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MeSH Review

Synthetic Biology

 
 
 

3. Basic Concepts

 

3.1 Bioinformatics and systems biology for synthetic biology

As synthetic biology develops and matures as a field, it has to develop its own ontology and tool box. For large scale synthetic biology applications, bioinformatics and systems biology models will be harnessed using engineering principles of computer aided synthetic modules and parts design. Trend for this has already started. Here we review developments in this interface discipline variously christened as synthetic bioinformatics or synthetic systems biology. We also enlist the direction and trends for this emerging field.

Bioinformatics uses biological information and computational methods to discover knowledge. The application of its methods results in a collective construction that is constantly being deposited in distributed information systems in which knowledge about interesting biomolecules is organized. It ultimately encodes relationships between composition (sequence), structure, functions, binding, abundance, etc. Bioinformatics products are, therefore, of prime importance for SynBio. Likewise, Bioinformatics methodologies, often resulting from the combination of others, are highly inspirational for SynBio. Bioinformatics is keen on evaluating similarities and screening results against defined discriminating criteria. It is also keen on integrating heterogenous data resources, often originated in vast data set, and distilling the most valuable results.

It is expected that bioinformatics will provide contributions to SynBio on a wide variety of levels, that can range from the selection and characterization of components (parts or modules), to the derivation of naturally inspired design rules, and to the creation of protocols for testing its products. Conversely, SynBio is constantly creating new challenges for bioinformatics practitioners - both developers and users, especially but not exclusively in the area of confluence with systems biology.

SynBio will open new possibilities of validating models and predictions that emerge from bioinformatics studies in the laboratory or even in organisms. The validity of in-silico predictions is often poorly tested because it involves inducing difficult changes in biological systems that aim at reproducing the changes that are easily induced in models. BioParts can play a very valuable role there, by making such changes possible by direct replacement of parts, such as a in the case of the activation/inactivation of a sector of a pathway.

 

4. Methods and Fundamental Techniques in Synthetic Biology

 

4.2 DNA synthesis

Chemical synthesis of DNA or oligonucleotide synthesis is an essential component of synthetic biology. Thanks to the advancements in automated DNA synthesizer, it is now possible to synthesize and assemble complete genes, regulatory elements, gene circuits or even entire genomes of micro organisms. Khorana and co-workers pioneered the chemical synthesis of DNA from oligonucleotides and demonstrated the first chemical synthesis of a yeast tRNA gene [1]. This process is also known as artificial synthesis of gene because no initial DNA template was used. Somatostatin was the first peptide and Leukocyte Interferon was the first protein-coding gene to be chemically synthesized and expressed in bacteria [2][3]. These studies demonstrated the potential applications of synthetic blology. Chemical synthesis of DNA is often more straight forward and economical than recombinant DNA cloning and is routinely employed in biotechnology.

 

4.3 DNA sequencing

First of all, DNA has to be extracted from the cell. This is achieved by mechanical or chemical means, followed by purification and separation of the DNA strands from binding proteins. Then they are incorporated into plasmid vectors, and transferred to bacteria or virus, which divides exponentially to form a high yield of DNA clones. The strands are separated by heating, followed by 'primer annealing'. Next, 'extension' of the strand is done using deoxynucleotides (dNTP), with the help of DNA Polymerase. In the 'termination' step, fluorescent-labeled dideoxynucleotides (ddNTP) are attached leading to termination of further chain growth as ddNTPs lack 3' hydroxyl group without which DNA Polymerase can not work. Each four ddNTPs are coded with a differently colored fluorescent dye. The ratio of dNTPs to ddNTPs are carefully chosen to obtain different chain lengths. In the next step, these mixtures are poured into a slender capillary tube which is subjected to an electrical field (capillary electrophoresis). The smallest pass through first and in this order the largest exits last. As they exit, a laser excites the fluorescent-labeled base and the event is read by a photocell which ultimately passes the signal onto a computer which decodes the 'terminator' base by showing a peak characteristic of that particular base (Fig.  ). Sequencing is done in this (smallest first etc.) sequence. Sequencing technology seems poised to another landmark shift as Life Technologies announced single day human genome sequencing at the cost of an ipad with its revolutionary ion proton technology  . Oxford nanopore has reportedly develop a nanopore based disruptive DNA strand sequencing technology  . These advances will provide a synergistic effect for advances in synthetic biology.

