getting started

[CodeWarrior]

I'll begin by apologising. I get the impression you guys get these
'what do I need to get started' emails on a regular basis and it must
be tedious. I'll try not to waist your time by being overly vague. I'm
interested in doing some home experimentation in biotech. I'm a final
year applied maths PhD student and would like to think I know the
basics, the theoretical basics that is, of biotechnology in the modern
age. My interest is in RNAi and transient gene expression through
insertion with Adeno-associated and Adenoviruses and Lipoplexes. This
is in the context of morphogenesis and homeostasis in polycellular
eukaryotes. Most probably samples taken from my self.

What I lack is any kind of experience with the experimental side of
biotechnology. What sort of lab can I expect to build oriented to that
sort of stuff. Funding wise my budget is in that grey zone of being
more than the average hobbyist can afford but a good deal less than a
well funded university lab. Can any one advise me on what I might need
to get started here?

regards
CW

[J. S. John]

On Fri, Apr 8, 2011 at 2:51 PM, CodeWarrior <code.w...@gmail.com> wrote:
> I'll begin by apologising. I get the impression you guys get these
> 'what do I need to get started' emails on a regular basis and it must
> be tedious. I'll try not to waist your time by being overly vague. I'm

No need to apologize. I think most of us have no idea on how to get
started since not all here are in the bio/chem field.

> interested in doing some home experimentation in biotech. I'm a final
> year applied maths PhD student and would like to think I know the
> basics, the theoretical basics that is, of biotechnology in the modern
> age. My interest is in RNAi and transient gene expression through
> insertion with Adeno-associated and Adenoviruses and Lipoplexes. This
> is in the context of morphogenesis and homeostasis in polycellular

Seems like you know more than you think. Think of yourself as a brand
new grad student and how would you go from there. The people around
here can advise you on where you should go. Judging from your area of
interest, which I don't know much about, this is not some trivial
subject like "how to grow GFP __{insert plant name}____". Without
doing some research, I guess you are dealing with gene insertion with
viruses. Seems like something done in a university lab. Can this be
done at home? We're talking about viruses here so...

> eukaryotes. Most probably samples taken from my self.

Whoa, working with mammalian cells is on another scale in the first
place. Working with your own cells might be disastrous. What if you
culture your cells and you change their DNA. Then you accidently get
those cells into your own body. Bad things are going to happen. I
haven't worked with mammalian cells before and safety-wise, I think
they're on a different level than bacteria or fruit flies.

> What I lack is any kind of experience with the experimental side of
> biotechnology. What sort of lab can I expect to build oriented to that
> sort of stuff. Funding wise my budget is in that grey zone of being
> more than the average hobbyist can afford but a good deal less than a
> well funded university lab. Can any one advise me on what I might need
> to get started here?

Labs are not cheap. Even with used equipment, you're probably looking
at thousands of dollars. A nice microscope is at least $100. Also,
depending on the subject you choose, the price will go up.

You're applied math. Why not start with some modeling? Can that be
done? It would be cheaper and less troublesome. I am very interested
in modeling but have very little experience with that.

Good luck. You might want to reconsider the self-samples part though.

-JJ

[Forrest Flanagan]

It's actually not that dire. Are you looking to turn some genes off?

In my opinion, a laminar flow hood, an incubator, and an some micropipetters would be good to start off. Where on the globe are you located? I might have some tips for outfitting a lab if you're in the US.

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[Bryan Bishop]

On Fri, Apr 8, 2011 at 2:15 PM, J. S. John <phill...@gmail.com> wrote:

Whoa, working with mammalian cells is on another scale in the first
place. Working with your own cells might be disastrous. What if you
culture your cells and you change their DNA. Then you accidently get
those cells into your own body. Bad things are going to happen.

It is much more likely that his cell cultures will die. IMHO, that's a good problem to have (it means you have a cell culture in the first place).

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

[Cathal Garvey]

The more likely hazard is that the cells will contract something that your immune system would normally fight off with trivial effort, acting as a vector for transmission of a disease-causing load of whatever disease agent we're discussing. So your cell cultures get sick, and you catch it from them.

