[caters (OP)]

I have a culture medium with vitamins, minerals, and sugars. I have a question. I have my chromosomes from DNA extraction that I separated using several rounds of differential centrifugation. I have them in separate test tubes with DNA replication enzymes such as helicase and DNA polymerase as well as DNA ligase. I have put them in an incubator with human body settings. I know that after 1 day I will have this number of chromosomes:
2x1 + 2x2 + 2x3 + 2x4 + 2x5 + 2x6 + 2x7 + 2x8 + 2x9 + 2x10 + 2x11 + 2x12 + 2x13 + 2x14 + 2x15 + 2x16 + 2x17 + 2x18 + 2x19 + 2x20 + 2x21 + 2x22 + 2x23. How long does 1 round of DNA replication take. Will the total number of chromosomes be an even larger power of 2? How many rounds of DNA replication can occur within 24 hours. (this whole DNA replication thing is at average speed
also will I have a different power of two of each chromosome if these chromosomes have these numbers of base pairs?:
Chromosome 1: 247,199,719
Chromosome 2: 242,751,149
Chromosome 3: 199,446,827
Chromosome 4: 191,263,063
Chromosome 5: 180,837,866
Chromosome 6: 170,896,993
Chromosome 7: 158,821,424
Chromosome 8: 146,274,826
Chromosome 9: 140,442,298
Chromosome 10: 135,374,737
Chromosome 11: 134,452,384
Chromosome 12: 132,289,534
Chromosome 13: 114,127,980
Chromosome 14: 106,360,585
Chromosome 15: 100,338,915
Chromosome 16: 88,822,254
Chromosome 17: 78,654,742
Chromosome 18: 76,117,153
Chromosome 19: 63,806,651
Chromosome 20: 62,435,965
Chromosome 21: 46,944,323
Chromosome 22: 49,528,953
X Chromosome: 154,913,754

[Ophiolite]

Caters, you have been posting this on just about every science forum on the web, often preceded by a claim that you are going to produce a functioning eukaryote cell from scratch - as your title indicates. You have been told on every forum that I have seen that a) this is not something we have yet been able to do; b) if you were able to do it you would be worthy of the Nobel prize; c) it is very unlikely that you are going to be the one to do it.

May I ask you why you are persisting with this?

[caters (OP)]

because I have some questions to which I want an answer.

[Ophiolite]

Do you seriously believe that you can achieve the goal? If so, what had led you to this belief, given that it has not yet been achieved and, as far as I know, is quite distant from being achieved?

[caters (OP)]

I do beleive I can do it. I have heard of IPS(induced pluripotent stem) cells. That was what led me to thinking"Okay I will make a stem cell". I then heard in a science chat about the minimal genes experiment for prokaryotes and I thought"Maybe they are trying to make one from its molecular components" and that is what has led me to this Eukaryotic Cell from Scratch experiment. That question at the very top is my current question and I want it answered. And because of how we have advanced so far in technology I don't think that making a eukaryotic cell from scratch is all that far away from being done for real.

[caters (OP)]

and if I make a stem cell(which I am planning to do) should I take the average of all the gene expressions and say "Okay this is the expression in a pluripotent stem cell." or should I take the median or what should I do as far as gene expression?

[caters (OP)]

please reply soon.

[evan_au]

DNA+raw materials = DNA + raw materials. It does not make a functioning cell.

Throw in a PCR machine (Polymerase Chain Reaction) and you will have:
 DNA+PCR + raw materials = more DNA + raw materials. It does not make a functioning cell.

This is because DNA contains instructions to make thousands of proteins.

• But it does not actually make any of these proteins by itself.
  
• It requires a amazingly complex cellular structure made of these thousands of proteins, which break down the raw materials, read the DNA instructions, feed them into cellular factories which can make more proteins, fold them correctly, and then direct them to the right part of the cell to carry on the process.
  

The cell which reads the DNA instructions and carries them out has basically the same complexity as the DNA itself.
So just having the DNA and raw materials will not make a functioning cell.

Having DNA without a functioning cell around it to interpret it
         ...is about as useless as having...
a computer program without a computer to run it on.

[caters (OP)]

I have a test tube with RNA polymerase and ribosomes to put the DNA in after PCR to make the protein. I can then use microtools to put the protein where it goes. IT WILL WORK.

[Ophiolite]

If it were that simple do you no think that scores of researchers would already have done it?

Now, it is not directly relevant to your ability to achieve your objective, but I would be interested to know what your scientific background is: degree qualifications, post-grad work, publications, etc. I am just curious what foundation would lead you to such a mistaken expectation.

[evan_au]

Eukaryote cells have the DNA inside a separate membrane, which keeps the DNA away from the mincers of enzymes and other pieces of cellular machinery. The information inside the DNA is transcribed into RNA, which is transported out of the nucleus into the surrounding cell where it is transported into the ribosomes where it is used to make proteins. The proteins are then transported to where they are needed, and waste materials are transported right outside the cell membrane, where they can't damage the delicate operation of the cell.

Now, if you disrupt these delicate membranes and their transporter mechanisms which partition the cell into various functional zones, a Eukaryotic cell will not function. In fact, we have words for these types of actions: antibiotics and antiseptics. They kill the cell.

