Development of a genetic frame for the creation of...
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Development of a genetic frame for the creation of concentration-sensitive bacterial sensors Introduction The Team Previous iGEM works Genetic frame PoPS Amplifiers Future Research Human Practices Acknowledgments Wiki Wiki >10 000 pb Argüelles, Campos, Cevallos, Contreras, Gálvez, Huerta, Núñez, Ostos, Reynoso, Siller Our proposal incorporated parts from these two previous iGEM projects as well as new BioBricks of our own design. The complete construction is presented in Figure 1. GFP YFP RFP (Cherry) pBAD Strong MuMor M uC MuC MuMor ΦR73δ ΦR73δ pBAD Weak Pm Po Mom pBAD Wild Type pBAD Wild Type pBAD Weak Figure 1: Complete genetic frame Figure 2: Sensor with different concentrations of arabinose (increasing from top to bottom) Figure 3: Reporter part of the construct Figure 6: MuMor sensitivity tuner characterization Figure 7: MuC sensitivity tuner characterization Our genetic frame consists of two main parts: the sensing part and the reporting part. In this case, we decided to use the pBAD family of promoters to detect arabinose as our target compound and fluorescent proteins GFP, RFP and YFP as reporter molecules because these BioBricks are well characterized and this would enable us to dedicate our time and efforts into characterizing the PoPS amplifiers we designed for this project. However, our proposal was designed with flexibility of modification in mind, and because it is compatible with the BioBrick Standard, modifying this construction is not very difficult and will enable the production of a wide variety of whole-cell Bacterial The sensing part of our construction was made incorporating the pBAD family of promoters developed by British Columbia’s 2009 team. We also included genes to codify for three different Bacteriophage activators that will serve as PoPS amplifiers. The promoters were arranged so that they “jammed” depending on the concentration of arabinose in the medium, as described by previous iGEM teams. Therefore, depending on the concentration, only one of the activating molecules is produced. This process can be pBAD Strong pBAD Wild Type pBAD Weak pBAD Weak pBAD Wild Type MuMor MuMor ΦR73δ MuC pBAD Strong pBAD Wild Type pBAD Weak pBAD Weak pBAD Wild Type J A MME D ! ΦR73δ ΦR73δ M uC pBAD Strong pBAD Wild Type pBAD Weak pBAD Weak pBAD Wild Type J A M ME D ! J A M ME D ! M uC MuC Upon deciding that we wanted to make a concentration sensitive bioreporter we did some research of previous iGEM teams’ projects to see if there were any BioBricks British Columbia’s 2009 project had an objective similar to ours. They incorporated three parts into their genetic construction that consisted of a sensor, a “lock and key” mechanism to control the expression of certain genes, and a jammer to inhibit translation. We were interested mainly in the sensing part and the jammer We decided to build upon both of these projects in order to contribute to the task of making better and more flexible genetic constructions for whole cell bacterial reporters. We decided to create a new mechanism for the development of “intelligent biosensors”, capable of detecting different concentrations of a target Depending on the concentration detected, only one activating molecule will be produced. Then, the reporting part of our construction produces one Fluorescent Protein depending on the activator that resulted from the sensing part. The mechanism for this is simple; each activator corresponds to one promoter as GFP YFP RFP (Cherry) MuC MuMor ΦR73δ Pm Po Mom To fully validate that our intelligent biosensor works as expected, we still need to carry out more experimentation to make sure that only one of the reporter molecules is expressed. We’ve already proven that our PoPS amplifiers work as anticipated; but we haven’t been able to do the same for the the genetic frame, so we will do further testing of the whole cell reporter. Once we have evidence that it does work properly, modifications to the frame should prove useful for the We are so interested in the further development of whole cell intelligent biosensors because we believe that such organisms will be a major breakthrough in several areas: our first approaches were environmental purposes, since detection and approximate quantification of a specific pollutant, like the troublesome benzene, can be easily achieved. In the medical field we could create alternatives to expensive tests and even devices that detect the concentration of a compound, say glucose, and release a response, glucagon or insulin in this example, accordingly to the need. Finally, a very ambitious approach would