That said, I don't think it's a serious risk. When working with your own tissue explants, there is also the possibility as JS mentions that the cells, being your own, could be accepted if you jabbed yourself and might start growing inside you again. Considering how the in-vitro environment is intrinsically mutagenic to some extent, there is a small cancer hazard there. But not much of one.

The biggest problem is that what you're setting out to do will be A) Very hard to do DIYbio and B) Possibly illegal or inadvisable. After all, adenovirus and other mammalian cloning vectors are used because they *infect and transform mammalian tissues*, something that should be carefully considered when working with transgenic stuff at home. Do you want to accidentally transform yourself with GFP? You won't start glowing; your body will find and destroy the transformed cells efficiently and in short order, no doubt. But then you may be hypersensitised to GFP for the rest of your life, possibly developing an allergy.

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[Jonathan Street]

Using samples from yourself is a not uncommon occurrence in biology but does present some additional problems that must be considered.  Taking a cheek swab for a PCR experiment in an undergrad class is essentially innocuous.  Taking cells with the intention of growing them is more problematic.  If those cells were to re-enter your body your immune system would likely not recognise them as foreign and they could replicate.  Many tissue culture experiments use immortalised cell lines for convenience which in many ways are similar to cancerous cells.  This isn't a good combination.

The suggested use of viruses adds to this potential danger.  I don't personally have any experience with the use of viruses for transfections.  Some of the people in the lab I work in do though and from what I've heard, in the UK at least, there are regulatory hurdles to jump before such work can be performed.

If you did choose to go ahead I would encourage you to use established cell lines rather than your own.  If possible from another species than human.

In addition to the safety challenges working with mammalian cells is more financially demanding than prokaryotes.  In contrast to prokaryotes which can survive happily on potatoes and glucose mammalian cells typically need a variety of growth factors - either purified or from sera.  Both are expensive.

If you do decide to go ahead maintaining sterility will be your first challenge - you'll definitely need a laminar flow hood, filter all your solutions using 0.22 um filters and likely still need to supplement your media with antibiotics.  You'll also need an incubator and a source of CO2 - a 5% CO2 atmosphere is typically maintained in incubators for mammalian work.

[CodeWarrior]

Mr Flanagan is correct my 1st interest would be performing gene
knockdown experiments. Mostly on fibroblasts, keratinocytes and other
cells that produce or interact with ECM. Then as a next step something
akin to gene knock up experiments if there were such a thing. The
delivery of DNA (but not reverse transcription) to the nucleus or
possibly the delivery of mRNA to the endoplasmic reticulum. I wasn't
so much thinking of using live viruses so much as ripping the code for
the protein shell off one inserting it into a single celled organism
and some how replacing the code for loading the virus with a copy of
it's own DNA with a different desired sequence. I imagine something
closer to Lipofection might be a lot easier to implement but my
background reading leaves me wondering about the effectiveness of
liposomes as a vector. The approach taken by EnGeneIC seams
interesting something similar but more simplistic might work for in
vitro work I suppose. Once again experimentally I'm out of my depth.
Working with non eukaryotes would be quite pointless. As I said I'm
interested in studying ecm / cell interactions. I suppose I could use
the cells of any mammal really but animal testing laws are kind of
harsh in the uk. Using my own cells I should be on safe ground
legally.

I would hope the likelihood of accidental re insertion was low as I'd
like to keep the cells at least partially bound in the ecm. Basically
I want to poke the fibroblasts and similar cells genes and see if I
can get them to remodel the ECM in any interesting ways.

On Apr 8, 8:34 pm, Forrest Flanagan <solenoidcl...@gmail.com> wrote:
> It's actually not that dire. Are you looking to turn some genes off?
>
> In my opinion, a laminar flow hood, an incubator, and an some micropipetters
> would be good to start off. Where on the globe are you located? I might have
> some tips for outfitting a lab if you're in the US.
>

> On Fri, Apr 8, 2011 at 2:15 PM, J. S. John <phillyj...@gmail.com> wrote:
>
>
>
> > On Fri, Apr 8, 2011 at 2:51 PM, CodeWarrior <code.w4rr...@gmail.com>

[CodeWarrior]

Another thought. I had hoped that cells still bound in thin slivers of
ECM or something like that might not need so much in the way of growth
factors but if they did and given I was already resigned to using a
prokaryote as a machine for churning out my vectors for gene
expression modification presumable I could load one with the necessary
genetic material for pumping out recombinant growth factors that might
be needed? Thus producing a kind of support prokaryote to
symbiotically sustain the experiment?