Another challenge to life in a test-tube: PCR tends to amplify all the DNA at once. However, the DNA in a living cell is carefully coiled, which allows it to fit into a smaller volume inside the nucleus, but which also allows most of the DNA to be tucked away where it is not expressed; just the parts that are relevant for current cell functions is exposed and expressed into RNA. A series of feedback loops control which parts are currently needed, and are exposed for transcription into RNA and subsequently into proteins (and at what rate).

Another Computer Program Analogy
A computer program has some parts that are used very frequently, and other parts that are used very infrequently. Which parts are used at which time is carefully orchestrated, based on the program design, external inputs, and the prior history as recorded in data from previous executions. Together, they can achieve a complex result for the user.

Copying all the DNA and/or RNA at once is like executing all pieces of a computer program in a random order - the results will be totally scrambled! (The "Blue Screen of Death", for those who have ever used Windows XP...)

[caters (OP)]

I am doing gene by gene okay and I will have 1 chromosome pair separate from the ones I am doing PCR with so that I can put it in the nucleus when I am almost done with it.

[Ophiolite]

Any chance you could address my earlier queries? For your convenience here they are again.

If it were that simple do you no think that scores of researchers would already have done it?

Now, it is not directly relevant to your ability to achieve your objective, but I would be interested to know what your scientific background is: degree qualifications, post-grad work, publications, etc. I am just curious what foundation would lead you to such a mistaken expectation.

[caters (OP)]

well scientists are so busy doing other things that they dont have time for this eukaryotic cell from scratch.

Also several things have made me think I can do this. Here they are:
Just knowledge of the cell
making of organs from stem cells
IPS cells
Artificially made prokaryote
information about proteins for different things in the cell on Wikipedia
Ability to find DNA sequences and gene expression profiles for each protein

Now I have a question related to gene expression. Here is my question:
Gene expression profile for Mucin 1:

Kidney: 63.25
Tonsil: 7.55
Lymph node: 6.40
Thymus: 5.60
Bone marrow: 7.80
Adrenal gland: 6.45
Adrenal cortex: 8.65
Olfactory bulb: 5.60
Trachea: 22.20
Salivary gland: 6.50
Pituitary: 8.50
Fetal liver: 6.30
Fetal lung: 232.90
Fetal thyroid: 9.15
Uterus: 8.65
Adipocyte: 7.05
Pancreatic Islet: 27.25
Pancreas: 20.45
Seminiferous tubule: 12.35
Leydig Cell: 15.85
Interstitial: 11.85
What are these numbers? What are they measuring gene expression in? There are more numbers for this but I am just giving you examples.

[caters (OP)]

I don't really have any degree or publications since I am in high school and this is my first scientific experiment.

[dlorde]

In 2010, Craig Venter and his team made a bacterium with a synthetic genome (which he amusingly called 'Synthia'). It was a major project by the current experts in the field and took them 15 years. They didn't create a bacterial cell from scratch because that was way beyond their capabilities, and still is; they inserted the synthetic genome into the cell of another bacterial species whose genome had been removed.

If you can create just a viable bacterial cell from scratch (with an existing genome) you'll get a Nobel prize, never mind trying to create a eukaryotic cell and its genome. In practice, there's no need to create the cell itself, it would be far easier to enucleate an existing one and replace its genome, but even that is way more difficult than working with bacteria.

Ambition is valuable and praiseworthy, but what you're talking about sounds like fantasy; so I'd say 'no', you can't make a eukaryotic from scratch.

[caters (OP)]

oh but what if you need stem cell treatment because you are in a clinical trial. you would be treated with your own unique stem cells.

[evan_au]

Gene expression profile for Mucin 1...What are these numbers?

Each gene is expressed to a different degree in each tissue. The DNA is transcribed into RNA, which is transcribed into protein. The DNA, RNA & protein for Mucin 1 is almost identical in all cells of an individual (barring de novo mutations).

It is hard to measure the level of a protein amongst all the other proteins in a cell.
However, it is relatively easy to measure the rate of RNA transcription for a particular gene by creating a matching template for gene(s) of interest, and the RNA sticks to this template. It is possible to have a DNA Microarray which checks for thousands of genes at once.

what if you need stem cell treatment because you are in a clinical trial?

There are immense challenges in detecting, selecting and multiplying stem cells in an individual - then delivering them to the right location, getting them to recognise what type of tissue they are in, what type of repair it needs, and then converting into the right type of cell, and integrating with the remaining cells - all without causing cancer!

Being able to create a stem cell from raw materials would be a great help to the "multiplying" stage of this process.

However, with our current level of technology, it is simpler to locate existing stem cells in the individual, and multiply these, rather than build whole new cells from raw materials, and multiply the artificial cells.

this is my first scientific experiment

I think that for a High-School student, achieving PCR with home-grown equipment would be a significant achievement.

You can order DNA micro-arrays, so you could aim to detect DNA, and have a "CSI" theme...

Do it while you can - I expect that in a few years, PCR machines will be as common in biology labs as colour printers.

[dlorde]

... Being able to create a stem cell from raw materials would be a great help to the "multiplying" stage of this process.

However, with our current level of technology, it is simpler to locate existing stem cells in the individual, and multiply these, rather than build whole new cells from raw materials, and multiply the artificial cells.

I think the point is, the cells are already self-replicating; we don't need to create them from scratch, they have had a couple of billion years to evolve a way to do it for us. We're better off tinkering with the tyres and bearings than reinventing the wheel from scratch.