June 2009 Archives

Data and References for Longest Published sDNA

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Various hard drive crashes have several times wiped out my records for the longest published synthetic DNA (sDNA).  I find that I once again need the list of references to finish off the edits for the book.  I will post them in the open here so that I, and everyone else, will always have access to them.

longest sDNA 2008.png

Year Length Refs
1979 207 Khorana (1979)
1990 2100 Mandecki (1990)
1995 2700 Stemmer (1995)
2002 7500 Cello (2002)
2004.4 14600 Tian (2004)
2004.7 32000 Kodumal (2004)
2008 583000 Gibson (2008)

Total synthesis of a gene
HG Khorana
Science 16 February 1979:
Vol. 203. no. 4381, pp. 614 - 625

A totally synthetic plasmid for general cloning, gene expression and mutagenesis in Escherichia coli
Wlodek Mandecki, Mark A. Hayden, Mary Ann Shallcross and Elizabeth Stotland
Gene Volume 94, Issue 1, 28 September 1990, Pages 103-107

Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides
Willem P. C. Stemmer, Andreas Crameria, Kim D. Hab, Thomas M. Brennanb and Herbert L. Heynekerb
Gene Volume 164, Issue 1, 16 October 1995, Pages 49-53

Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template
Jeronimo Cello, Aniko V. Paul, Eckard Wimmer
Science 9 August 2002: Vol. 297. no. 5583, pp. 1016 - 1018

Accurate multiplex gene synthesis from programmable DNA microchips
Jingdong Tian, Hui Gong, Nijing Sheng, Xiaochuan Zhou, Erdogan Gulari, Xiaolian Gao & George Church
Nature 432, 1050-1054 (23 December 2004)

Total synthesis of long DNA sequences: Synthesis of a contiguous 32-kb polyketide synthase gene cluster
Sarah J. Kodumal, Kedar G. Patel, Ralph Reid, Hugo G. Menzella, Mark Welch, and Daniel V. Santi
PNAS November 2, 2004 vol. 101 no. 44 15573-15578

Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome
Daniel G. Gibson, Gwynedd A. Benders, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Holly Baden-Tillson, Jayshree Zaveri, Timothy B. Stockwell, Anushka Brownley, David W. Thomas, Mikkel A. Algire, Chuck Merryman, Lei Young, Vladimir N. Noskov, John I. Glass, J. Craig Venter, Clyde A. Hutchison, III, Hamilton O. Smith
Science 29 February 2008: Vol. 319. no. 5867, pp. 1215 - 1220

Another Step Toward DIYStemCells

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(18 June 2009: Lightly edited for clarity.)

The June 5 issue of Cell Stem Cells has a brief report describing the use of four proteins to reprogram human fibroblasts into induced pluripotent stem cells (iPSCs).  I think this is a pretty important paper, as it dispenses with any sort of genetic manipulation of the target cells or any use of plasmids to insert new "control circuitry", or any chemical manipulation whatsoever.

As expected, it is getting easier to produce iPSCs, and the authors of the paper ("Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins") note that their work demonstrates the elimination of "the potential risks associated with the use of viruses, DNA transfection, and potentially harmful chemicals and in the future could potentially provide a safe source of patient-specific cells for regenerative medicine".

Kim et al used four recombinant human proteins to turn human newborn fibroblast cells (purchased from ATCC -- see the Supplemental Data) into iPSCs, where each of the proteins was fused to a nine amino acid long "cell-penetrating peptide" (CPP) that facilitated the importation of the proteins across the cell membrane.  The procedure was not particularly efficient, but after multiple treatments the authors produced cells that could differentiate into many different kinds of human tissues.

Here are a couple of thoughts about the paper.  Note that in what follows I have only had a few sips of my first cup of coffee today, and my brain is still quite fuzzy, but I think I am mostly coherent.  You can be the judge.

First, the authors did not use mature cells from adults, so don't expect this paper to lead to replacement organs and tissues tomorrow.  The use of cells from newborns makes a great deal of sense for a first go at getting protein-based reprogramming to work, as those cells have already been demonstrated to be relatively easy to reprogram.  The published procedure required many weeks of effort to produce iPSCs, and authors note that they have quite a ways to go before they can produce stem cells at the same efficiency as other techniques.

Nonetheless, it works.

Second, the paper describes PCR-based cloning of human genes to add the CPP sequences, along with a fair amount of bench manipulation to generate cells that made each of the four reprogramming proteins.  All the sequences for those proteins are online, as are the sequences for the CPPs, so generating the corresponding genes by synthesis rather than cloning would now cost less than $10K, with delivery in 2-4 weeks.  In another year, it will probably cost no more than $5K.  (How long will it be before these proteins show up in the Registry of Standard Biology Parts?)