 

4.4 DNA Parts Assembly

DNA assembly challenge is to take a set of double-stranded DNA sequence fragments, and stitch them together in the user-defined order and orientation to yield a single, potentially circular, assembled DNA sequence.

The following is the development progress of DNA parts assembly, from which it is inferred that parts standardization, one-step (cut-ligation), and large-scale are the future.

1. Traditional MCS (Multiple Cloning Site) Approach

3. SLIC, Gibson, CPEC and USER assembly methods.

4. Golden Gate assembly method (and MoClo and GoldenBraid)

Self-explaining details for public are available in J5's webpage  . In addition, more professional guides and protocols are available from the journal The Methods in Enzymology. Volume 497, Pages 2-662 (2011), is dedicated to Synthetic Biology  .

 

4.5 DNA Part Analysis and Description Standards

 

4.6 DNA arrays

Gene expression in eukaryotes is a dynamic process and involves complex interactions of genetic regulatory networks (or GRN) [4]. We need to understand the design principles of these networks in order to advance synthetic biology. Micro arrays or gene chips are mainly made up of known cDNA (complimentary DNA) or oligonucleotide sequences of thousands of genes on a chip and extensively used for gene expression analysis; see animation  . This method is based on hybridization of labeled cDNA species derived from biological samples to the gene chips. This step is followed by fluorescent detection and quantitative analysis of data to reveal differential expression. Micro arrays enable simultaneous, quantitative monitoring of expression of thousands of genes in response to multiple experimental conditions on a single platform. The data sets of gene expression are subjected to a clustering algorithm to reveal genes that are co-expressed (simultaneously increased or decreased) and are regulated as discrete networks [5][6].

 

5.Biological networks and circuits

5.1 The concept of regulatory and metabolic circuits

5.2 Oscillators

5.2.1 Types of oscillators

5.2.1.1 The Elowitz and Leiber Represillator

5.2.1.2 The Atkinson Oscillator

5.2.1.3 The Metabolator

5.2.1.4 Circadian circuits

5.2.2 Designing oscillators (with examples)

5.3 Inverters

5.3.1 Designing inverters (with examples)

5.4 Chemotaxis and quorum sensing

5..4.1 Cell - cell communication systems

5.5 Design and characterization of synthetic gene networks

- coordinated expression

- stability of expression

- transcriptional cascades

- toggles

- logic gates

- signal amplifiers

- pulse generators

 

6. Applications of Synthetic Biology

 

6.1 Synthetic biology and health

Artemesinin, an antimalarial drug, is one of the prototypical drug that is being synthesized at a low cost using SynBio technique. Although the Chinese have been using this herbal medication (qinghaosu) containing the active principle for over 2000 years as an antipyretic, the pure form was prepared only recently. Now it is being synthesized in E.coli or as a precursor, amorphadiene in yeast which can be chemically converted to artemisinin [7] [8]. Sulfonamides (antimicrobials), many antimetabolite anticancer drugs, monoclonal antibodies like Trastuzumab (used in the treatment of breast cancer), Rituximab (used in the treatment of non-Hodgkin's lymphoma) are some of the many designer drugs synthesized using SynBio approach  .

 

6.2 Synthetic biology and environment

 

6.3 Synthetic biology and new sources of energy

A microbial platform for the direct production of bioethanol from alginate, a polysaccharide constituent of seaweed was reported [9] . The key advance in this platform is the bioengineering of enzymes involved in alginate metabolism and transport from Vibrio splendidus into E.coli for industrial applications.

 

6.4 Synthetic biology and new biomaterials

In a very recent development scientists at Wyss Institute created a DNA nanorobot that could harmlessly deliver anticancer drugs on target tumor cell surfaces with almost no collateral damage (i.e. without harming healthy cells)   (see video below). The barrel shaped molecule identifies specific target cells by latching onto the cell surface with the help of two aptamers present on the barrel surface, in much the same way a key fits to a lock. This (binding) causes unfolding of the barrel and consequent delivery of the drug which is kept inside the barrel to prevent the drug from interacting with other cells which do not express the 'key'. This bioengineering concept known as DNA origami may also be used in diagnostics apart from therapeutic purposes.

