Project

Introduction

Synthetic Biology is the engineering of Biology[1] . It attempts to make the construction of novel biological entities easier by introducing engineering concepts such as standardization, decoupling and abstraction[2]. Standardization refers to the introduction of shared units ([3],[5]) and DNA standards[4]. Decoupling means splitting up complex problems into simpler ones and can be best realized by introducing an abstraction hierarchy, ranging from "Parts" [2] (DNA, genes and proteins) and "devices" (a number of parts combined to process inputs to a functional output [6]) to "modules" (a set of interconnected devices that performs complex tasks [7]). Parts, devices and eventually modules would ideally be made freely available from a central registry. The Registry of Standard Biological Parts (http://www.partsregistry.org/) is an early implementation of such a collection of parts. Many parts in the registry are constructed from teams participating at iGEM([8],[9]), the world's premiere student competition in synthetic biology & biotechnology.

A further lesson learned from engineering is that the only efficient way to construct systems of high complexity (such as computers or airplanes) is simulating these systems on the computer prior to construction[7]. The construction of systems behaving predictably is indeed possible in biology, as two impressive publications demonstrate: Basu et al. |[10] show that programmed patterns can be formed by bacteria with transcriptional repressors of different strength at hand. Very recently, Ellis et al. |[11] built scalable timers in yeast from a library of promoters of different strength. For systems to behave predictably, it is an obvious requirement to have well-characterized parts and devices at hand. A very high standard for device characterization has been proposed [12], but most registry entries remain uncharacterized.

The majority of the teams uses E. coli as chassis. Concerning eukaryotes within the iGEM competition most projects focused on yeast as chassis. There are very few teams which actually worked with mammalian cells. One of them was the Slovenia team of 2006 whose chassis was the human embryonal kidney cell line HEK293. Apart from iGEM there are a few labs that are concerned with synthetic biology in mammals. Research was focused on regulatory cascades, epigenetic toggle switches and even entire ecosystems with synthetic intra- and interspecies crosstalk [13]. However, no systematic approaches to Synthetic Mammalian Biology (SMB) exists, especially in the iGEM community. SMB therefore requires the accomplishment of a variety of tasks that have successfully been approached in bacteria, such as promoter measurement [5], the definition of mature cloning standard and, finally, the creation of an encompassing collection of parts and devices. All in all, SMB is a new and open field. The potential applications of SMB are manifold and include virotherapy to disease [19] and drug development [20].

We decided to approach some of the most pressing issues of SMB systematically and, wherever possible, in analogy to the well-accepted work that was done in bacteria. We analyzed all cloning standards proposed so far[14] and found Tom Knight's [http://dspace.mit.edu/handle/1721.1/45139 BB_2 proposal] to be the most mature. Therefore, we suggest do make it commonly used in SMB. We adopt the defining publication of promoter measurement in bacteria [5] to mammalian cells by applying state-of-the art promoter measurement techniques [15]. Recognizing gene regulation as a most central issue in SMB, we reviewed literature on the design and production of synthetic promoters([16],[17],[18]) and were inspired to the development of two entirely novel procedures - one for the design, one for the synthesis of promoters.

References

[1] "Synthetic Biology. Applying Engineering to Biology". Report of a NEST High-Level Expert Group. European Commission, Directorate-General for Research. Available online at http://www.synbiosafe.eu/uploads///pdf/EU-highlevel-syntheticbiology.pdf

[2] Endy, D. Foundations for engineering biology. Nature 438: 449-453 ()2005)

[3] Endy, D. Deese I. Adventures in synthetic biology (comic).Nature 438: 449-453 (2005)Available online at [http://www.nature.com/nature/comics/syntheticbiologycomic/index.html nature.com]

[4] http://www.partsregistry.org/Assembly:Standard_assembly (Website, accessed 10/15/2009)

[5] Kelly, J.R., Rubin A.J., Davis J.H., Ajo-Franklin C.M., Cumbers J., Czar M.J., de Mora K., Glieberman A.L., Monie D.D., Endy D. Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering 3 (2009)

[6] Voigt, C. Genetic parts to program bacteria. Current Opinion in Biotechnology 17: 548–557

[7] Andrianantoandro E., Basu S., Karig D.K., Weiss R. Synthetic biology: new engineering rules for an emerging discipline. Molecular Systems Biology: 0028 (2006)

[8] https://2009.igem.org/About (Website, accessed 10/15/2009)

[9] Goodman, C. Engineering ingenuity at iGEM. Nat Chem Biology 4:13. (2008)

[10] Basu, S. A synthetic multicellular system for programmed pattern formation. Nature 434:1130-1133

[11] Ellis T., Wang X., Collins J.J. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nature Biotechnology 27: 465-471 (2009)

[12] Canton, B., Anna Labno A., Endy D. Refinement and standardization of synthetic biological parts and devices. Nature Biotechnology 26: 787-793 (2009)

[13] Weber, W. & Fussenegger, M. Engineering of Synthetic Mammalian Gene Networks. Chemistry and Biology 16: 287-297 (2009)

[14] http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats (Website, accessed 10/19/2009)

[15] Ducrest A-L., Amacker M., Lingner J., Nabholz M. Detection of promoter activity by flow cytometric analysis of GFP reporter expression. Nucleic Acids Res. 30, e65 (2002).

[16] Venter, M. Synthetic promoters: genetic control through cis engineering. Trends in Plant Science 12, 118-124 (2007). (and the references cited therein)

[17] Ogawa, R. Construction of strong mammalian promoters by random cis-acting element elongation. Biotechniques 42, 628-632 (2007).

[18] Tornoe, J. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 297, 21-32 (2002).

[19] Dorer, D.E., Nettelbeck, D. Targeting cancer by transcriptional control in cancer gene therapy and viral oncolysis. Advanced Drug Delivery Reviews 61, 554-557 (2009).

[20] Weber W., Fussenegger M. The impact of synthetic biology on drug discovery. Drug Discovery Today 14, 956-963 (2009)