
chemical called Luciferin. The species that have luciferase also have
pathways to build Luciferin using energy and simple building blocks.
When Luciferase is used in the lab the scientist adds (commercially
available) Luciferin to the sample instead of building in the complete
pathway to make Luciferin. This way all they need is a plasmid
carrying the Luciferase protein. The problem is that the Luciferin
only lasts a short while before it gets used up. So, in short, you
could add a plasmid carrying the Luciferase gene to yeast and as long
as there is Luciferin in the growth media it will emit light.
> I assume there is
> an extant anabolic pathway in some organism, how do I find it?
Good question. I wish I knew a good way to do this.
> Is the
> main body of the problem dealing with codon bias?
If you're referring to engineering a metabolic pathway into an
organism, codon bias can be a problem but it's a relatively easy one
to solve. The difficult part is to figure out what proteins you need
and also how to get them expressed in the correct amounts. Too little
and the pathway won't work; too much and it can be toxic to the cell.
> Lets say I engineer
> the pathway, can a plasmid then transfer the neccessary data or will i
> need something like a lamda phage?
Plasmids are too small to carry all the genes and promoters necessary
to code the entire pathway. A Yeast Artificial Chromosome would be
more suited for that job.
http://en.wikipedia.org/wiki/Yeast_artificial_chromosome
> And last but not least, are you interested in working on this with me?
I can help out intellectually. If I were to actually do any physical
work on the project it would automatically become property of Caltech.
:(
> One more, wouldn't this using this plasmid acheive the desired result?
> http://www.addgene.org/pgvec1?f=c&identifier=1329&atqx=BAC%20Lux&cmd=findpl
That plasmid would, in the presence of Galactose, express Luciferase.
Since it's being advertised as a yeast vector it has probably been
codon-optimized for Saccharomyces cerevisiae. To make it emit any
light you would still need a source of Luciferin.
Cory
Myriam Abdelaziz
Myriam Abdelaziz
Photographer
New York + 1 917 637 94 19
Paris + 33 (0) 6 64 14 55 96
Cairo + 201 204 259 35
www.myriamabdelaziz.com
Bryan Bishop
> My name is Myriam Abdelaziz. I am a freelance photographer based in New
> York.
Hi there. :-) Do you have a flickr stream we can check out?
> Sorry to bump into the blog but further to the AP article published on dec.
> 25th, I am interested in your activites and trying to get in touch with
> members of DIYbio for further details...
You're already in contact, we all participant via the mailing list and
sometimes IRC. We'd be happy to publicly discuss what's going on, and
maybe point you to some items in the mailing list archive to see what
the trends have been.
> Could you please send me the contact info of a person I can speak to.
> Thank you very much for your help,
All of us would be qualified, but I think Mac, Jason and a few others
have been doing most of the media waltzing, so check them out.
John Cumbers
John Cumbers, Graduate Student
NASA Ames Research Center
Mail Stop 239-20, Bldg N239 Rm 373 Moffett Field, CA 94035, USA.
cell +1 (401) 523 8190, fax +1 (650) 604-1088
Graduate Program in Molecular Biology, Cell Biology, and Biochemistry
Brown University, Box G-W Providence, RI, 02912, USA
John Cumbers
John Cumbers, Graduate Student
NASA Ames Research Center
Mail Stop 239-20, Bldg N239 Rm 373 Moffett Field, CA 94035, USA.
cell +1 (401) 523 8190, fax +1 (650) 604-1088
Graduate Program in Molecular Biology, Cell Biology, and Biochemistry
Brown University, Box G-W Providence, RI, 02912, USA
Mary Wahl
You can find classic textbooks on NCBI Books (http://
www.ncbi.nlm.nih.gov/sites/entrez?db=Books). These books are free but
you can't read them page by page: you have to search for keywords. Try
searching for keywords like "recombinant" or "cloning" in the genetics
book by Griffiths.
Also I wanted to point out to you that "luciferase" and "luciferin"
are generic terms that refer to the enzyme and its substrate,
respectively, that are used in the bioluminescence pathway. V.
fischeri luciferase (luxA and luxB) is not homologous to firefly
luciferase, which is not homologous to the cnidarian Renilla's
luciferase. (If you want to convince yourself of this, search NCBI
Protein for the amino acid sequence of each species' luciferase, then
try an alignment.)