> On 8 April 2011 20:34, Forrest Flanagan <solenoidcl...@gmail.com> wrote:
>
>
>
> > It's actually not that dire. Are you looking to turn some genes off?
>
> > In my opinion, a laminar flow hood, an incubator, and an some
> > micropipetters would be good to start off. Where on the globe are you
> > located? I might have some tips for outfitting a lab if you're in the US.
>

> > On Fri, Apr 8, 2011 at 2:15 PM, J. S. John <phillyj...@gmail.com> wrote:
>
> >> On Fri, Apr 8, 2011 at 2:51 PM, CodeWarrior <code.w4rr...@gmail.com>

[Nathan McCorkle]

You sound pretty keen for a math major. And without any resources to
begin with, you're going to spend a good two to three thousand USD to
really be comfortable with any of your projects. What you need:

--Incubator with non-dry environment... you could build this yourself,
but unless you know how to build electronics and program, you can
probably find one at an auction or on ebay. (as for the rest of the
equipment)

--laminar flow hood - again you can build this, there are guides
online... some as jerry-rigged as a clear plastic tub and a HEPA
filter siliconed to a hole cut in the backside. UV light inside it
turned on when you aren't doing work is good measure IMO too.

--pipettors, pipets syringes and fine needles (a lot of antibiotics
come in sure-seal bottles), scalpels (you mentioned tissue), culture
dishes and multi-well plates, inverted stereo microscope

--growth media (MEM, DMEM, RPMI, serum-free), serum (bovine, fetal
calf, horse, goat, probably your own??), antibiotics/antimycotics
(streptomycin, penicillin, amphotericin)
(google 'DMEM antibiotics', first 4 links deserve a reading)

--cells - can be ordered as live mice with known genotype, frozen cell
lines (often cancer cells are of use), extracted from live organisms

I don't think eating chicken or eggs is illegal in the UK, so if you
bought a live chicken and cooked dinner (as many people do) with all
but the heart, which you threw in some DMEM to keep going, people may
think you're weird but there should be no fuss IMO... you could pierce
your ear, and throw the chunk you remove into some media likewise, how
else were you thinking of getting your own cells?

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Matt DiLeo]

Keep in mind for all this molecular biology stuff, a pipet isn't
something you can just pick up, start pressing and expect to work.
Before you spend your first couple thousand on enzymes and kits, make
sure you can pipet reproducibly and without contamination. The failure
to move liquids accurately and without picking up DNA / microbes from
the environment is a major cause of failures in the lab. A nice drill
that I learned from my postdoc PI is to pull up 1000 uL of water, and
lay 10 100 uL drops on a flat, hydrophobic surface like wax, then move
each 100 uL drop into 10 uL drops. you'll be amazed how fast you run
out of water when you first start. A general awareness of your
surroundings is also critical - e.g. to notice if you swing the tip of
your "clean" pipet into something on your bench while looking for a
tube you're about to use. Likewise, when working in a flow hood, you
don't want to reach over your "sterile" surface to pick up stuff
behind it. In my last lab, the manager would send undergrads home with
a latex glove and a screw tap tube and make them practice opening and
closing it with one hand while they watched tv in the evenings. These
types of practical details are mundane but really critical.

> > For more options, visit this group athttp://groups.google.com/group/diybio?hl=en.

[General Oya]

Try and get your hands on this text I found for the bio-printer projects.

Culture of Cells for Tissue Engineering
http://www.wiley.com/WileyCDA/WileyTitle/productCd-0471629359.html

It covers safety concerns, equipment, protocols for biopsy and (de/re)differentiations and reagents.

I think you'll find it useful.

