Eukaryotic Cell from Scratch

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Eukaryotic Cell from Scratch

I am making a human totipotent stem cell from scratch. It will have my DNA in it.

The tools I am using are:

normal lab equipment

Computer to look up proteins

Microtools which are so small you need a microscope

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

Big computer to keep track of my processes

In the culture medium I have vitamins and minerals, as well as sugars and fats.

The process is like this:

DNA extraction------------Chromosome separation--------------DNA replication and formation of liposome---------Looking up proteins for organelle of interest---------------PCR--------------Transcription and translation------------repeat until organelle is complete--------------put organelle inside the cell------------repeat until cell is complete-------------ATP injection------------Action potential to start life.

I have some questions.

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?

That is for the whole cell.

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?

That is the PCR and Protein synthesis.

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?

That is for the ATP formation.