Third, the authors did not use purified reprogramming proteins to generate iPSCs, but rather used whole cell extracts from cells that produced those proteins.  Thus the concentrations of the reprogramming proteins were limited to whatever was in the cell extract.  This might critically affect the efficiency of the reprogramming.  Presumably, the authors are already working on generating cultured cell lines to produced the reprogramming proteins in larger quantities.  But if you wanted to do it yourself, it looks like you might "simply" have to order the appropriate sequences from Blue Heron already cloned into the human expression plasmid pCDNA3.1/myc-His A, which is available from Invitrogen.  This would add a couple of hundred dollars to the cost because Blue Heron would have to play around with a proprietary plasmid instead of the public domain plasmids they usually use to ship genes.  You would then follow the recipe from the Supplementary Data to transform a protein production cell line to make those proteins.  Or perhaps you have a favorite recipe of your own.  Here is something I don't get -- it looks like that particular expression plasmid adds a His tag to the end of the gene, so I don't understand why Kim et al didn't try a purification step, but maybe that is underway.

Fourth, if you wanted to do this at home, you could.  You should expect to fail many times.  And then you should expect to fail some more.  And then, assuming your human cell culture technique is up to snuff, you should expect to eventually succeed.  You might want to wait until the inevitable paper showing how to do this with adult differentiated skin cells is published.

And then what?

You will have an autologous stem cell line that you can use to produce tissues that are, immunologically speaking, identical to those in your body.  What should you do with them?  I would suggest you show them off at cocktail parties, brag about them on Facebook, and then destroy them with bleach and an autoclave.  In lieu of an autoclave a microwave would probably do just fine.

But I expect that at least some of you will try to follow a recipe to generate some sort of human tissue, or even to simply inject those cells in your own bodies, which will result in all kinds of crazy teratomas and other tumors.  To quote Harold Ramus, "that would be bad".  So don't do that.  Just because DIYStemCells are cool doesn't mean you should actually use them yourself.  But I know some of you will anyway.  That is the future of biological technologies, for better or worse.
Last week The Economist ran an online debate considering the motion "Biofuels, not electricity, will power the car of the future".  I was privileged to be invited as a guest contributor along with Tim Searchinger of Princeton University.  The two primary "speakers" were Alan Shaw of Codexis and Sidney Goodman of Automotive Alliances.  Here is my contribution to the debate, in which I basically rejected the false dichotomy of the motion (the first two 'graphs follow):

The future of transportation power sources will not be restricted to "either/or". Rather, over the coming decades, the nature of transportation fuel will be characterised by a growing diversity. The power sources for the cars of the future will be determined by the needs those cars address.

Those needs will be set for the market by a wide range of factors. Political and economic pressures are likely to require reducing greenhouse gas emissions and overall energy use per trip. Individuals behind the wheel will seek to minimise costs. But there is no single fuel that simultaneously satisfies the requirements of carbon neutrality, rapid refuelling, high-energy density for medium- to long-range driving and low cost.
I find it interesting that the voting came down so heavily in favor of electricity as the "fuel" of the future.  I suppose the feasibility of widespread electric cars depends on what you mean by "future".  Two substantial technology shifts will have to occur before electric cars displace those running on liquid fuels, both of which will require decades and trillions.

First, for the next several decades, no country, including the US, is likely to have sufficient electricity generating resources and power distribution infrustructure to convert large numbers of automobiles to electric power.  We need to install all kinds of new transmission lines around the country to pull this off.  And if we want the electricity to be carbon neutral, we need to install vast amounts of wind and solar generating capacity.  I know Stewart Brand is now arguing for nuclear power as "clean energy", but that still doesn't make sense to me for basic economic reasons. (Aside: at a party a few months ago, I got Lowell Wood to admit that nuclear power can't be economically viable unless the original funders go bankrupt and you can buy the physical plant on the cheap after all the initial investment has been wiped out.  Sweet business model.)

Second, the energy density of batteries is far below that of liquid hydrocarbons.  (See the Ragone chart included in my contribution to The Economist debate.)  Batteries are likely to close the gap over the coming years, but long distance driving will be the domain of liquid fuels for many years to come.  Yes, battery changing stations are an interesting option (as demonstrated by Better Place), but it will take vast investment to build a network of such stations sufficient to replace (or even compete with) liquid fuels.  Plugging in to the existing grid will require many hours to charge the batteries, if only because running sufficient current through most existing wires (and the cars themselves) to recharge car batteries rapidly would melt those wires.  Yes, yes -- nanothis and nanothat promise to enable rapid recharging of batteries.  Someday.  'Til then, don't bother me with science fiction.  And even if those batteries do show up in the proverbial "3 to 5 year" time frame, charging them rapidly would still melt most household power systems.

In the long run, I expect that electric cars will eventually replace those powered by liquid fuels.  But in the mean time, liquid fuels will continue to dominate our economy.