Tuesday, May 27, 2008

Is Synthetic Biology Really New, or Old Wine in a New Bottle, or harbinger of a Brave New World?

Sohan P. Modak
May 27, 2008
Henry Harris, in 60s, developed the technique of cell fusion to form heterokaryons by fusing nucleated chicken erythrocyte with Hela cell by assisting the cell fusion by UV-inactivated Sendai virus. Heterokaryons contain genetically different nuclei in a single cell. In contrast, homokaryons are produced by fusing two cells of the same genotype giving rise to bi- or multi-nucleate cells. This principle was exploited by Rao and Johnson [http://www-rcf.usc.edu/~forsburg/ccslides/sld001.htm] to fuse cells from different phases of cell cycle to examine the effect of intracellular environment on chromosomes of nuclei in different phases of cell cycle (Figure 1).

One of the most important spin-off from cell fusion experiments is the onset of Somatic Cell Genetics, i.e. genetic studies of cells other than germ cells. Somatic cells are diploid and differentiated. Therefore Somatic cells from different tissues of the same individual should be genetically identical but may acquire tissue-specific differences during differentiation. Most differentiated cells do not grow readily in culture as most maintain tissue-specific or differentiated traits but have lost their ability to divide. Such cell populations may contain a subset that retains division potential but a exhibit finite lifespan, unlike those derived from tumors or transformed in vitro with viruses, e.g. Adenovirus, Epstein-Barr virus, etc. or treatment with carcinogens and become immortal.
Early cell fusion experiments revealed that in hybrid cells, say human and rodent cells, most human chromosomes were selectively eliminated while retaining the rodent chromosomes and establishing a novel karyotype. In case of human-chicken hybrids, most chicken chromosomes were eliminated which allowed pinpoint the chromosomal location of expressed chicken phenotype like isocitrate lyase. Thus cell hybridization technique permits mapping genes to specific chromosomes or chromosomal sets, a forerunner of genome mapping. Another example includes mapping thymidine kinase on human chromosome 17. A huge amount of work was contributed by teams at Karalinska Institute, Sweden.
The next step involved producing chromosome-specific hybrids where in single chromosomes are transferred to cells. By prolonged treatment of cells with colcemid or colchicine, again a technique pioneered by Karolinska group, nuclear membrane reconstitutes around metaphase chromosomes in the absence of mitosis and gives rise to mini nuclei that can then be separated and fused with cells from another species giving rise to hybrids bearing full karyotype of the recipient cell and one chromosome from the donor specie, again allowing specific gene mapping studies.
In 50s and 60s, nuclear transplantation experiments were pioneered by Briggs and King in USA and Gurdon, Blackler, and Fischberg in UK. These demonstrated that intra-generic nucleo-cytoplasmic combinations perform better than those across genera. These experiments opened new frontiers in cell tinkering were when it was shown that and led to preparing enucleated somatic cell cytoplasm which is then fused with a foreign nucleus thereby creating a Cybrid wherein genomic DNA and cytoplasmic mitochondrial DNA are of different origin. Cybrids were developed by treating monolayer cultures with cytochalasin, which depolymerizes the cytoskeleton and the loosened nucleus can then be removed by centrifugation leaving behind cytoplasmic mass. Such cytoplasmic bodies well delimited by the plasma membrane and containing all intracytoplasmic organelles are then fused with isolated nuclei from a different cell type either in the presence of polyethylene glycol, or Sendai virus, or simply microinjected into the cytoplasm. Hence one could form cybrids between HeLa cytoplasm and mouse or chicken or rat nucleus.
This principle is now being applied to exchanging zygotic DNA with that from somatic cell of another species to generate a novel set of hybrid stem cell lines that would have direct applications in replacing diseased/damaged cells in tissues of patients suffering from degenerative diseases or in accident cases.