6.5 Nanobiotechnology

6.6 Synthetic genomics

6.6.1 Genome re-writing and refactoring

6.6.2 Genome transplantation

6.6.3 Synthetic genomes and 'synthetic' organisms

6.7 Other applications

 

7. Discussion

Given the power that SynBio has in its armory; it is no wonder that manipulating microorganisms, therapeutic or otherwise administration on animal or human subjects are certain to raise bioethical and religious concerns. Properly educating the public is very necessary to gain trust and obviate any hurdle that might arise. Moreover, the fear of bioterrorism will always be lurking around. Protection from such biohacking attempts should be anticipated and methodically countered, if not prevented. Emergence of highly virulent strains as a consequence of therapeutic misadventure is also possible.

The future of SynBio looks very bright. With the discovery of green fluorescent protein (GFP) and the subsequent synthesis of its analogs which fluoresce with many different colors of the electromagnetic spectrum  , have empowered us with diagnostics, e.g. detecting urinary tract infection (UTI); to map and trace the neuronal circuitry etc. GECIs (genetically-encoded calcium indicators) are SynBio derived molecules which respond to calcium ions, a reliable indicator of Action Potential in excitable tissues such as neurons. We can picture them in 'real time', in action, thus gaining valuable insight to their functional aspects. Optogenetics  , at present, allows us to manipulate genetically modified rats using light shone into their brains using very slender fiber-optic probes. It has the potential to generate courage, erase fear memories and many more  .

Bionic prostheses e.g. cochlear implants  , articular prostheses where tissue-engineered chondrocytes would adhere to in a more biologically friendly manner   are not far beyond. Functioning cardiomyocytes (cardiac muscle cells) has been created from bone marrow cells of rats; then cultured and repositioned into the damaged portions of the heart of the same rat the bone marrow cells came from (autologous transplantation)  . This improved myocardial functions of these rats when compared to the control population. This is a milestone in transplantation research since it steers clear of the 'stem cell controversy'. Nowadays scientists are attempting at 3D sculpting of animal including human tissues (bioparts) by 'ink-jet printing' using individual cells, which promises potential benefits  . A real case in point is that of Luke Massella who received an engineered bladder 10 years ago 'printed' by Dr. Anthony Atala, a surgeon  . Liposomal drug delivery systems have made lipid-insoluble drugs to manoeuvre past the lipid bilayer to reach their target location possible  . Biosensors capable of detecting heavy metals, TNT have also been developed. Construction of logic gates such as AND, NAND gates from the four bases of DNA and then using these parts to construct a biological DNA computer is also bound to happen. Though slow, they will be biologically compatible. Biosensors that sense blood glucose level may be incorporated in a positive feedback driven insulin pump and then implanted in a patient of Insulin Dependent Diabetes Mellitus (IDDM) patients.

SynBio is also ready to modify gene expression by altering epigenetics; the modification of DNA methylation or acetylation status of DNA. This is poised to alleviate or cure cancer.

 

7.1 Biosafety and biosecurity

 

7.2 Bioethics in synthetic biology

 

7.3 New approaches towards synthetic life

Protocells, non-DNA genetic information, non-protein enzymes etc.

 

7.4 Future of synthetic biology

 

8. Resources

There are many excellent sources of synthetic biology on the Internet. A comprehensive source of Internet links was developed by Dana Antonucci-Durgan of the Stony Brook University Library (USA)  .

 

8.1 DNA synthesis, sequencing

- INTERPRETING DNA SEQUENCE  

- DNA - Protein Sequencing & Synthesis Facilities  

- Tech Summary: Illumina's Solexa Sequencing Technology (Forum)  

 

8.2 Software Tools

(A list and description of the different tools can be found by clicking on the heading. see also 4.1 Computational modeling)

 

8.3 Educational materials: videos, reviews, journals, mashups, news, comics

 

8.3.1 Videos:

- Synthetic Biology Explained  

- Andrew Hessel´s Introduction to Synthetic Biology  

- Synthetic Biology on the BBC  

- Decoding Synthetic Biology - KQED QUEST  

- J. Craig Venter on Synthetic Biology at NASA Ames  

- Drew Endy on Synthetic Biology  

- Drew Endy on Engineering Biology  

- The New Biology  

- Anthony Atala: Printing a human kidney  

- DNA nanorobot  

 

8.3.2 Reviews:

- Five hard truths for synthetic biology  

- Science/AAAS | Special Issue: Synthetic Biology  

- Nature Biotechnology December 2009, Volume 27 | Focus on Synthetic Biology  

- Journal of the Royal Society Interface | Synthetic Biology Focus Issue  

- EMBO reports special issue  

 

