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caters 02-25-2014 09:35 PM

Eukaryotic cell from scratch
 
I am making this stem cell from scratch or in other words starting with the molecular components.

Here are the tools I am using:

Regular lab equipment for things like PCR

Normal size computer for researching proteins and their DNA sequences and expression

Much larger computer to keep track of my progress

Microtools which are so small you need a microscope and are perfect for putting proteins in membranes

Microgoggles which when you wear them give your eyes the power of a microscope

and

An electrode to send an action potential to start life after I put ATP inside the cell.

Now in the culture medium I have vitamins and minerals as well as sugars.

The process has a little cycle inside the process(kind of like the Krebs cycle is the cycle in respiration.

It goes like this:

DNA Extraction--------------Chromosome separation-----------Replication of DNA and formation of liposome---------------Looking up proteins for the organelle of interest-----------PCR------------Transcription and Translation--------------Forming the organelle------------Putting the organelle inside the cell--------------Repeat from looking up proteins until cell is complete--------ATP injection-------Sending action potential

Now I have some questions, some of them related to particular cellular processes and some of them related to this whole cell from scratch thing.

Q1 I am wanting to make an animal cell from DNA, RNA, Protein, and Fatty Acids as well as Glucose and other sugars. I am wondering. Should I start with the cell membrane or the nucleus and nucleolus(both are very important)?

Q2 The pace of DNA replication is 8 2/3 hours for every chromosome and 2097152 of each chromosome in 1 week. Is that just an estimate because I know that it is 8 2/3 hours per replication origin.

Q3 Once I know how many proteins of a particular type should I do this:

Go to BioGPS

Look up the proteins

Make sure I have human selected(since I am making a human stem cell)

Average the mRNA expression to get stem cell mRNA expression

Multiply by 100 to get the amount of protein

Q4 I have heard of amplicons used in PCR. Do I really need one to copy a whole gene or just primers, DNA, and enzymes?

Now the ATP formation:

I have heard that with glucose you get 2 ATP from glycolysis and 1 NADH(which is = 3 ATP), 2 NADH from pyruvate -> acetyl CoA, 6 NADH and 2 FADH2( each of which is = 2 ATP) from the krebs cycle so that from glucose you get 38 ATP.

I have also heard that an 18 carbon fatty acid gives you 146 ATP.

I have not heard of ATP numbers for amino acids(which can enter in glycolysis, pyruvate -> acetyl CoA, or the Krebs Cycle)

How do fatty acids give you more ATP than glucose?

Does forming nucleotides from Glucose 6 Phosphate give you ATP?

What about Gluconeogenesis? Does that have an investment phase and a payoff phase like glycolysis does?

Does forming fats and phospholipids from acetyl CoA give you ATP?

Does forming amino acids from intermediates in the Krebs cycle give you ATP?

Now the PCR to protein synthesis:

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?


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