The first highly successful of protoplast fusion was demonstrated by Carlson’s team in 1972between species Nicotiana lansdorfia and Nicotiana glauca that led to a spurt of activity in developing methodologies to produce viable plant heterokaryons as well as and homokaryons. Bacterial protoplast fusion was first reported by Rollin Hotchkiss’s group in 1976 (Schaeffer et al., PNAS, 73, 2151-2155). The principle claim of these experiments was that, similar to plants, whole genomes could be transferred among bacteria. Thus, intra-stain and intragenic genome transfers with resulting genome hybrids do give stable progeny With this background one should examine the recent advances in genome transfers. In June 2007, Carole Lartigue from Venter Institute transplanted entire chromosome of Mycoplasma capricolum (JVC1.10) into Mycoplasma mycoides (LC) to change the bacterial species phenotype to Large Colony (LC)., and showed transformation one type of bacteria into another type dictated by the transplanted chromosome (C. Lartigue et al., Science, Genome Transplantation in Bacteria: Changing one species to another. Science, 317, 632-638, 2007). In parallel, scientists at Craig Venter Institute set out to construct the entire 582,970 base pair genome (3.6 x 108 daltons) located on a single chromosome of Mycoplasma genitalium (JCVI-1.0), a sexually transmitted infectious agent in humans, and Gibson et al. reported successful completion (Gibson et al., Complete synthesis, assembly and cloning of a Mycoplasma genitialium genome., Science, 319, 1215-1220). Until now it has been possible to synthesize 30,000 base pair-long DNA in vitro as longer fragments are highly susceptible to errors and mechanical damage. According to the JCVI team the copy is perfect, but they voluntarily have modified a gene necessary for the bacteria to infect people. The entire chromosome-length double stranded molecule of synthetic DNA containing specific identification markers that do not affect the information content of the genome, was then successfully transferred to enucleated Mycoplasma capricolum. Clearly CVI team has taken a quantum jump in synthesizing a fully readable genetic script and transferred it into a compatible environment. Here the crucial issue is that the donor genome should be able to take control of the living cell cytoplasm and convert it into a new life form. Basically, what Craig Venter’s team has achieved is to translate the earlier work on intraspecific chromosomal and cellular hybrids to a stage where entirely new species can be created by synthesizing and transferring a artificially written DNA script in an readable form. Craig Venter has now declared that his next step is to synthesize organisms that use atmospheric CO2 to produce methane and reduce dependency on fossil fuels.
One the prime issue facing the Molecular Geneticists concerns the fact that ghe genetic grammar is still not fully understood. For example, while we know the basic triplet code and some of the punctuation marks such as the beginning and end of a genetic coding sentence as well as some of the alphabet blocks/words acting as signaling elements for retrieval of the genetic information and converting it into a polypeptide, there is a huge lacuna in conceiving, let alone understanding the a potentially large number of signaling elements or punctuation marks in form of words longer than triplets which are located in the non-coding regions of the genome. Therefore, transferring a readable genome synthesized in vitro into a closely related (intra-genic) organism is one thing, but creating one that would be compatible to completely different intracellular environment and control take over the biochemical and biological processes leading to survival and replication is a dream for the future. In the interim, however, the time has come to produce designer organisms that are otherwise very closely related to the existing ones but more useful. For example, would it be possible to synthesize a non-virulent strain of Mycobacterium tuberculosis that competes out the virulent strain? Such are the possibilities of Synthetic Biology. Needless, to caution that there are always individuals , organizations or even Governments that could use this technology for Biological and Economic Warfare! Whatever the ethical and socio-considerations, successful generation of new species would require far detailed understanding of the sthat ignals that govern allele-specific, gene battery-specific and temporal and positional regulatory signals and signal networks that qualitatively and quantitatively control specific gene expression. Well, we are still far away from that stage. For example the present methodology of gene annotation is at best tedious, clumsy as it requires knowledge of the amino acid sequence of the final product of gene expression before it can be retraced via bottoms-up approach to the genome. It is a trap that has led searchers to wild goose chase for a bona fide coding sequence from among many partials, homologues, Paralogues and requiring expensive and time consuming validation by wet lab analyses.
Can we not think of a vastly different Top-down approach to develop a gene annotation methodology?

