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Team:Tec-Monterrey/Introduction
From 2010.igem.org
Introduction to Synthetic Biology
The term Synthetic Biology has been used to describe a number of different disciplines since its first appearance in literature in the decade of the 1970s. Waclaw Szybalski has been credited as the person who coined the term in 1974 to refer to the modification of organisms through the subtraction and addition of genes. (Benner S. , 2008) In 1980 Barbara Hobom used the phrase in a similar fashion, mainly to report advances in recombinant DNA technology. (Benner & Sismour, 2005) The logic behind the use of Synthetic Biology for this type of situations was that the genetically modified bacteria were natural organisms (biological) that were modified by human intervention (in other words, synthetically). Today, however, we tend to refer to the genetic modification of bacteria and other organisms as “genetic engineering” or “bioengineering”.
In the year 2000 Erik Kool used the term Synthetic Biology during a conference in San Francisco in reference to the synthesis of unnatural organic molecules that have functions in living organisms. This use of the term Synthetic Biology was applied mainly to scientific efforts to “redesign life”, and it was considered an extension of the area of “biomimetic chemistry”. The larger goal of this branch of science is to construct systems (synthetically) that behave following Darwinian rules of evolution (biologically) with the hope that these artificial systems will permit a better understanding of our own natural world. (Benner & Sismour, 2005) However, new uses of the term Synthetic Biology have become more common and relatively few people still apply this phrase to refer to these artificial chemical systems.
In recent years, the engineering community has adopted a new use for the term Synthetic Biology. This group of scientists and industry professionals seek to create standard interchangeable parts from living organisms that might serve as construction units for the development of living devices that carry out functions not readily found in nature. In this case, the parts are natural (biological) but their arrangement and assembly are not (synthetic); so far the term has been well received and has become one of the most important branches within modern genetic studies (Benner & Sismour, 2005). One of the greater promises of this new discipline is the ability to create gene networks that emulate digital circuits and devices. Through the use of an industry standard that enables easily exchangeable parts, scientists will be more able to “program and design” cells according to some of the basic principles of modern computing (Friedland, Lu, Wang, Shi, Church, & Collins, 2009).
In the last decade a group of scientists defined a new standard for the creation of standard interchangeable parts in Synthetic Biology. Tom Knight from the MIT Artificial Intelligence Laboratory defined what he called the Standard Biobrick Sequence Interface. This initial Interface consists of a circular vector or double stranded DNA that contains the sequence of interest. This sequence is flanked by upstream EcoRI and XbaI restriction sites and downstream SpeI and PstI restriction sites. In order for the parts to be interchangeable, the mentioned restriction sites must be unique within the whole vector; this enables a simple two enzyme digestion to release the fragment and unite it with another one with a ligation reaction. (Knight, 2003) In 2006, Ira Phillips and Pamela Silver refined Knight’s Sequence Interface in order to prevent a change in the reading frame when ligating two Biobricks. The change in the interface was small, but it effectively reduced the mixed site that results from a ligation to contain only 6 base pairs instead of the 8 base pairs that resulted from using the first Interface. (Phillips & Silver, 2006) This new Standard Biobrick Sequence Interface has enabled a free exchange of DNA parts with the certainty that they are compatible with other Biobricks that use the same standard.
As a result of this standardization, the field of Synthetic Biology has begun to expand within the international scientific community. The results of one research team can now be easily shared with others enabling a fast growth within the discipline. Numerous countries and institutions have increased their support to synthetic biologists because of the great promise of the field and the applications it can have in the future.
Bacterial Reporters
One of the many applications within the area of Synthetic Biology are whole-cell bioreporters. Bioreporters are defined by Jan Roelof van der Meer and Shimshon Belkin as: “microorganisms, cell cultures or cell lines, often genetically engineered, with an activity that reflects changes in environmental conditions in a dose dependent manner”. (van der Meer & Belkin, 2010) They can be very useful to detect the presence of a certain type of compound or organism within a medium.
One of the first uses of bioreporters was the now called Ames test, developed in the 1970s. This test uses a strain of Salmonella enterica subsp. enterica that containes an auxotrophic mutation (that is, a mutation that inhibits the capacity of a strain to produce an essential substance so this nutrient must be administered artificially in order for the microorganism to survive) that makes it incapable of synthesizing histidine. The strain can then be exposed to metabolically active proposed carcinogens in order to detect if they are mutagenic. If the strain grows in medium that doesn’t contain a supply of histidine, it means that it developed a new mutation that enabled it to synthesize histidine once again, proving the substance is indeed a mutagen (van der Meer & Belkin, 2010) . Salmonella is considered a bioreporter in this case, because it detects mutagens and reacts to their presence. However, it is not a very efficient biosensor because it depends on the probability that the mutagen will have its effect on the region that codes for the synthesis of histidine.
Over the course of the following years, as scientists gained more knowledge of molecular biology and genetic engineering, whole-cell bioreporters became more common and more precise. Instead of relying on mutations, they began to take advantage of naturally occurring gene regulation mechanisms that enabled the expression of proteins and molecules when the organism was in the presence of a certain substance. Finally, molecular biologists began to genetically modify microorganisms to make bioreporters according to their necessities.