By my count we are up to five different bioluminescence systems
simultaneously under discussion. It does not make sense to talk about
how difficult the project would be because we have not made an
informed decision about which system to use. FYI, a bacterial
bioluminescence system has already been transferred to yeast (see one
application in Sanseverino 2005, http://aem.asm.org/cgi/reprint/71/8/4455.pdf).
This system includes the bacterial luciferin biosynthesis pathway so
the live cells luminesce: no need to lyse the cells and add a
luciferin. Some luciferin anabolic pathways might take years to port
into yeast...but not that one, evidently! At least, not for you: the
corresponding author is pretty much obliged to send you any strain
that they have published if you request it nicely. Clearly this
bacterial bioluminescence would be an appropriate system to use.
Cory Tobin
> bioluminescence system has already been transferred to yeast (see one
> application in Sanseverino 2005, http://aem.asm.org/cgi/reprint/71/8/4455.pdf).
> This system includes the bacterial luciferin biosynthesis pathway so
> the live cells luminesce: no need to lyse the cells and add a
> luciferin. Some luciferin anabolic pathways might take years to port
> into yeast...but not that one, evidently! At least, not for you: the
> corresponding author is pretty much obliged to send you any strain
> that they have published if you request it nicely. Clearly this
> bacterial bioluminescence would be an appropriate system to use.
Wow, great find! The system would need some modifying so it doesn't
rely on estrogen but it would make for a great starting point.
I don't know much about beer (except how to drink it). Anyone know
what species of yeast is commonly used? Just curious.
Cory
Scott Kerr
>what species of yeast is commonly used? Just curious.
For lagers you use Saccharomyces pastorianus (according to wikipedia this a hybrid species made up of cerevisiae and Saccharomyces bayanus)
White labs distributes many different varieties.
I have not done enough home brew to really do a side by side test but presumably they all have some sort of mutations to cause subtle flavor differences. This along side the seemingly endless other variables in the brewing process that cause flavor differences.
Actually now that I think about it, making your own beer is probably a great way to get for DIYbio beginners looking for a new project. You learn all about sterile technique (or what the local bacteria population smells/looks/tastes like until you learn sterile technique) and figuring out creative ways to do protocols with minimal equipment.
Additionally, many micro brews "bottle condition" their beers, this is the process of carbonating the beer after the cap has been put on the bottle. In each beer there is a small amount of yeast that finishes off the last bit of sugar in the beer and in the process creates the carbonation. I have not personally tried this but the if you check out some home brew forums there should be some protocols on how to capture this yeast and start a culture. Homebrewers do it all the time, DIYbio should have no problem
tyso...@gmail.com
digs the idea of glowing beer I guess :) I kid, I understand why
everyone keeps suggesting that, it is a heck of a lot easier than
doing the luminescent. And I will probably create one with GFP first
just for practice, but I will keep hammering away at my goal. At the
moment I just need to study how Luciferin and luciferase are built
within the cell. And at the moment it must remain within the realms of
a thought experiment until I come home, sorry I know i keep mentioning
it but i really miss it. Also I do intend to do extensive testing of
any byproducts created, I understand the value of caution, esp with
the tenuous position of the biohack movement in regards to regulation.
Does anyone think it might be easier moving it into cyanobacteria
instead? just a thought.
Jim H
find literature references. Also, don't think you'd want to drink it.
tyso...@gmail.com
> find literature references. Also, don't think you'd want to drink it.
>
Touche
Thank you to Mary and Cory. I know I ask many questions. As Mary
pointed out it would be easier to realize this project by settling on
a luciferase system and further helped by giving us all the paper on
people who have done it already. So the question now becomes how to
alter the promoter. If I understand the paper correctly the plasmids
which would need to be kept are the pUTK404 and the pUTK401/407(which
would need to be altered to a new promoter) as well as the plasmid for
ampicillin resistance.
So..... the question then becomes what promoter to select so that
transcription is continuous. Could we use the same promoter that
pUTK404 uses? From the paper I don't think that was estrogen
dependent. Any other Ideas?
If I am wrong about any of these PLEASE correct me.
tyso...@gmail.com
404?
Cory Tobin
> 404?
I don't think they are for sale. You can request them from the
corresponding author of that paper Mary linked to. Gary Sayler. His
contact info is on the bottom of the first page.
-Cory
tyso...@gmail.com
Once I get back I will try and put together a protocol, vet it, and
post it.