Revision as of 22:23, 24 October 2010 by Peterbrazda (Talk | contribs)



Eukaryotic synthetic biology has huge potential, yet it is still in need of more diverse molecular tools for defined gene regulation. Nuclear receptors are a conserved family of proteins responsible for sensing lipids; they may be viewed as lipid activated transcription factors. We have successfully developed a kit with a variety of lipid responsive domains (from H.sapines, D.melanogaster and C.elegans) for the rational construction of synthetic transcription factors. The domains respond only to predefined lipids and selectively activate predetermined gene expression. To characterize theses domains, we used standardized protocols for comparable measurements. In vivo gene expression was measured as a function of ligand concentration using luciferase activity.

The potential for these tools is immense; e.g. from the ultra sensitive detection of lipid contaminants in the environment to the opportunity of titration specific gene expression canges in patients undergoing gene therapy.


Synthetic Biology

Synthetic biology is a relatively new area of biological research; similar to many other new scientific fields it has many definitions. The best way to express the meaning of synthetic biology is to understand the desired end-result: engineering of complex biological systems. These systems are best thought of as analogues of everyday machinery: cogwheels, levers, timers, button and buzzers (in this case a clock), only in the case of biological systems (molecular ones) cells, DNA, proteins, lipids, sugars and RNA are the “parts” of the system.

Similar to mechanical engineering (or every other engineering branch) there is a need for standards (consensus way of doing things), abstraction (simple and unified way of thinking about the parts of a system), and modularity (how these parts interact to become the device, or several devices into a system). Thus a good definition for synthetic biology could be engineering of molecular (for the time being) biological systems according to preset standard parts. The international genetically engineered machines and associated parts registry are, to date, one of the largest registries for standard parts in use for synthetic biology. Free information can be found in the registry regarding parts, devices and modules all inputted by various teams worldwide.

Eukaryotic synthetic biology is still in its infancy. The large kingdom of metazoans includes all multicellular eukaryotes such as mammalians, arthropods and nematodes. No standard chassis (framework) exists for the animal kingdom which makes them far less popular then the famous E.coli. The protein modules derived from metazoans (like Drosophila or C. elegans) are functional in yeasts also. Very few iGEM teams (or even labs outside iGEM) have chosen to toggle the animal chassis (two notable examples are team Heidelberg 2009 and team Slovenia 2006). The amount of available compatible parts is limited, which severely restricts the options of creating complex biological devices. Nearly no imagination is required for designing tools, since their analogues already exist in the bacterial chassis. The possible use of such systems is unlimited. Field’s such as of environment, medicine, energy and research all gain to profit from the development of animal synthetic biology.

Systems requiring gene expression input in eukaryotic synthetic biology systems require a way to standardize gene expression, a complicated task. The way from gene to protein contains many steps of possible error: transcription factor binding, promoter strength, recruitment of auxiliary proteins, nuclear RNA synthesis and many more steps finally leading to translation, folding, cleaving and delivery (but hey, you have to start somewhere).

Our team was interested at designing eukaryotic synthetic biology tools related to PoPs. PoPs (polymerase per second), the flying Dutchman of synthetic biology, is a number which represents the rate (base pair per second) at which RNA polymerase crosses past a given DNA position. Currently, no in vivo technique for measuring PoPS directly exists; it can be estimated indirectly by measuring other parameters (eg protein expression or enzyme activity). Nevertheless it is still a useful abstraction for thinking about transcription-based logic devices and it allows the engineer to define devices. Our aim, was not only to infer PoPs but to devise a way to titrate it remotely.

New Tools for Synthetic Biology: Nuclear Receptors

Nuclear receptors, best viewed as transcription factors which can be activated by extracellular cues, are unique in their ability to allow direct remote PoPs titration (both activation and repression). These receptor classes bears high homology to each other throughout the animal kingdom and are modular into distinct domains. All of these features attracted our attention to find a way to incorporate these tools in the parts registry, and characterize them in standardized methods.

The ligand binding domains (LBD) are the segment of the receptor which changes its conformation upon lipophilic ligand binding; this also causes the exposure of a powerful transcriptional activation domain (which attracts the transcriptional machinery). This was our segment of interest since it links ligand binding with transcriptional activity.

Our team has generated a library of ligand binding domains for the rational construction of synthetic titrateable transcription factors. In our model hybrid receptor, this LBD was fused to a Gal4 DNA binding domain. The sequance to which Gal4 binds is CGG-N11-CCG, where N can be any base. Although Gal4 is a yeast protein not normally present in other organisms it has been shown to work as a transcription factor in a variety of organisms such as Drosophila, and human cells, highlighting that the same mechanisms for gene expression have been conserved over the course of evolution.

The examination of these chimeric receptors was through a version of the two hybrid assay.


First we had to generate expression vectors that can be used in mammalian expression systems. We generated two such expression systems: one is an adaptation of the widely used pCDNA3.1 expression vector by removing the MCS (multiple cloning site) of this vector and replacing it with a cloning site compatible with the RFC25 compatible parts.

The other expression vector is a fully SB (Synthetic Biology) compatible mammalian expression vector generated from pSB1A3. We inserted into the pSB1A3 the TRE (tetracycline response element) a strong CMV (cytomegalo virus) promoter and a polyA element that is a terminator of transcription in mammalian cells. These four elements are available as individual parts in the parts registry and can be used in combination with other parts too.

The functionality of these vectors was assesed by Gal4 Western Blot.

We moved the composite parts generated in pSB1C3 into the expression vectors and transfected them into COS- 1 cells. The new “hybrid” receptors were designed to enhance the gene expression of a luciferase enzyme through a GAL4 sensitive promoter (which was also transfected). The luciferase activity was assessed photometrically for several ligand concentrations and normalized to a constitutively expressed beta-gal activity. The final result was plotted on a dose response curve. The EC50 was then extracted from the curve.

Plans, Possibilities

The remotely activated transcription factor concept can be used to construct highly complex synthetic biological systems, for us it was a very appealing concept. Physicians may use fruit fly hormones in humans to titrate specific genes in gene therapy patient in selected tissue, such as dopamine receptor genes in schizophrenia. Worm species that can synthesize a different color reaction based on the amount of environmental pollutants in the soil they live. Scientists may induce stem cell pluripotency by titrating the exact amount of oncogenes needed for a fibroblast to turn into an embryo.

In our system we would like to induce apoptosis by increasing the amount of proapoptotic proteins such as BAX by producing an expression vector containing TRE-minimal CMV promoter, BAX or PUMA and polyA tail. The pro-apoptotic proteins are able to express only in a controlled form using the Tetracyclin-controlled transcriptional activation system. The overexpression of these proteins can lead to Mitochondrial Outer Membrane Permeabilisation (MOMP) leading Cytochorome c release which is a point of no return in the mitochondrial pathway of Programmed Cell Death (PCD). The goal is to “switch off” the cells, which were genetically modified by us, after they “finished their work” in the recent local environment combining the components of the tetracycline-inducible system in self-contained retroviral and plasmid vectors.

Furthermore human, arthropode or C. elegans nuclear hormone receptor composite constructs can be expressed in an inducible form using Tetracyclin-controlled transcriptional activation system rather than using a vector which ensures constitutive expression of the parts. In our system Tetracycline Response Element (TRE) is recognized and bound by the Tetracycline repressor (TetR) protein. The TRE consists of 7 repeats separated by spacer sequences and placed upstream of CMV minimal promoter that has basal expession in the abscence of bound TetR. Tetracycline derivatives (e.g. doxycycline) bind TetR and render it incapable of binding to TRE, thereby forcing the expression of target genes.