The potential commercial and academic applications of this technology, led to even more research in the area and in the 1990s several whole cell bioreporters were developed using genetic engineering. Some reporters were developed to detect nutrients in soil, such as sugars and proteins. Jaeger and his team published an article in 1999 in which they detailed the construction of a bioreporter that detected sucrose and tryptophan in soil using promoters that were sensible those compounds (Jaeger III, Lindow, Miller, Clarck, & Firestone, 1999). That same year, another group reported their results for a biosensor that produced Green Fluorescent Protein in the presence of L-arabinose using a pBAD promoter (Shetty, Ramanathan, Badr, Wolford, & Daunert, 1999). There were also reports of biosensors used to detect certain types of contaminants such as Toluene and its derivatives (Willardson, et al., 1998).
Bacterial bioreporters are the most common type of whole-cell bioreporters used today. Bacteria are preferred because they are easy to manipulate genetically and they are able to survive in a wide diversity of media. This versatility makes them very attractive because they have a number of applications in different areas. Biosensors can be great diagnosis tools in the medical industry, especially for compounds that are hard to detect using other methods. And, recently many people have begun to see their potential in environmental science because they can detect the presence of contaminants that could be hard to analyze using conventional methods. Kiyohito Yagi reports that biosensors can be especially useful to detect heavy metals, and genotoxic materials (Yagi, 2007).
The advantage of using a biological sensor instead of a chemical or physical sensor is that biological components have great specificity, sensitivity and portability, since they usually don’t require large and expensive equipment. However, within the industry, there are many types of biological systems that can be used as sensors, such as enzymes, antibodies, sub-cellular components and the whole cell. The advantage of choosing a whole-cell bioreporter is that they are far more versatile than enzymes and other biological alternatives. Enzymes often require long purification steps in order to reach the level of purity required for these types of tests, which often increase the price of the system. Besides, enzymes only work on certain temperatures and pH conditions. Using microorganisms permits a much greater flexibility. Since bacteria grow in such a great diversity of environments, it is easy to choose an appropriate strain to carry out the job that is required. They are also cheaper because they don’t require sophisticated purification techniques and they can be grown using inexpensive medium. A final advantage of whole-cell biological sensors is that they can analyze samples through processes that require many enzymes, something that is not as easy when using other systems (Yagi, 2007).
Modern bacterial biosensors produce a substance, generally a protein, which is considered the “output” that results from the detection of the substance of interest. The amount of reporter protein is taken as a proxy for the cellular response to the target. Reporter proteins are preferably easy to detect, quantify and not present in the native organism (van der Meer & Belkin, 2010). Today, whole-cell biosensors exist for a wide variety of target substances, ranging from potentially dangerous compounds and contaminants to many types of carbohydrates and heavy metals. However, there have been few mentions of biosensors that not only detect the presence of a substance but that are also able to distinguish different concentrations of the target compound and react differentially depending on the amount detected. One reason for this is that the amount of genetic engineering required to achieve such a goal is considerable, and for many purposes it is not practical. However, Synthetic Biology may offer a reasonable alternative by providing a standard genetic frame to achieve a biosensor capable of reacting differentially to various concentrations of a substance of interest.
References
Benner, S.(2008). Biology from the bottom up. Nature, 692-694.
Benner, S. A., & Sismour, A. M. (2005). Synthetic Biology. Natrue Reviews Genetics, 533-543.
Friedland, A. E., Lu, T. K., Wang, X., Shi, D., Church, G., & Collins, J. J. (2009). Synthetic Gene Networks That Count. Science, 1199-1202.
Jaeger III, C., Lindow, S., Miller, W., Clarck, E., & Firestone, M. (1999). Mapping of Sugar and Amino Acid Avalability in Soil around Roots with Bacterial Sensors of Sucrose and Tryptophan. Applied and Environmental Microbiology, 2685-2690.
Knight, T. (2003). Idempotent Vector Design for Standard Assembly of Biobricks. 1-11.
Phillips, I. E., & Silver, P. A. (2006). A New Biobrick Assembly Strategy Designed for Facile Protein Engineering. 1-6.
van der Meer, J. R., & Belkin, S. (2010). Where microbiology meets microengineering: design and applications of reporter bacteria. Nature Reviews Microbiology, 511-522.
Shetty, R. S., Ramanathan, S., Badr, I. H., Wolford, J. L., & Daunert, S. (1999). Green Fluorescent Protein in the Design of a Living Biosensing System for L-Arabinose. Analytical Chemistry, 763-768.
Willardson, B. M., Wilkins, J. F., Rand, T. A., Schupp, J. M., Hill, K. K., Keim, P., et al. (1998). Development and Testing of a Bacterial Biosensor for Toluene-Based Environmental Contaminants. Applied and Environmental Microbiology, 1006-1012.
Yagi, K. (2007). Applications of whole-cell bacterial sensors in biotechnology and environmental science. Applied Microbiology and Biotechnology, Vol. 73, p. 1251 - 1258