be to develop a biosensor that detects the concentration of a given substance or substances and reacts by producing molecules that interact with such target. This way, bioprocesses In summary, we know that the potential of these biosensors is enormous and the applications are endless. They surely represent one of those cases in biology where imagination is the only limit and that’s why we keep on working to prove that the genetic frame works as it is supposed to and we also encourage future iGEM Teams One of the greatest complications that we had to deal with during the summer was the Mexican Customs Department. We were surprised to discover that our government doesn’t have a defined protocol for the importation of biological material, and even if our packages were correctly labeled, they were delayed or permanently detained. On speaking to other researchers in our country, we learned that we were not alone on this difficulty. Scientists with enough money pay specialized agencies to arrange the importation of their biological material, which may cost more than the actual material to be imported, while researchers without an ample budget simply rely on luck and hope that their package will go unnoticed if undeclared. Clearly, this complication not only slowed down our iGEM project; it Synthetic biology is an emerging science in our country. We know that it is only a short matter of time before more Mexican teams participate in future iGEM competitions, and we believe that we can ease their experience by sharing our own. As part of our iGEM Human Practices collaboration, we created an Importation Manual, which is a step-by-step guide to receiving donations of synthetic DNA – such as the iGEM Kit or the BioBricks from the Registry. We hope that this information will be valuable also for any researcher working in the field of We also made a list of recommendations to improve the process of importation of biological material into our country, which we voiced to our local Congresswoman: 1. Easy on-line guides available to avoid hiring an agent. 2. Every Custom Agent should have a minimal understanding of scientific language. 3. Committee to review current laws. 4. New tariff classification for biological products. 5. Simple declaration forms based on biohazard levels. 6. Reduction of taxes on biological material for research. 7. Acceleration of the overall process. development of biosensors that work for many types of compounds by using a different family of promoters (which can be achieved via mutagenesis just like the 2009 British Columbia Team did) or that have a different set of reporter molecules easier to detect (like the BioBricks developed by 2009 Cambridge Team). Cambridge’s 2007 project which consisted of many sub projects. The one that we were most interested in was the PoPS amplifier or sensitivity tuner that is of great Mor and C are proteins encoded in the genome of Mu bacteriophage, and they have similar characteristics, like size, sequence and secondary structures and domains. The Proteins References Objectives Project idea: intelligent biosensor The first family of activators used was the Mor-Pm family. Pm promoter is the middle promoter of the Mu bacteriophage, where it initiates the transcription of C protein. The second family had four promoters: Plys, Pi, Pp and Pmom. We chose Pmom because it is the most characterized. It is activated by the C protein. The final bio-bricks were: Pm (BBa_K427003), Pmom (BBa_K427004) The Promoters These devices were designed with two purposes in mind: increase the PoPS output of the pBad promoter (any promoter would work) and change its almost linear production into an almost Boolean one. These parts have the following structure: activator protein, transcriptional stop and The Sensitivity Tuners We characterized both sensitivity tuners using Hill equations. In order to do this, we had to build another two parts which were only used to characterize the sensitivity tuners: BBa_K427007 for the MuC sensitivity tuner and BBa_K427008 for the MuMor sensitivity tuner; such parts consisted of a bidirectional terminator (BBa_B0014), PBad Weak (BBa_K206001), the sensitivity tuner and GFP reporter (BBa_E0840). Graphs for the characterization can be seen in Figures 6 and 7. Characterization Pmom MuC MuMor Pm To develop a genetic frame, compatible with the BioBrick standard, for the creation of a concentration-sensitive bacterial sensor. In the process we also developed and characterized BioBricks for two new families of phage Biosensors are intact, microbial organisms capable of producing a measurable signal when there is a physical or chemical agent present in their environment. Fluorescent