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Genetic engineering - Help

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Genetic engineering - Help

Postby lwpolkadot » Sun May 02, 2010 6:27 am

I'm extremely interested in genetics, molecular biology, and synthetic biology. A friend and I have recently had the desire to genetically re-engineer a snail. There is a species of algae known as Pyrocystis fusiformis, which is bio-luminescant. I want to extract the exact genotype, which has already been found so i don't have to spend 10 years searching for the exact enzyme. Well, i want to extract the DNA From the algae and either inject into the nucleus with a microsyringe, or do a fusing technique.

Further information: I have a saltwater fish tank meaning I've got plenty (thousands) of snail eggs. I'm not certain as to what the exact species of snail it is, does this post a problem? I have the equipment to duplicate DNA and to fuse cells with DNA, I just need help with the process.

We also need to know how to isolate a single cell, without damaging it. Maybe of a plant?

I would like to have a step by step, or simple explanation. Like i mentioned above I'm merely a high school student, 9th grade.
Last edited by lwpolkadot on Tue May 04, 2010 12:05 am, edited 1 time in total.
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Postby JackBean » Sun May 02, 2010 6:50 am

Why not take GFP, that would be easier...

You're high school student, but have stuff for DNA manipulation?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby lwpolkadot » Sun May 02, 2010 7:35 am

What do you mean by your remark? I don't mind if you just meant it to be witty
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Postby kolean » Sun May 02, 2010 8:53 pm

I think he meant, what kind of equipment do you have? That way we can know what you are capable of doing. (though JackBean is very witty :wink: , oh yeah, 8) )

Though I do not know if algae DNA can be incorporated into the snail's genome. Recombination has to happen (which happens in the gametes of the organism) and do not know if there is sufficient homology between snail and algae DNA'genome for that to happen.

Most take the gene of interest (such as your bioluminescence gene) and put it into a plasmid, with a promoter that is from the organism that you intend to put it into, and a promoter can be tissue specific or ubiquitous in making the gene express the interested protein in that organism. But, as you said, it can take awhile to find it (but I bet some biotech company has already isolated the bio-luminescent gene you desire. usually a google search will show a biotech company that has it).

And what do you mean by fusing technique? fusing cells together? or DNA?
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Postby lwpolkadot » Sun May 02, 2010 9:39 pm

Well, The equiment i have is not super high tech. It has been used before for the kind of things i'm trying to do.
If the DNA cant incoorporated itself in the snails genome what other organism could i perform this upon? And as JackBean stated above: Why not take GFP, that would be easier... Well i think that may be what i will do.
I know that 'fusing technique' was somewhat of a vague description.. The instrument spins the cells and the dna together at 530 rpms' and hopefully some of the dna punctured through the cell wall, and into the nucleus. I can know more about the machine monday.
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Postby JackBean » Sun May 02, 2010 10:12 pm

LOL, that's lolobiond. I've heared about several protocols to transform cells, but I think never about centrifuge.

The homology between your gene and snail's DNA is no problem, just add some overhangs in PCR. If you can't do PCR, than don't do anything of that.

By using GFP I meant, that there are plenty of plasmids with GFP, so you can just pick one, but for your luminiscent gene, you probably had to search it by yourself. Although it shouldn't be so hard, if you know sequence (as you said).

Do you know The Big Bang Theory? :-P
http://www.biolib.cz/en/main/

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Re: Genetic engineering - Help

Postby kolean » Mon May 03, 2010 3:37 pm

Do you just want to express the bio-luminescent gene in the snail?

How are you able to amplify the gene? Do you have access to PCR? Do you have primers for the gene of interest?
Do you a plasmid vector?

How would you go about transfecting the snail egg with the plasmid? Centrifuging?

Very amibitious high school freshmen!
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Postby lwpolkadot » Tue May 04, 2010 12:00 am

Yes, i have a PCR, and a Plasmid Vector, and primers. Primarily all i want to do is have the luminescent gene from the GFP or the biolumenescant algae to show itself.

Yes, Centrifuging is what i had in mind. If that won't work, please tell me? What might be an easier way to go about this? I thought about injection, but i learned that that whole process was quite intricate.

If i did proceed with the centrifuge, would the gene necesarilly attach itself with the snails DNA, or even go into the cell at all?

Oh, Kolean, Thanks! We try to do more, and learn more. Whats the point of lazing around?
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Postby jonmoulton » Tue May 04, 2010 3:42 pm

Centrifuging isn't used as a transfection method. Look into either reagents (like lipofectamine2000) which are a standard lab method or look into physical methods like electroporation (this takes some equipment: if you are electronically handy you can rig up a waveform generator and send a few pulses through your culture -- there is lots written on this).
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Postby lwpolkadot » Thu May 06, 2010 9:27 pm

what would be the easiest mean of transfection? And what is needed?
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Postby JackBean » Thu May 06, 2010 9:44 pm

IMHO heat or Li treatment, but these are probably not used for animal cells.
http://www.biolib.cz/en/main/

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Postby Orome » Fri May 07, 2010 8:14 am

I hate to disillusion you but the project you requires a completely equipped lab, lots of experience, at least 3 years of work and last but not least a lot of money. But nevertheless if this is not a problem for you, this is the way I would work:
1. go into literature (http://www.ncbi.nlm.nih.gov/pubmed/) and try to find out if someone has done something similar to your project, if not – forget it, just to establish a transfection protocol for a new species takes a few years

2. try to identify your snail species and look up if the genome is sequenced (http://www.genomenewsnetwork.org/resour ... e_p1.shtml)

3. if the sequence is not known it will be hard to design a construct for homologous recombination (in this case you can only try to transiently transfect your snail eggs – what means that the effect is probably lost in the mature animal)
If the sequence is known, design primers that amplify the specific gene of Pyrocystis fusiformis (I don`t know the gene, if it is to big on genomic level you have to switch to mRNA level) and contain flanking regions of a certain locus in the snail genome and enzyme restriction sites

4. Extract DNA from Pyrocystis fusiformis, do PCR with your Primers (if you are on mRNA level do RT-PCR before using your primers)
Clone it into a appropriate expression vector (should be a shuttle vector that can be expressed in bacteria and eukaryots – that means it contains origin of replication, several selection markers, MCS, poly A signal, maybe a strong promoter that works in both bacteria an eukaryots, fuse your gene of interest with GFP would be great – maybe with …)
Probably you have to perform several steps of subcloning.
Amplify your vector in e.coli, pick transformed clones, do miniprep, sequcence your vector to check if everything has inserted correctly
5. Establish a transfection protocol for the snail eggs (electroporation, CaCl2, chemicals like lipofectamin or fugene, microinjection…)
6. Transfect the the snail eggs with your vector (could also be a viral vector), culture the cells and select them for positive transfection

So go to an institute and ask them for help ;)
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