Saturday, February 28, 2009

Synthetic Biology: Engineer’s Approach to Biology


Contemporary biology recognizes the genes and proteins responsible for a particular cellular phenomenon, but today at this hour the focus is on deciphering the connectivity between those genes and proteins. Mathematical models best describes these circuits, which in reality resemble the electric circuits. In this scenario, biology is looked at through engineer’s perspective, providing the framework for the construction and analysis of the underlying sub modules that constitute the network. Thus synthetic biology creates a platform on which prediction and evaluation of dynamics of cellular processes is facilitated. In this review we will take a look at synthetic biology and the varied facets it offers.

Keywords: Synthetic biology, oscillator, reprissilator, cellular noise, biobricks.

“…the clock ticks life away…” teens hum on the tunes of Linkin park. (Popular band)
Though the lyrics refer to the passing time in context of digital clock or watch…the man created version. The same words hold true for the nature’s version of the clock…the biological clock as we may call it.
In order to understand how exactly the clock ticks or works…one may have to break open a clock and look at its components…best way to get a better insight into its working, is to try creating one’s own clock out of similar parts!
The previous is what contemporary biology deals with, using genetic and biochemical techniques to isolate genes and proteins involved in feedback loops of gene expression, that are necessary for clock functioning of biological clocks e.g. circadian rhythms.( Cyran, S. A. et al., 2003)
But the later, is the one which helps us answer insightful questions pertaining to the clock like-

1. What sets the period of the oscillation?

2. How does the clock operate reliably in diverse cellular conditions? and

3. What features of its design are responsible for its reliable operation?

The above mentioned i.e. creating a new clock; is the way Synthetic Biology approaches the biological dead ends! Several synthetic genetic clocks have now been constructed in bacteria and mammalian cell lines too.( Fung, E. et al, 2005; Tigges Marcel et al, 2009) These circuits are simpler versions of the actual naturally found biological clocks.

What exactly is synthetic biology?

Marc W. Kirschner (Department of Systems Biology Harvard Medical School) sheds light on it…

“Synthetic biology is the study of the behaviour of complex biological organization and processes in terms of the molecular constituents. It is built on molecular biology in its special concern for information transfer, on physiology for its special concern with adaptive states of the cell and organism, on developmental biology for the importance of defining a succession of physiological states in that process, and on evolutionary biology and ecology for the appreciation that all aspects of the organism are products of selection, a selection we rarely understand on a molecular level. Systems biology attempts all of this through quantitative measurement, modeling, reconstruction, and theory. Systems biology is not a branch of physics but differs from physics in that the primary task is to understand how biology generates variation. No such imperative to create variation exists in the physical world. It is a new principle that Darwin understood and upon which all of life hinges. That sounds different enough for me to justify a new field and a new name”

Synthetic biology deals with ‘programming’ of the cell. Reprogramming a cell involves the creation of synthetic biological components by adding, removing, or changing genes and proteins. Design, fabrication, integration, and testing of new
Cellular hardware lies at the core of this field. But the tools and methods necessary for same are derived from experimental biology. The process begins with the abstract design of devices, modules, or organisms, and is often guided by mathematical models. The synthetic biologist then tests the newly constructed systems experimentally. However, such initial attempts rarely yield fully functional implementations because of incomplete biological information. Rational redesign
Based on mathematical models comes for rescue in such situations.

Newer approaches to address and deal with synthetic constructs are also being developed-

1. One can just apply directed evolution to genes comprising a simple genetic circuit and what you get is evolution of improperly matched non-functional components to functional ones.( Yokobayashi Yohei et al, 2002)

2. In silico evolutionary procedure is also being used to create gene networks performing basic tasks. Main highlights of this procedure are that small functional modules with diverse functions can be created.( Franc¸ois Paul et al, 2004)

3. Yet another approach could be just to couple simple models into complex networks with behaviour that can be predicted from individual components. Thus properties of regulatory sub-systems can be used to predict behaviour of larger more complex regulatory networks.( Guido Nicholas et al, 2006)

Designing constructs…

Modeling and construction of many and varied gene regulatory circuits are reported till date. Oscillators being the most popular, are constructed by coupling positive and negative feedback loops, such that the whole system oscillates or moves back and forth between the two steady states. Also lots of variations are also possible here, in terms of the components that make the whole system oscillate.

