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PCR that leads to protein synthesis


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#1 caters

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Posted 14 February 2014 - 02:49 PM

For the cell I am making I have the DNA replicated and 1 diploid set of chromosomes separated from all the rest and used telomerase to extend the telomeres(So that I would not have programmed cell death or apoptosis when I put the nucleus in the cell).

 

Now I am making the membrane proteins(just starting) and I need to copy whole genes(exons and introns) leaving the promoter and termination sequence where it is(some of the ensembl genes either start or stop at an exon and no intron at the beggining or end).

 

Will just the normal PCR do this or do I need to do some other form of PCR?

 

Also I have in a test tube splicing enzymes(the ones that get rid of the introns), RNA Polymerases 1, 2, and 3(1 forms tRNA I do beleive and 3 I think forms rRNA(I might have this backwards) but I do know that 2 forms mRNA), Whatever enzyme it is that attaches an amino acid to tRNA, Ribosomes, and the enzymes that do glycosylation(attaching sugars to Asparagine mainly) and other post translational modifications if needed.

 

Now how can I get the amino acids I need to put in the solution with all those enzymes? Will I need to put protein rich food in an acidic environment with pepsin and other proteases to break it down into individual amino acids? If so the pH should be 2 but isn't HCl + H2O + Pepsin really acidic with a pH lower than 2? Isn't pepsin itself an acid in water? If so how much HCl, H2O, and Pepsin would I need?

 

And how am I going to separate the amino acids from the HCl, H2O, and Pepsin if I do it that way?

 

How long would it take for the protein to become amino acids?



#2 hobglobin

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Posted 15 February 2014 - 04:14 AM

Not sure if I understood you right, but you'd be a Nobel prize candidate if you could develop an artificially cell....and well single amino acids you can buy just like other chemicals.


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#3 caters

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Posted 15 February 2014 - 11:37 AM

yeah but isn't it possible that the amino acids are denatured in some way which is not good yet protein rich food + HCl + H2O + Pepsin preserves the amino acids from denaturation(because of the acid) and so the stomach-like environment is better for getting amino acids? You just have to separate them somehow.



#4 bob1

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Posted 15 February 2014 - 12:25 PM

You do realize that this has been tried before right?  It doesn't work - mostly because we don't know enough about how proteins form from their component aminioacids into the 3D structures, and whether those pathways are mediated by 3rd party proteins.  PCR will not lead to transcription by itself  We also don't know how membranes form around the cell fully.

 

You could try using a pre-digested cell lysate instead of pepsin etc., then at least the 3rd party enzymes are there already.  You could otherwise by amino acids from suppliers like Sigma-Aldrich.



#5 caters

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Posted 15 February 2014 - 12:27 PM

PCR will lead to transcription once I put the results of PCR in the test tube with the RNA polymerases, ribosomes, tRNA, and enzymes that help in that process.



#6 bob1

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Posted 15 February 2014 - 12:28 PM

Maybe - read up on in-vitro transcription ...



#7 caters

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Posted 15 February 2014 - 12:40 PM

that is basically what I am doing between PCR and putting the protein where it goes only it has translation as well.



#8 Ameya P

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Posted 22 February 2014 - 04:04 AM

Caters, 

 

I have seen a few of your posts around the forum here and am really interested to know what is prompting you to take up this experiment. 

 

To successfully install 'Life' into this setup of yours, you will need either need 'divine intervention' or at least that extremely rare and random event that scientists credit life to, to reoccur in your lab. 

 

Please tell me this is a Lab at some University, where you are carrying out these experiments. 


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#9 caters

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Posted 22 February 2014 - 08:41 AM

Several things are prompting me to make a cell from its molecular components.

 

1. Stem Cell Research and the fact that I hate that they have to get pluripotent stem cells from embryos and lower the pregnancy rate.

 

2. iPS Cells I know about but I don't know if that is good or bad.

 

3. I know so much about biology even though there is a lot more to learn

 

and lastly there is a field of synthetic biology and this would be included in it.

 

 

This lab is at a university yes.

 

and

 

The thing that will trigger life if not the ion channels is the electrode I have to send an action potential(you know sort of like the electrodes in FES that trigger the neurons there to fire and let out acetylcholine which makes muscles function as muscles).



#10 bob1

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Posted 22 February 2014 - 12:50 PM

What makes you think that a pulse of electricity will power a cell? As far as I am aware most of the active energy in a cell comes from ATP being hydrolysed to ADP and AMP.  This has little or nothing to do with electricity.



#11 caters

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Posted 22 February 2014 - 06:08 PM

pulse of electricity from neuron makes the next neuron fire so I think that doing the same thing to my cell would make it come to life. Now yes ATP is important but so are ions.



#12 phage434

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Posted 22 February 2014 - 09:00 PM

I 'spect you need a large pulse -- perhaps from a lightning bolt. Be sure to tie the proto-cell down well to the table, else it might become violent and escape.



#13 bob1

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Posted 23 February 2014 - 11:36 AM

pulse of electricity from neuron makes the next neuron fire so I think that doing the same thing to my cell would make it come to life. Now yes ATP is important but so are ions.

Yes - that's an action potential - the next neuron is already alive and functioning before the pulse hits, it isn't the pulse that brings it to life.



#14 caters

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Posted 23 February 2014 - 03:37 PM

Some ATP will be in the cell from an injection just before I send the action potential and then it will go do lots of glycolysis and fatty acid and amino acid metabolism to really raise the ATP levels.

 

Just ATP though doesn't bring it to life either so it is important that not only does the cell have ATP but also have an action potential sent by the electrode.

 

And Phage434 just an action potential will do it. In fact lightning would kill your cells unless you have AF or VF(atrial fibrillation and ventricular fibrilation), then it would act like a defibrilator making your heart go back to normal.



#15 Tabaluga

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Posted 23 February 2014 - 06:08 PM

As an aside, do you know how much voltage a lightning has ? Up to 200 Million Volts. Do you know how much voltage is applied at defibrillation ? Around 2000-4000 Volt. A lightning strike often induces VF or asystolia, which is one of its deadly mechanisms, and a lot more thermic damage. It does not "make your heart go back to normal" when you have AF/VF.

On another note, when experimenting with electricity, please always consider your safety.

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