Ryan

[mad_casual]

I know you said "Working with non eukaryotes would be quite
pointless.", but, have you cultured *any* cells in a pure culture,
bacterial, yeast, eukaryotes, or mammalian? Have you transformed
*anything*? Immortalized a cell line? Passaged or divided a cell line?
I don't mean to say you're not talented, but the question "Where
should I go?" is sometimes best answered with the question "Where have
you been?"

Additionally, I don't know you personally, your ultimate goal, or what
your budget/timeline is, but you're potentially talking about years of
work by someone trained at the Ph.D. level with a pre-existing lab.
Additionally, your lack of practical knowledge combined with the
assumption that you will just be able to waltz into a lab and do
whatever you're trying to do is a little presumptuous. I'm not saying
you can't do it, and I wholly support the 'poke it and see what
happens' attitude, but you're asking about building and testing a jet
fighter without apparently having built a car or flown a plane.
Probably best right now to get your hands wet any way you can.

[J. S. John]

On Sat, Apr 9, 2011 at 1:21 PM, mad_casual <ademl...@gmail.com> wrote:
> whatever you're trying to do is a little presumptuous. I'm not saying
> you can't do it, and I wholly support the 'poke it and see what
> happens' attitude, but you're asking about building and testing a jet
> fighter without apparently having built a car or flown a plane.
> Probably best right now to get your hands wet any way you can.

That is so true. Unless you did something in a lab, you won't truly
appreciate how hard it is to get something as simple as bacterial cell
cultures to work. Sometimes the skills are not really lab related like
steady hands and keen eyesight. You know how hard it is at first to
fill electrophoresis gels wells without contaminating nearby wells?
You got a PhD in applied math but that is as practical when it comes
to lab work as a PhD in EE is.

Maybe you can get a teaching post for a lab class? I know the TAs
usually try to reproduce the experiments beforehand or set it up. I
don't know about your University but you could try.

[CodeWarrior]

I was thinking about shave biopsies of things like moles (I should
point out these are long term moles I have had for years that I must
admit to my shame I've been picking on a regular basis). Also very
small punch biopsies. In terms of self manufacture of equipment I have
some skills and know my way around bread board but unless the cost
savings are large I'd rather use second hand / ready made equipment
for reliability's sake. Money wise a few 1000£ (GBP) is well with in
the realms of possibility. In terms of long term goals my interest is
in modelling morphogenesis and homeostasis mathematically and then
potential applications in manipulating there processes in vivo. I
actually sent round a few proposals for post doc work round
universities to this effect but got no responses. I've become rather
despondent with the academic process over the last 2 and a bit years
working as a mathematician in a department of engineers. Felt rather
marginalised by the average experimentalists difficulty in relating to
my work and the lack of contact with other students and staff with
maths background. Never the less I feel I've got some interesting new
models which I hope will improve insight into how polymeric liquids
flow. I doubt it'll be well received though since the maths involved
is totally alien to the engineers. Now I'd like a shot at doing the
same thing for biology. But frankly I don't think I'm going to get it.
And unlike polymer engineering the experimental data I need and the
means to sanity test my results are not available. There are no well
established mathematical models for tissue homeostasis and genesis to
compare my own with for calibrating and validation. The only way to do
it is experimentally. I've basically worked in isolation through out
my PhD so it doesn't make much difference to me if the research I do
after my PhD is in or outside of main stream academia so long as I get
to work on projects I find interesting which frankly I might not
unless I'm snapped up by some ambitious uni that likes blue skies
research. So yes I'm a mathematician and therefor prone to thinking
I've a right to meddle in any field I can write an equation for. It
would be much nicer if I could have a supportive university to get
experiments done for me by colleagues but that doesn't look very
likely right now. So long as I have time and tenacity, so long as I
can grind enough money out with some sort of day job and pursue my
interests it's got to be better than working on uninspiring short
sighted projects under some universities umbrella and feeling
despondent all the time. Thankyou all for your advise be assured I am
taking it on board.

> > For more options, visit this group athttp://groups.google.com/group/diybio?hl=en.