proteins were chosen to be the measurable signals since they offer the There are several advantages to using a bacterial bioreporter instead of a traditional physical or chemical sensor, for example, bacteria can offer the same specificity and sensitivity that traditional sensors offer, but they are much more portable and grow in Further research and development in the areas of genetic engineering and synthetic biology have resulted in many more applications such as detection of contaminants (Willardson, et. Al., 1998) and sugar and amino acid availability in soils (Jaeger, et. Even though the reporters have gotten more sophisticated and sensitive, we realized that there isn’t much mention of a single bacterial bioreporter capable of detecting different concentrations of a substance and reacting differently depending on the concentration. Therefore, we decided to pave way for the Figure 4: MuC sensitivity tuner (BBa_K427005) Figure 5: MuMor sensitivity tuner (BBa_K427006) 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. 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. friends and teachers. synthetic biology in Mexico. also delays the progress of science in our country. involving enzymatic chain reactions can be improved. Reporters. seen in Figure 2. indicated in Figure 3. phage promoter. The final BioBricks are shown in Figures 4 and 5. utility when making whole cell bacterial reporters. because they were both working as expected. activators. advantage of being read on a real-time basis. al., 1999). inexpensive media. creation of a concentration-sensitive bacterial sensor. to come up with novel implementations of this system. The development of this project wouldn’t have been possible without the backing of our university and the guidance of our instructors: Dr. Mario Moisés Álvarez and M. Sc. Sergio García Echauri. We also appreciate the support given to us by our sponsors, and the people that surrounded us during our work, specially our families, that we could use. We found two projects that were of interest to us: compound and reacting differently depending on the concentration. The final bio-bricks were: Mor (BBa_K427002) and C (BBa_K427001). iGEM TEC DE MONTERREY 250 pb 1000 pb 2500 pb 5000 pb 4300 pb 9500 pb 9400 pb 1600 pb 10000 pb 500 pb 300 pb 900 pb GEM Listen to them!
Transcript of Development of a genetic frame for the creation of...
Development of a genetic frame for the creation of concentration-sensitive bacterial sensors
Introduction
The Team
Previous iGEM works
Genetic frame
PoPS Amplifiers
Future Research
Human Practices
Acknowledgments
Wiki Wiki >10 000 pb
Argüelles, Campos, Cevallos, Contreras, Gálvez, Huerta, Núñez, Ostos, Reynoso, Siller
Our proposal incorporated parts from these two previous iGEM projects as well as new BioBricks of our own design. The complete construction is presented in Figure 1.
GFP YFP RFP (Cherry)
pBAD Strong
MuMor MuC
MuCMuMor ΦR73δ
ΦR73δ
pBAD Weak Pm Po MompBAD Wild Type
pBAD Wild Type pBAD Weak
Figure 1: Complete genetic frame
Figure 2: Sensor with different concentrations of arabinose (increasing from top to bottom)
Figure 3: Reporter part of the construct
Figure 6: MuMor sensitivity tuner characterization
Figure 7: MuC sensitivity tuner characterization
Our genetic frame consists of two main parts: the sensing part and the reporting part. In this case, we decided to use the pBAD family of promoters to detect arabinose as our target compound and fluorescent proteins GFP, RFP and YFP as reporter molecules because these BioBricks are well characterized and this would enable us to dedicate our time and efforts into characterizing the PoPS amplifiers we designed for this project. However, our proposal was designed with flexibility of modification in mind, and because it is compatible with the BioBrick Standard, modifying this construction is not very difficult and will enable the production of a wide variety of whole-cell Bacterial
The sensing part of our construction was made incorporating the pBAD family of promoters developed by British Columbia’s 2009 team. We also included genes to codify for three different Bacteriophage activators that will serve as PoPS amplifiers. The promoters were arranged so that they “jammed” depending on the concentration of arabinose in the medium, as described by previous iGEM teams. Therefore, depending on the concentration, only one of the activating molecules is produced. This process can be
pBAD Strong
pBAD Wild Type pBAD Weak
pBAD WeakpBAD Wild Type
MuMor
MuMor
ΦR73δ MuC
pBAD Strong
pBAD Wild Type pBAD Weak
pBAD WeakpBAD Wild Type
JAMMED!
ΦR73δ
ΦR73δ
MuC
pBAD Strong
pBAD Wild Type pBAD Weak
pBAD WeakpBAD Wild Type
JAMMED
!