 In case of E.coli itself it is possible to construct oscillatory circuit using IPTG, lacI protein and arabinose regulatory sites, together.( Stricker Jesse et al, 2008; M Rachael et al, 2002) Similar is the deal with reprissilator in which three transcriptional repressor systems build an oscillator, but the twist is that the period of oscillation is shorter than cell-division cycle, so the state of oscillator needs to be transmitted to the next generations. Thus such reprissilator make possible, design and construction of artificial genetic networks with new functional properties from generic components that naturally occur in other contexts.( Elowitz et al, 2000)
 In the same E.coli one can also create oscillations by exploiting glycolytic flux i.e. construction of a metabolator! In this acetyl phosphate acts as a signalling metabolite and under its control the two metabolite pools interconvert.( Fung, E. et al, 2005)

 Toggle switches are another kind of circuits tried and tested in E.coli which require only transient induction and hence can function as a cellular memory unit. This can very well be exploited in industries, since it permits high induction of recombinant proteins without the high cost of large quantities of inducer. (Gardner Timothy et al, 2000)

 Establishment of communication between bacteria by constructing an artificial quorum sensor has not only enabled intraspecies but also intrespecies communication possible, leading to various behaviours and phenotypes. It facilitates achievement of co-operative transcriptional response.(Garcia-Ojalvo Jordi et al, 2004; Bulter Thomas et al, 2004)

 Mammalian cells are also being used for construction of oscillators which give self-sustained, tunable autonomous and robust oscillations, thus opening new vistas for future gene and cell therapies.( Tigges Marcel et al, 2009)

The untold story…

Synthetic biology is not just messing around with the genome (as it may seem to be from the above text) to make useful constructs, basically its not tinkerer’s approach but engineers approach to a problem.

One can say that success of synthetic biology is essential to understand life…this is because in this whole process of modelling, construction and testing the understanding of the system itself is achieved in a better way. This holds true not only for the insight into working of the cell cycle (a mitotic oscillator is at work there) (Goldbeter Albert, 1991) but also in understanding of the sleep-wake cycle or the circadian rhythms.

It is only while working with the synthetic constructs a phenomenon of “noise” comes into light. Noise is the one which can actually collapse your circuit or rather may give your circuit an insignificant and non-functional look! Trying to reduce the noise level in one’s own construct(Orrell David et al, 2004) leads to design enhancement and also appreciation of the noise resistant constructs found in the nature built circuits!( Vilar, J. M et al, 2002)

Fascinating shades…

A giant leap of synthetic biology is to “write the genome” as Craig Venter puts it! As a step in this direction not only have they constructed synthetic whole genomes(Smith Hamilton et al, 2003) but also expanded the genetic code with a functional quadruplet codon.(Which can incorporate unnatural amino acids into proteins.)(Anderson Christopher et al, 2004)

In order to bring all these constructs and related research in laboratories around the world under a common umbrella standard protocols and registry of the constructed parts has been developed on the World Wide Web It actually involves efforts to develop “tool box” of standardized genetic parts with known performance characteristics—analogous to the transistors, capacitors, and resistors used in electronic circuits—from which bioengineers can build functional Devices and, someday, synthetic micro organisms. The registry is made up of components called “BioBricks,” short pieces of DNA that constitute or encode functional genetic elements. Examples of BioBricks are a “promoter” sequence that initiates the transcription of DNA into messenger RNA, a “terminator” sequence that halts RNA transcription, a “repressor” gene that encodes a protein that blocks the transcription of another gene, a ribosome-binding site that initiates protein synthesis, and a “reporter” gene that encodes a fluorescent protein. A BioBrick must have a genetic structure that enables it to send and receive standard biochemical signals and to be cut and pasted into a linear sequence of other BioBricks. Further, work on the lines of improvising the repository of biobricks and methods for their easier handling is being done, actively.( Peccoud Jean et al, 2008; Shetty Reshma et al, 2008) Also work on the lines of developing an organism with minimum essential genes(Glass John et al, 2005), so that the organism can be exploited efficiently for integrating the constructs, is in progress.

Excellent examples of intelligent use of the toolbox involves -

1. Engineering a metabolic pathway for the synthesis of artemisinic acid in yeast, which is the immediate precursor of the drug artemisinin( a natural product) that is highly effective in treating malaria, thus reducing the cost of the drug.( Ro Dae-Kyun et al, 2006)

2. Construction of a sensory synthetic kinase that allows a lawn of bacteria to function as a biological film, such that the projection of a pattern of light on to the bacteria produces a high-definition (about 100 megapixels per square inch), two-dimensional chemical image. Thus using spatial control of bacterial gene expression to 'print' complex biological materials, for example, to investigate signalling pathways through precise spatial and temporal control of their phosphorylation steps.( Levskaya Anselm et al, 2005)

It is crystal clear that the field of synthetic biology has the potential to bring about epochal changes in science and a few decades from now it may have a profound influence on the definition of life, itself!

Perhaps the most intriguing problem right now is to observe how the designed circuit operates in the context of a complete organism. There are no dotted lines inside the cell isolating circuits from one another. The ultimate test for this synthetic approach is to delete natural circuits and replace them with synthetic counterparts within organisms. This will lead to interference of the synthetic circuits with the rest of the cell. Obviously these circuits would be less functional than their natural counterparts. But at this stage one can learn more by putting together a simple, though inaccurate, pendulum Clock, than one can by disassembling the finest Swiss timepiece.

(these days i am going gaga... over synthetic after my paper ppt my review follows...again on synthetic biology! nothing like it if get to work in relation to the field...hopefully!)

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