[Cathal Garvey]

Ambition is admirable, and I'd encourage you to follow your ambitions to the fullest extent, provided you're aware of how much work and risk is involved. You may not be able to jump into advanced cell culture without working first with simpler projects to learn key skills, but if you're determined, it's your learning curve. :)

I'd also encourage you consider and revise your plans according to hazard and legality: not everything is appropriate to DIYbio in the same way that not everything is appropriate for hobby electronics tinkering (unlicensed radio, high voltage, and driving homemade vehicles on the motorway without a license come to mind).

There's a point, in other words, when your project is either hazardous or illegal enough to warrant finding alternate means to do it. You could still do it yourself (DIY), but it may hotel getting a license and building a licensable premises. I've hit this wall in Ireland, where you must get a license to work with GMOs of any sort, no matter how inane or innocuous the alterations. So, I'm getting one. If it's possible you need a license where you live to work with potentially infectious human cell lines, make sure you've done all the research.

Regarding self-biopsies.. I'd suggest having them cut out entirely rather than shaving or punching only a little. Pre malignant growths can be stimulated to full malignancy by biopsy, so for small things like moles it's better to chop than shave.

Sent from my Phone
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> You sound pretty keen for a math maj...

> On Fri, Apr 8, 2011 at 9:04 PM, CodeWarrior <code.w4rr...@gmail.com> wrote:

> > Another thought. I...

> > For more options, visit this group athttp://groups.google.com/group/diybio?hl=en.

>
> --
> Nathan McCorkle
> Rochester Institute of Technology

> College of Science, Biotechnology/Bio...

[Patrik]

If your main interest is in modeling morphogenesis, there are far
easier organisms to work with than your own cells. There's a lot of
work on morphogenesis (and some pretty good mathematical models) in
developing fly embryos, sea urchins, etc. Heck, I bet even plant cells
are much easier to work with.

Since we talked about slime molds on some other threads recently...
There was a really cool paper in Science a couple weeks ago, showing
that slime molds actually develop the equivalent of a polarized
epithelium during their multicellular fruiting stage - which is pretty
mind blowing, considering they spend most of their life cycle as
single-celled amoeba!

http://www.the-scientist.com/news/display/58047/
http://www.sciencemag.org/content/331/6022/1336.abstract

[CodeWarrior]

thank you to everyone. you've all been very helpful and given me a lot
to think about and research. Especially the individual who sent me
that informative pdf. One last thing. can any one recommend good
budget suppliers for the uk? Not that I'm afraid to spend money but
I'd rather not waist it.

[Cathal Garvey]

For lab-grade chemicals, I love mistralni.co.uk.

For glassware and basic lab stuff, try the lab and measurement section on http://www.brouwland.com/ (do not omit the www on that site or it borks).

Alternative source for glassware, which is highly complementary to Brouwland, is this ebay seller: 

stores.ebay.ie/Laboratory-Stuff-and-Fishy-Things

You'll need a pressure cooker as big as you can find, which is best bought locally due to size and weight. Make sure it has safety valves in case the main pressure valve gets blocked somehow. I'd advise buying some autoclave indicator tape from the ebay seller above, it's a special sort of tape that turns black striped when you have properly sterilised something by pressure-cooking; without that tape you can often make errors in sterilising that can cost whole experiments.

Because you're planning to work with mammalian cell lines, there will be *many* things that you have to buy pre-sterilised from expensive suppliers because there are simply no other sources. Some things you *can* find via outside sources, including things like amino acids (L-glutamine comes to mind), but you'll have to sterilise those as well and they rarely tolerate pressure cooking. Therefore, you'll also need 0.2um sterile filters, which sterilise liquids by simply having too small a filtering size for bacteria to get through. However, don't expect to find these cheaply at all. You might be able to use a household water sterilisation gravity filter if you're doing large volumes of stuff, but given the price of most of the reagents we're discussing "large volumes" simply aren't feasible.

Um, good luck. I don't think this is going to be very affordable, but it'll be interesting to see if you pull it off. Don't forget that if you're in the UK, you might be better served joining a DIYbio group, of which there are several already, and learning through and with those folks. However, membership will probably include conditions on what you can and can't do under the auspices of the group, which might mean your intended project would get disqualified on safety grounds.