8.3.3 Journals:

- ACS SyntheticBiology  

- Journal of Biological Engineering  

- Journal of Computer-Aided Molecular Design  

- Journal of Synthetic Biology  

- Molecular Systems Biology  

- Systems and Synthetic Biology  

 

8.3.4 Mashup:

- The SynBioLogist  

- SynBioFromLeukipposInstitute  

 

8.3.5 News:

-Synthetic Biology on GEN  

 

8.3.6 Comics:

- Adventures in Synthetic Biology  

 

8.3.7 iGEM Foundation and Competition  :

The International Genetically Engineered Machine foundation   is dedicated to the advancement of synthetic biology by creating summer undergraduate competition to build biological systems in living cells from a kit of biological parts (BioBricks from the registry of standard biological parts). This competition began as a month long course at M.I.T in 2003 and grew to a summer competition in 2004 and became very popular with 165 international teams participating in 2011.

 

8.3.8 Community:

This section includes influential people, institutions, companies, social and professional networks such as Facebook, LinkedIn, Mendely and Twitter. (For details please click on the heading.)

8.4 Graduate Studies in Synthetic Biology

A list of universities with graduate programs in Synthetic Biology.  

 

8.5 Influential Articles

Please click on the heading and find some of the most cited and influential papers in SynBio.

References

  1. Studies on polynucleotides. 103. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast. Khorana, H.G., Agarwal, K.L., Büchi, H., Caruthers, M.H., Gupta, N.K., Kleppe, K., Kumar, A., Otsuka, E., RajBhandary, U.L., Van de Sande, J.H., Sgaramella, V., Terao, T., Weber, H., Yamada, T. J. Mol. Biol. (1972) [Pubmed]
  2. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Itakura, K., Hirose, T., Crea, R., Riggs, A.D., Heyneker, H.L., Bolivar, F., Boyer, H.W. Science. (1977) [Pubmed]
  3. Total synthesis of a human leukocyte interferon gene. Edge, M.D., Green, A.R., Heathcliffe, G.R., Meacock, P.A., Schuch, W., Scanlon, D.B., Atkinson, T.C., Newton, C.R., Markham, A.F. Nature. (1981) [Pubmed]
  4. Emerging properties of animal gene regulatory networks. Davidson, E.H. Nature. (2010) [Pubmed]
  5. Unbiased reconstruction of a mammalian transcriptional network mediating pathogen responses. Amit, I., Garber, M., Chevrier, N., Leite, A.P., Donner, Y., Eisenhaure, T., Guttman, M., Grenier, J.K., Li, W., Zuk, O., Schubert, L.A., Birditt, B., Shay, T., Goren, A., Zhang, X., Smith, Z., Deering, R., McDonald, R.C., Cabili, M., Bernstein, B.E., Rinn, J.L., Meissner, A., Root, D.E., Hacohen, N., Regev, A. Science. (2009) [Pubmed]
  6. Systematic discovery of TLR signaling components delineates viral-sensing circuits. Chevrier, N., Mertins, P., Artyomov, M.N., Shalek, A.K., Iannacone, M., Ciaccio, M.F., Gat-Viks, I., Tonti, E., DeGrace, M.M., Clauser, K.R., Garber, M., Eisenhaure, T.M., Yosef, N., Robinson, J., Sutton, A., Andersen, M.S., Root, D.E., von Andrian, U., Jones, R.B., Park, H., Carr, S.A., Regev, A., Amit, I., Hacohen, N. Cell. (2011) [Pubmed]
  7. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D., Keasling, J.D. Nat. Biotechnol. (2003) [Pubmed]
  8. Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Westfall, P.J., Pitera, D.J., Lenihan, J.R., Eng, D., Woolard, F.X., Regentin, R., Horning, T., Tsuruta, H., Melis, D.J., Owens, A., Fickes, S., Diola, D., Benjamin, K.R., Keasling, J.D., Leavell, M.D., McPhee, D.J., Renninger, N.S., Newman, J.D., Paddon, C.J. Proc. Natl. Acad. Sci. U. S. A. (2012) [Pubmed]
  9. An engineered microbial platform for direct biofuel production from brown macroalgae. Wargacki, A.J., Leonard, E., Win, M.N., Regitsky, D.D., Santos, C.N., Kim, P.B., Cooper, S.R., Raisner, R.M., Herman, A., Sivitz, A.B., Lakshmanaswamy, A., Kashiyama, Y., Baker, D., Yoshikuni, Y. Science. (2012) [Pubmed]
 
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