Any high level biologists here?

>>8123113
Wait... to add to this, maybe switch out an existing glutamate receptor with a mouse neural AMPA receptor?

>>8123113

have you tried looking up the methodology of papers to base this on? Review the literature to find a suitable method.

Have you found a supervisor and spoken about this idea?

I'm not a molecular biologist but your method is complicated and a fair bit of error can be introduced. Needs to be simplified.

>>8123132
The original proposal/presentation for my design is independent, so I can't really get outside help until the proposal (15 minute presentation + paper etc.). Once I propose, I can get a team and we work on the idea and get professor help.

I haven't found anything about anyone inserting neuronal DNA into E. coli, because most researchers just go straight to the source and get slices of the hippocampus or use mutated mice. I can't do either of those unfortunately.

>>8123138
hmmm I think inserting DNA into E.coli and extracting any protein for an exp is gonna be a lot more pricey and complicated than just getting some tissue and extracting from it. Would it be possible for you purchase purified forms of the protein?

>>8123113
like this?
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC28322/

but also what the fuck is your research question. Good science is grounded anon, you need to find a model, think about how it can be improved or further characterized, then test.

File: nah.png (513KB, 600x782px) Image search: [Google]

513KB, 600x782px

>>8123144
just looked it up, looking at around $400 dollars per 20 ug

>>8123157

hey beats trying to do it yourself and you can get right into your experiment without worrying about fucking anything up.

>>8123113
>>8123148

This anon is right.
Can you give us more detail as to what exactly your question is? Walk us through your proposal. That not only gives us more of an ability to engage with your idea, but also give you some feedback on the presentation itself.

>>8123163

But it could potentially be good for anon to attempt purifying it himself. It gives more room to fuck up but it's kinda part of the undergrad experience to learn through fucking up a few times.

>>8123167
Are we actually bored enough to do some pseud's homework tn? I think I might be.

>>8123174

fair enough, still interested in op's idea so we can get a better idea of what could be done.

Also OP this may be a bit paranoid but I would understand if you did not want to share the exact details as it may be "copied" or "stolen"

>>8123167
>>8123148
This paper looks very useful, thank you anon.

For my senior thesis, next year (and likely this summer) part of our engineering exit is working ona design project. We get 6-10 months and our own lab with limitations and obviously limited funds. This means that anything I'm interested in will have to be scaled down to a reasonable time frame and cost.

I want to know the role of CaMKII in phosphorylating stathmin, which is implicated in regulating fear and learning in the hippocampus as a function of its microtubule dynamics regulatory processes. For as little funds and time as I'll have, I was thinking that something as easy as site directed mutagenesis to maybe change the binding affinity for the calcium/calmodulin complex.

Further down the line, I want to see the rate at which mutant CaMKII affects localization of AB oligomers at GluA2 (which has been implicated as the site at which AB oligmers bind and traffic AMPAR receptors in long term potentiation.

>>8123180
honestly, my idea isn't too revolutionary, so I'm not too concerned. It's not even research I'd be able to finish now.

>>8123189
To add to this

>Further down the line, I want to see the rate at which mutant CaMKII affects localization of AB oligomers at GluA2 (which has been implicated as the site at which AB oligmers bind and traffic AMPAR in long term potentiation.

This is because CaMKII also kinases existing AMPARs so it would be difficult to isolate the effect its modification has on stathmin by measuring other things, such as concentrations of glutamate or even calcium. (For example, in LTP AMPARs are phosphorylated by CaMKII increasing activity in itself).

>>8123196
Sorry to keep adding to this, but I keep remembering things to add.

I was initially thinking of using drosophila (especially because it would maybe let me test learning through olfactory shock conditioning), but modifying drosophila DNA seems like it might be out of my reach, and I have had experienced with e. coli cloning, so I figured using that would be easier.

>>8123189
That's graduate level work anon.

>>8123210
what'd you do with the coli cloing?

>>8123217
I was working in a professors lab and we were just making proteins he needed for research he was working on. Honestly don't even remember all of them, it was more just mechanical work, ie. pcr, inserting the plasmid, running gels to make sure we didn't fuck up etc.

>>8123216
so am I reaching too far? Do you think site directed mutagenesis of CaMKII would be too much?

>>8123113
Is this feasible without a Methyl-2difetamine reaction? I believe you'd have to oxidize it first through carbon-electrodilution as well. Then you'd have to bubble the solution through a mixture of 2fentaoxide and protocarbonate.

>>8123232
I don't know Chemical Engineering (presuming that's what you are) but what you're doing sounds like a legit paper I could find in some journal.

>>8123189
>>8123232

It might not be the best option due to the time constraint, but I feel like for trying to create mutants that change the binding affinity random mutagenesis is a much better approach than site-directed.

That said, I don't think that's too difficult or too time-consuming for undergraduate work.

>>8123294
thanks anon, open to any criticism.

I was thinking of doing site directed at specific binding domains (like an arginine for a histidine). Why random? Also, I've never done any mutagenesis, so are there any resources you could point me to?

>>8123294
>change the binding affinity random mutagenesis is a much better approach than site-directed.

That is interesting but not translational.

>I was thinking of doing site directed at specific binding domains (like an arginine for a histidine). Why random? Also, I've never done any mutagenesis, so are there any resources you could point me to?

You are better off doing site-directed mutagenesis and trying with different conserved domains, albeit not just the binding sites.

You can do random mutagenesis by increasing the temperature 1 or 2 degrees during PCR. It is not likely useful in this context, however.

>>8123338
I've done random to create mutants for both activity and stability before. Sometimes you can get some pretty crazy good results from a combination of mutations both near or totally remote from sites you would normally target with site-directed.

Site-directed seems to be the best way to elucidate the function of a particular amino acid or domain of a protein, while random seems to be the best way to engineer proteins with more useful properties.

Site directed protocol involves designing mutagenic primers, that are made to fuck with only the one nucleotide you want to change. Protocol:
http://www.ncbi.nlm.nih.gov/pubmed/24011050
Bit more of an explanation here:
http://2014.igem.org/wiki/images/b/b5/Wageningen_UR_protocols_Site_Directed_Mutagenesis.pdf

Random mutagenesis can be done a number of different ways, but I've mostly done it by altering PCR procedure by including a higher concentration of Cl+ ions and introducing Mn++ ions at various concentrations.

General protocol can be found here:
http://www.ncbi.nlm.nih.gov/pubmed/20676978

A paper I read recently that utilized random mutagenesis in a pretty cool way can be found here:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4182741/

Pretty much they did a bunch of random mutagenesis of the lux operon (which codes for bacterial luciferase), isolated a few mutants that had a higher quantum yield than the wild type, and then shuffled the DNA of the mutants via DNase digestion and recombination by PCR, finding a particularly good mutant that performed much better than wild-type.

>biologist
Kek

>>8123138
Getting E. Coli to express a human membrane protein sounds awful.

>>8124863
the paper in

>>8123148

did it 20 years ago