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[Nathan McCorkle]

There is a lot of filtering that you would have to do, but is blood
enough to grow cells on? Could you "feed" your own experiments? If
that is too gruesome, what about getting it from a flock of healthy
goats? It seems that the only bad thing about this is that using a
needle everyday isn't healthy for the animal's (or your) blood vessels
and thus decrease the overall fitness of your blood source (which is
not cool, because goats are friendly, and you don't want to put
yourself at undue risk).

It seems like DIYbio should think about easy ways to produce feedstock
for these more touchy cells... even for minimal media recipes for
bacteria, there are a lot of special components. Maybe some sort of
transgenic yeasts or bacteria that secrete only certain amino acids,
so a cell free growth media would have /mostly/ one amino acid.
Purifying the salts seems like the only next step to having pretty
pure aminos... mix them with some phosphate buffers and serum (which
would be acquired some other way).

Our bodies make everything our cells need, so a bio-fermentor solution
to this problem IS possible.

--

[jlund256]

> There is a lot of filtering that you would have to do, but is blood
> enough to grow cells on? Could you "feed" your own experiments? If
> that is too gruesome, what about getting it from a flock of healthy
> goats? It seems that the only bad thing about this is that using a

No, no human blood, a hundred times no. Same with livestock. Blood
serum is a common additive to mammalian cell culture and is collected
from slaughtered animals. Usually as an additive at 5% or 10% or
media volume to provide protein growth factors and whatnot. That
would be the source, but really, mammalian cell culture isn't worth
the trouble.

Insect cell culture probably isn't worth the trouble either, but is
cheaper/easier/safer, the provides most of the cell types, genes, and
biology happening in mammals.

Or try a squid. I hear they have neurons big enough to stick a straw
into.

> It seems like DIYbio should think about easy ways to produce feedstock
> for these more touchy cells... even for minimal media recipes for
> bacteria, there are a lot of special components. Maybe some sort of
> transgenic yeasts or bacteria that secrete only certain amino acids,

There's a strange interest on this list in taking existing lab
protocols, and replicating them as DIY projects by replacing the
reagents known to work with baroque and complicated DIY replacements.
Well, these are mostly thought experiments, or just notions. These
proposals nearly always seem unlikely to work and difficult to
implement. You would spend months and years assembling ingredients
and never bake a cake.

Instead, I suggest looking for interesting things to study and/or cool
experiments to do, and find the simplest and fastest way to do them.
The best plants and animals to study are those at close at hand.

Jim Lund

[Cathal Garvey]

Insect cell culture interests me, but I know nothing yet of it. Based solely on the fact that few intracellular parasites (viruses, bacteria) exist that shuttle between human and Insect hosts, Insect cell culture makes a lot more sense.

I do think that, by the time you have established a DIYbio cell culture lab that works, you could probably have completed a college degree in cell biology and have gotten a position studying it somewhere better equipped, possibly for the same or less cost. Some things aren't well suited to DIYbio just because the economies don't make sense, although there's a case to be made for do-it-with-others bio when this is the only barrier.

It stands out at me though that plant cell and tissue culture is an untapped area for us: it's not much harder than bacterial culture and it can be more practical and more rewarding than mammal or insect because you can optionally regenerate entire plants from your experiments. Indeed, it's often used to mass propagate valuable isolates industrially.

I studied plant tissue culture and biotech in college and have a little practical experience, so I'd be able to offer some pointers to people with an interest in this area, although there might be someone else lurking around here with true practical experience!

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On 13 Apr 2011 07:43, "jlund256" <jlun...@gmail.com> wrote:

> There is a lot of filtering that you would have to do, but is blood

> enough to grow cells on? Cou...

No, no human blood, a hundred times no.  Same with livestock.  Blood
serum is a common additive to mammalian cell culture and is collected
from slaughtered animals.  Usually as an additive at 5% or 10% or
media volume to provide protein growth factors and whatnot.  That
would be the source, but really, mammalian cell culture isn't worth
the trouble.

Insect cell culture probably isn't worth the trouble either, but is
cheaper/easier/safer, the provides most of the cell types, genes, and
biology happening in mammals.

