Re: [DIYbio] A newbie says hello!

[Avery louie]

Hello, and welcome!

Where are you located?  There may be a local group.

--A

On Sep 3, 2012 9:10 AM, "HadyE" <had...@hotmail.com> wrote:

Hi there. I have been reading a ton about SynBio and have become extremely interested. I have been trying to get research positions in the bioengineering department at my university to no avail, but I don't want to wait on a professor to get started with this incredible field. I'd really appreciate it if you guys and gals could point me in the right direction. If you would help me find out about equipment, techniques, good reading material, example experiments; any useful information at all, that would be fantastic. Hopefully, I will be able to contribute more and more to this forum and the field at large over time. Thanks for having me aboard!

Hady

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[Katherine Gordon]

I am in Great Falls, Montana and my name is KateG, is there a local DIYbio in my area?

[Avery louie]

Not that I know of, but maybe someone will see this.

[Bryan Bishop]

On Mon, Sep 3, 2012 at 9:10 AM, Katherine Gordon <kthrn...@gmail.com> wrote:

I am in Great Falls, Montana and my name is KateG, is there a local DIYbio in my area?

http://diyhpl.us/wiki/diybio/groups

other things you asked for:
http://diyhpl.us/wiki/diybio/faq
http://diyhpl.us/wiki/diybio/faq/books/

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

[Mega]

Hi,

It would be good for us to know you state-of knowledge...

Are you familiar to basic operation such as restriction, ligation, transformation? Know what plasmids are? Ever done a transformation/PCR?
You studied biology?

Synbio essentially is something different, because you'll get synthesized most of / all you need and just transform it.  It's all about $$$, nowadays, because synthesis of a medium-sized plasmid will likely cost you about 3,000$.

Best,

[HadyE]

As far as my knowledge of biology goes, I had been studying a lot of biology my first two years of college. I understand how helicase unzips DNA, Polymerase replicates it, how RNA Polymerase transcribes DNA, and how Ribosomes translate DNA to Protein. I took two semesters of Molecular Cell Biology. I'm no expert by any stretch, but I do have a basic understanding of molecular biology. What can I do as far as DIYBio is concerned, since Synbio is a bit pricey? Would you guys mind sharing the different things youve made at your benches?

Thanks,

Hady

[HadyE]

Thanks for the links Bryan I'm looking into them now.

Hady

[Bryan Bishop]

On Tue, Sep 4, 2012 at 6:04 PM, HadyE <had...@hotmail.com> wrote:

What can I do as far as DIYBio is concerned, since Synbio is a bit pricey? Would you guys mind sharing the different things youve made at your benches?

I suggest starting with the FAQ :-) http://diyhpl.us/wiki/diybio/faq There's also projects listed there. If you don't find the answers satisfactory, please edit the wiki or suggest which answers need expansion.

[Dakota]

I just got pGREEN and pVIB plasmids and I'm working on mini/maxi preps to make sufficient quantities to ship out to people as free "starter kits".  I have tried my hand at acquiring the Lux operon and luciferase operon from the 2010 iGem team but to no avail, even via my old school.  The old team members got back to me in 1 day, and even forwarded directly to their old supervisors, but to no avail seemingly.  

I really think there is a potential for a parts registry that is NOT via iGEM and that serves the DIY community and whoever else, be it universities or private sector or...

 So far we have meager offerings, a few plasmids + pUC19 with random inserts.

But...something has to start somewhere.  

Since you are from Montana, maybe check out http://www.hometrainingtools.com/

They are based in Montana, and I just ordered $150 worth of chemicals and equipment from them.  The company was started by an old chem engineer, and who knows...maybe he'd be into donating some stuff to the DIY cause?

I'm going to be posting a review of their borrosocilate glass and chemicals when I receive them, which will hopefully be within the next 2 days.  

My plan is to eventually have a starter series of videos but, if you ever want to chat on google chat or skype drop me a line. 

PS.  I love fly-fishing, and am jealous that you live in Montana!  Check out Dr. Gary Strobel's work at the Univeristy of Montana, he is my hero.

[Cathal Garvey]

Whether or not it works 100%, my plasmid was ~3.6kbp and I got it for
~�1000 from Epoch, so it's a bit cheaper than you think! ;)

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PGP Public Key: http://bit.ly/CathalGKey

[HadyE]

I'm actually from missouri.

Hady

[HadyE]

I was actually hoping to just do a basic experiment to make a luminescent protein in a vial, just to see if my technique is correct. I'll be searching, but if anyone has any pointers or experience, please, by all means let us know.

Hady

[Andreas Sturm]

Well, that sounds great.

If you want to make something glowing (there's actually no luminescent protein, but a pathway that makes light by oxidation of aldehydes), you can get a plasmid called pVIB from Carolina Bio for 10$. Insert it into bacteria (optimized for E.Coli), and they will glow.

Here you can find fotos of my first try, with pVIB
http://diyspartanbiotech.wordpress.com/2012/05/11/transforming-e-coli-with-pvib-plasmid/

When I plated them on new, fresh selection plates they were much brighter, it truely was enlightening!

On Thu, Sep 6, 2012 at 12:55 AM, HadyE <had...@hotmail.com> wrote:

I was actually hoping to just do a basic experiment to make a luminescent protein in a vial, just to see if my technique is correct. I'll be searching, but if anyone has any pointers or experience, please, by all means let us know.

Hady

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[HadyE]

Wow Mega, thats awesome! You've just sparked my interest, and I'd like to try that! 

I am trying to make a list and I have no doubt I am forgetting something. Here is what I have so far.

-MicroLive Bacteria EColi Culture.

-pVIB plasmid 1 µg

-BioBrick Assembly kit

-Aldehyde compound?

What else may I need? Any other experiments anyone can think of?

Thanks a ton,

HadyE

[Dakota]

Here is the teacher's guide that goes along with all the plasmids sold from carolina in .pdf format, it gives detailed instructions on pVIB as well as the other common teaching plasmids.  It's a good read, and comes with some nice pictures of what the colonies should look like after successful transformation.    

http://basepair.library.umc.edu/sri/Carolina_transformations_manual.pdf

[HadyE]

Great, thank you.

[HadyE]

Thanks for the awesome replies everyone. I'll hop to it soon. If anyone has anymore knowledge they'd like to drop on me, I'm your student as always.

HadyE

[Andreas Sturm]

About the things you'll need,
you've written bio-brick assembly.

Actually, the plasmid is ready-to-use. Just do a heat shock or PEG transformation, and it makes the bacteria glow...


In short:

-> Grow E. Coli to exponential phase,
-> strike a colony into (arounhd 1 molar) CaCl2 solution (do you have eppendorf tubes?)
-> add plasmids,
-> incubate on ice for 10 mins  (plasmid binds to membrane through cacl2)
->, heat shock, 45°C      30-45 sec  (cell membran gets porous -> plasmids get in)
-> ice 5-10 mins   (recovery)

Now one out of 1000 bacteria (or so) has taken up one plasmid. Now you have to kill the 999 that don't have the plasmid. Therefore, the plasmid carries not only lux, but also an antibiotic resistance (ampicillin).

Plate the CaCl2-bacteria solution on LB-Amp agar petri dishes (you'd better buy pre-poured lb amp agar petri dishes. Or get them from university. You can make them yourself, but they can totally spoil your transformation. I used bird-medicine ampicillin for my first transformation, with the result that it was much work, and finally the ampicillin didn't work. It was a lawn of bacteria, and, of course you don't see the one out of 1000 glowing. )