Team:Tec-Monterrey/Geneticframe

1. Development of the genetic frame for an “Intelligent Biosensor”
2. Phage Activators

Development of the genetic frame for an “Intelligent Biosensor”

After deciding we wanted to make a concentration sensitive biosensor, we began to investigate the different ways we could achieve our goal. After an initial research concerning biological reporters and the mechanisms currently used to make them, we began to read past iGEM projects to look for useful BioBricks that could be applied to our work. Two past iGEM projects were of our particular interest: British Columbia’s 2009 project and Cambridge’s 2007 project.

• British Columbia’s project

  In the past iGEM competition The University of British Columbia team developed a project that resembled our initial proposition. They wanted to produce an E. coli that expressed different Fluorescent Proteins depending on the concentration of arabinose in the medium. Basically, they proposed a system that used three different sensitive promoters to activate a “Lock and Key” mechanism that enabled the expression of the Fluorescent Protein. There were 3 promoters: the wild type pBAD promoter that is sensitive to arabinose, and two others called pBAD weak and pBAD strong that were designed by the team via mutagenesis. As their name implies, the weak promoter is sensitive only to higher concentrations of arabinose and the strong promoter is sensitive to lower concentrations of arabinose compared to the wild type. Finally, to prevent the expression of all the fluorescent proteins when there was enough arabinose in the medium to activate all three promoters, they developed a “jammer” that consisted in placing a reverse promoter on the other side of the fluorescent protein. Thus, when both promoters were activated, the production of fluorescent protein ceased.

  (For more information about this project, you can consult their wiki)

• Cambridge’s project

  In 2007, the University of Cambridge iGEM team worked in developing both intracellular and intercellular communication pathways as well as a Gram-positive platform for synthetic biology. We were particularly interested in the intracellular project. It consisted of a PoPS amplifier made with transcriptional activators and promoters that were obtained from bacteriophage components. The Cambridge team decided to use activators from bacteriophage components because they are not common in the cellular signals of bacteria. In this particular case, they used activator and promoters from the bacteriophage PSP3 and fR73.

  (For more information about this project, you can consult their wiki)

After reading about these projects we decided that we could adapt our idea and build upon the achievements of those two previous teams. We wanted to contribute in a significant way, so we began by analyzing their results and identifying points that could use more work. We found that, in general, British Columbia’s proposal was complicated and apparently ineffective. The problem seemed to be in the “lock and key” mechanism, which didn’t seem to behave as expected. On the other hand, we found that their “jammers” could be very useful and they seemed to work as expected.

So we began to design of a different genetic frame to create “intelligent biosensors”, one that preferably didn’t contain a “lock and key” mechanism. Our solution is composed by a combination of parts of the two previously mentioned projects. We incorporated the “jammer” mechanism in addition to PoPS amplification systems in order to create a flexible genetic construction for concentration sensitive bio-reporters. Our proposed solution can be seen in Figure 1.

In theory, this genetic construction is able to detect different concentrations of a compound provided that you have three different promoters that are sensitive to different concentrations of the substance. Based on the amount of compound detected, the bacteria will produce one of three reporter proteins. For our project, we decided to use the same sensors and reporter proteins that the University of British Columbia team used in their 2009 iGEM project. Therefore we began with 3 promoters: pBAD wild type, pBAD weak and pBAD strong; each promoter was able to detect arabinose at different concentrations. Our reporter proteins were GFP, YFP, and RFP (Cherry). We arranged the constructions so that GFP is produced when the cells were induced with low concentrations of arabinose, YFP is produced when induced with medium concentrations and RFP (Cherry) is produced when there is a high concentration. The mechanism that enables this behavior will be explained in two parts, the first part involves the detection of the compound and the second part concerns the production of the reporter protein.

The first part of the genetic construction is responsible for the detection of arabinose. This is achieved by using a series of promoters from the pBAD family that were developed and characterized by British Columbia. Each promoter is activated at a different concentration of arabinose, the strong promoter requires a higher concentration of arabinose than the weak promoter. When the promoter is activated, it enables the expression of a bacteriophage activator that works as a PoPS amplifier. The first amplifier we used was the one provided by the Cambrdige 2007 team. However, for our construction to work, we required two additional amplifiers. So we did some more research on bacteriophage activators and constructed two new PoPS amplifier systems in order to finish our construction. We consider these new amplifiers to be a valuable addition to the Registry of Standard Biological Parts, because they come from different bacteriophage families, this means that they produce different activator proteins and they can be used at the same time without causing interference.

Eventually we realized that if we didn’t have a mechanism that disabled the expression the PoPS amplifiers, when the cells were induced with high concentrations of arabinose, all three reporter proteins would be produced. Once again, incorporating the previous advances of the 2009 British Columbia Team, we were able to overcome this obstacle. The solution consisted in implementing their “jammer” systems, in order to suppress the expression of the PoPS amplifiers as the concentration of arabinose in the medium increases. The full mechanism is explained in the following figures:

• Figure 2: shows the sensor part of the final construct
• Figure 3: shows the sensor when there is a low concentration of arabinose
• Figure 4: shows the sensor when there is a medium concentration of arabinose
• Figure 5: shows the sensor when there is a high concentration of arabinose

The second part of the construction is in charge of producing the reporter proteins. According to our research on biological sensors, the reporter molecule must be easy to detect and it must be a substance that is not naturally produced by the organism. Fluorescent proteins are great reporter proteins because fulfill both of these requirements. The team from British Columbia University used three different fluorescent proteins in their project in the 2009 competition: Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP), and the Cherry variety of the Red Fluorescent Protein (RFP Cherry). We ultimately decided to use the same three molecules because they have been widely characterized and there is plenty of information regarding their fluorescence spectrum and behavior.

When the sensing part of our construction produces one of the bacteriophage activators, this molecule enables the expression of one of the reporter proteins. Because these promoters are produce have very high PoPS numbers, the amount of reporter molecule produced is elevated, even though the arabinose concentration isn’t. The following figure is a detail that explains the mechanism behind the production of reporter proteins.

Figure 6: shows the reporter part of the final construct.  

Phage Activators

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