Or try a squid.  I hear they have neurons big enough to stick a straw
into.

> It seems like DIYbio should think about easy ways to produce feedstock

> for these more touchy c...

There's a strange interest on this list in taking existing lab
protocols, and replicating them as DIY projects by replacing the
reagents known to work with baroque and complicated DIY replacements.
Well, these are mostly thought experiments, or just notions.  These
proposals nearly always seem unlikely to work and difficult to
implement.  You would spend months and years assembling ingredients
and never bake a cake.

Instead, I suggest looking for interesting things to study and/or cool
experiments to do, and find the simplest and fastest way to do them.
The best plants and animals to study are those at close at hand.

Jim Lund

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[Jonathan Street]

It seems like DIYbio should think about easy ways to produce feedstock

for these more touchy cells... even for minimal media recipes for
bacteria, there are a lot of special components. Maybe some sort of
transgenic yeasts or bacteria that secrete only certain amino acids,

so a cell free growth media would have /mostly/ one amino acid.
Purifying the salts seems like the only next step to having pretty
pure aminos... mix them with some phosphate buffers and serum (which
would be acquired some other way).

Amino acids aren't the problem.  They're easily available and cheap.  The difficult part is the insulin, transferrin, EGF, FGF and/or the myriad other growth factors that are routinely used in mammalian cell culture.  There is a trend to move away from, or at least reduce the need for, serum by supplementing media with purified growth factors.  I assume these come from transgenic expression systems.  There may well be a DIYbio project in manufacturing these proteins but it would be a long route to mammalian cell culture if that is your aim.

Our bodies make everything our cells need, so a bio-fermentor solution
to this problem IS possible.

No they don't:
http://en.wikipedia.org/wiki/Essential_amino_acid
http://en.wikipedia.org/wiki/Scurvy
http://en.wikipedia.org/wiki/Rickets
etc etc

I studied plant tissue culture and biotech in college and have a little practical experience, so I'd be able to offer some pointers to people with an interest in this area, although there might be someone else lurking around here with true practical experience!

I recall someone on this list previously mentioning they funded their university education by cloning orchids.  Simon perhaps?

[General Oya]

We're currently culturing SF-9 insect cells (on passage 10 in T-225's). If we were licensing them as compared to using them in an educational environment, we'd have to pay Life Sciences around $75,000 a year just for the priviledge.

I'd love to culture some Golden Orb Spider spinnerette cells... but I'll still need to figure out how to immortalize, unless the Chinese have already created a Master Cell Line.

I think we'll need to figure out a few things about mammalian cell culture if we really want to produce functional bio-printer projects. Once again, I've found the Cell Culture for Tissue Engineering text to be quite helpful.

JCVI may actually be on the precipice of building a community lab capable of Cell Culture here in Maryland. We'll have to see how this project develops.

Ryan

[Cathal Garvey]

At a cellular level immortalisation in insects is probably pretty similar to mammals. Mostly it's a matter of providing telomerase, I think?

Sent from my Phone
www.twitter.com/onetruecathal
www.indiebiotech.com

On 13 Apr 2011 17:45, "General Oya" <gener...@gmail.com> wrote:

We're currently culturing SF-9 insect cells (on passage 10 in T-225's). If we were licensing them as compared to using them in an educational environment, we'd have to pay Life Sciences around $75,000 a year just for the priviledge.

I'd love to culture some Golden Orb Spider spinnerette cells... but I'll still need to figure out how to immortalize, unless the Chinese have already created a Master Cell Line.

I think we'll need to figure out a few things about mammalian cell culture if we really want to produce functional bio-printer projects. Once again, I've found the Cell Culture for Tissue Engineering text to be quite helpful.

JCVI may actually be on the precipice of building a community lab capable of Cell Culture here in Maryland. We'll have to see how this project develops.

Ryan

On Wed, Apr 13, 2011 at 6:05 AM, Jonathan Street <streetj...@gmail.com> wrote:
>>

>> It seems...