4 Comments:

Blogger DokC said...

This post has been removed by the author.

June 1, 2008 2:32 AM  
Blogger DokC said...

Dear Spm,
nice post. I have a couple of thoughts on it though.

1. What is the purpose of synthetic biology? Is it something beyond and outside the scope of regular biological research? Infact, could we even consider synthetic biology to be at the stage of "synthetic" biology. As you pointed out, appears apparent- tinkering with existing sequences and modules without understanding the "newton's laws" - or grammar if you prefer- does not to my perception- conform with Synthetic Biology.

The closest work at the genetic regulatory pathways level I have seen in this is the Repressilator work (Elowitz and Leibler, 2000). And more recently work by Noireaux V, Bar-Ziv R, Libchaber A. (2003) on "Principles of cell-free genetic circuit assembly".

2. The synthetic biology claims to work in a top-down method, but the tools used continue to be those of classical genetics. It is well known in the quantitative sciences (physics, maths, statistics) that to solve such inverse problems, i.e. inferring the rules of the system from the measurements of it, that a very strict formalism needs to be developed (equations) and then worked on using quantitative observations. The studies I mentioned before- from the Libchaber and Leibler labs do have the disadvantange of not trying the top down approach- but atleast they understand it. The top down approach was attempted in the early days of gene-chip data analysis, but the "genetic reductionism" of these studies led to its own demise.

So the moot question to me still remains, how can we begin to understand the "laws of gene-protein regulation" - top down or bottom up- from such relatively complex cell-reprogramming experiments.

Concerning the Ethical and Sociological implications of this technology- i.e. functional manipulation of the existing rules and genetics of organisms to make "monster bugs", there is a a meeting called in Novemember in my own institute-
Systems and Synthetic Biology: Scientific and Social Implications, 7-8 November 2008, EMBL Heidelberg
.

Sounds to me like a long haul from biophysics to syntheic biology.

Cheers,
Chaitanya

June 1, 2008 2:38 AM  
Blogger Ramray Bhat said...

This was a nice post; I should have noticed it earlier. The various concerns raised are very real and probably underscore some stark epistemic reductionisms, biologists are in the habit of making. Carrying on from where dokc left of, I must say there are a few flights in terms of conceptual advancements which synthetic biology has presented the community with, in the last decade. In fact I must concede, that given the dizzying pace with which biology progresses, the glamour which was associated with this field even only five years ago has waned. Once again dokc has nicely pointed out the repressilator model by Elowitz and Leibler which probed for the connection between interactive gene regulatory networks (not the Davidsonian type, but a mathematically simplified one), stochasticity and ohenotypic readouts.
A second significant paper was one in which Elowitz, by then a leader in the field and Steven Strogatz coauthored showing how quorum sensing in terms of gene expression can be exhibited by damping of expressional stochasticity.
A third interesting paper this time from Elowitz lab showed how stochasticity played into an "excitable" gene network to bring about phenotypic plasticity.
Sever papers by Alex Oudenaarden, Jim J Collins and Jeff Hasty (not to mention several of their colleagues) teasing apart the contributions of intrinsic and extrinsic noise to alteration of cellular fates.
I ubderstand dokc's concerns regarding the use of similar tools to solve these problems but at least as he rightly puts it, they are well aware of the problems and are taking the "noise" bull by its horns instead of just ignoring it. Moreover using the theory of non linear dynamics they are trying to intuite the laws of the system on a cellular or even cell colonial level rather than just keep gnawing at the intricacies of the molecular network as several other classical developmental geneticists are wont to do.

August 30, 2008 10:28 PM  
Blogger ravi said...

dear sir,

it is really difficult to understand the possibilities of future by manipulating the cell genomics with species... but it is ultimate curiosity of man to explore which is not there which can lead to disastrous or new innovation to deal with present probms...
i think future is very unpredicatble b'coz of this new ideas that we have!

July 15, 2009 7:18 AM  

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