[Nathan McCorkle]

On Wed, Apr 13, 2011 at 6:05 AM, Jonathan Street
<streetj...@gmail.com> wrote:

I guess what I meant is that the body takes easily acquirable
feedstock and turns it into everything our bodies need. So imagine if
you could take an animal with the smallest genome, and start
attempting to strip off working genetic modules... there'd be no need
for all the macro sensors like eyes, ears, touch, smell, but the
immune system and skin and ECM would be good things to keep, and maybe
a mouth too, with a mastication inducer as well.

And for the non-essentials, some other organism produces them, so pull
in those operons/modules and forget about it being a problem.

Alternatively, if sequencing and synthesis were in the DIY realm, we
could just do all this in vitro. Bootstrap with some purified ribosome
and RNA polymerase, and you could theoretically have an in-vitro
"cell" or "organism"... conveniently with digital control systems.

--

[Nathan McCorkle]

On Wed, Apr 13, 2011 at 5:48 AM, Cathal Garvey <cathal...@gmail.com> wrote:
> Insect cell culture interests me, but I know nothing yet of it. Based solely
> on the fact that few intracellular parasites (viruses, bacteria) exist that
> shuttle between human and Insect hosts, Insect cell culture makes a lot more
> sense.
>
> I do think that, by the time you have established a DIYbio cell culture lab
> that works, you could probably have completed a college degree in cell
> biology and have gotten a position studying it somewhere better equipped,

sure if that's what you want to do... if you want to work for
yourself, and be close to your community, what I am speaking of would
be something like a sustainable lab co-op... you wouldn't have to deal
with Life Sciences $75k/year licensing type things... I think this
could only be possible by using bio-tech to our advantage, by having
it do more work and allow us to rely less on preparative chemistry
techniques for purification.

[Nathan McCorkle]

On Wed, Apr 13, 2011 at 2:43 AM, jlund256 <jlun...@gmail.com> wrote:
>> There is a lot of filtering that you would have to do, but is blood
>> enough to grow cells on? Could you "feed" your own experiments? If
>> that is too gruesome, what about getting it from a flock of healthy
>> goats? It seems that the only bad thing about this is that using a
>
> No, no human blood, a hundred times no.  Same with livestock.  Blood
> serum is a common additive to mammalian cell culture and is collected
> from slaughtered animals.  Usually as an additive at 5% or 10% or
> media volume to provide protein growth factors and whatnot.  That
> would be the source, but really, mammalian cell culture isn't worth
> the trouble.

doesn't blood infuse all the nutrients into the tissue it passes?
couldn't you then just dialyse blood to leave all the big stuff
inside, and enrich the dialyte with serum? (this is a real question,
not rhetorical)

> Insect cell culture probably isn't worth the trouble either, but is
> cheaper/easier/safer, the provides most of the cell types, genes, and
> biology happening in mammals.
>
> Or try a squid.  I hear they have neurons big enough to stick a straw
> into.
>
>
>> It seems like DIYbio should think about easy ways to produce feedstock
>> for these more touchy cells... even for minimal media recipes for
>> bacteria, there are a lot of special components. Maybe some sort of
>> transgenic yeasts or bacteria that secrete only certain amino acids,
>
> There's a strange interest on this list in taking existing lab
> protocols, and replicating them as DIY projects by replacing the
> reagents known to work with baroque and complicated DIY replacements.
> Well, these are mostly thought experiments, or just notions.

Brute force is how knowledge has been acquired since the beginning of
time. Linux isn't always as plug-n-play as Windows, but those
programmers who built it just didn't want to pay for licensed
technology they couldn't take apart or have the potential to improve.

> These
> proposals nearly always seem unlikely to work and difficult to
> implement.  You would spend months and years assembling ingredients
> and never bake a cake.

sure, but there is something to say for that, because what if after
that time you are really good at baking lots of cakes? Would you buy
cakes from a baker that used all local ingredients and had wholesome
business practices?

Even if you did this without some crazy scheme using crazy unheard of
systems, you don't think its possible to beat those prices?

>
> Instead, I suggest looking for interesting things to study and/or cool
> experiments to do, and find the simplest and fastest way to do them.
> The best plants and animals to study are those at close at hand.
>
> Jim Lund
>