JAMMED
!MuC
MuC
Upon deciding that we wanted to make a concentration sensitive bioreporter we did some research of previous iGEM teams’ projects to see if there were any BioBricks
British Columbia’s 2009 project had an objective similar to ours. They incorporated three parts into their genetic construction that consisted of a sensor, a “lock and key” mechanism to control the expression of certain genes, and a jammer to inhibit translation. We were interested mainly in the sensing part and the jammer
We decided to build upon both of these projects in order to contribute to the task of making better and more flexible genetic constructions for whole cell bacterial reporters. We decided to create a new mechanism for the development of “intelligent biosensors”, capable of detecting different concentrations of a target
Depending on the concentration detected, only one activating molecule will be produced. Then, the reporting part of our construction produces one Fluorescent Protein depending on the activator that resulted from the sensing part. The mechanism for this is simple; each activator corresponds to one promoter as
GFP YFP RFP (Cherry)
MuCMuMor ΦR73δ
Pm Po Mom
To fully validate that our intelligent biosensor works as expected, we still need to carry out more experimentation to make sure that only one of the reporter molecules is expressed. We’ve already proven that our PoPS amplifiers work as anticipated; but we haven’t been able to do the same for the the genetic frame, so we will do further testing of the whole cell reporter. Once we have evidence that it does work properly, modifications to the frame should prove useful for the
We are so interested in the further development of whole cell intelligent biosensors because we believe that such organisms will be a major breakthrough in several areas: our first approaches were environmental purposes, since detection and approximate quantification of a specific pollutant, like the troublesome benzene, can be easily achieved. In the medical field we could create alternatives to expensive tests and even devices that detect the concentration of a compound, say glucose, and release a response, glucagon or insulin in this example, accordingly to the need. Finally, a very ambitious approach would be to develop a biosensor that detects the concentration of a given substance or substances and reacts by producing molecules that interact with such target. This way, bioprocesses
In summary, we know that the potential of these biosensors is enormous and the applications are endless. They surely represent one of those cases in biology where imagination is the only limit and that’s why we keep on working to prove that the genetic frame works as it is supposed to and we also encourage future iGEM Teams
One of the greatest complications that we had to deal with during the summer was the Mexican Customs Department. We were surprised to discover that our government doesn’t have a defined protocol for the importation of biological material, and even if our packages were correctly labeled, they were delayed or permanently detained. On speaking to other researchers in our country, we learned that we were not alone on this difficulty. Scientists with enough money pay specialized agencies to arrange the importation of their biological material, which may cost more than the actual material to be imported, while researchers without an ample budget simply rely on luck and hope that their package will go unnoticed if undeclared. Clearly, this complication not only slowed down our iGEM project; it
Synthetic biology is an emerging science in our country. We know that it is only a short matter of time before more Mexican teams participate in future iGEM competitions, and we believe that we can ease their experience by sharing our own. As part of our iGEM Human Practices collaboration, we created an Importation Manual, which is a step-by-step guide to receiving donations of synthetic DNA – such as the iGEM Kit or the BioBricks from the Registry. We hope that this information will be valuable also for any researcher working in the field of
We also made a list of recommendations to improve the process of importation of biological material into our country, which we voiced to our local Congresswoman: 1. Easy on-line guides available to avoid hiring an agent. 2. Every Custom Agent should have a minimal understanding of scientific language. 3. Committee to review current laws. 4. New tariff classification for biological products. 5. Simple declaration forms based on biohazard levels. 6. Reduction of taxes on biological material for research. 7. Acceleration of the overall process.
development of biosensors that work for many types of compounds by using a different family of promoters (which can be achieved via mutagenesis just like the 2009 British Columbia Team did) or that have a different set of reporter molecules easier to detect (like the BioBricks developed by 2009 Cambridge Team).
Cambridge’s 2007 project which consisted of many sub projects. The one that we were most interested in was the PoPS amplifier or sensitivity tuner that is of great
Mor and C are proteins encoded in the genome of Mu bacteriophage, and they have similar characteristics, like size, sequence and secondary structures and domains.
The ProteinsReferences
Objectives
Project idea: intelligent biosensor
The first family of activators used was the Mor-Pm family. Pm promoter is the middle promoter of the Mu bacteriophage, where it initiates the transcription of C protein. The second family had four promoters: Plys, Pi, Pp and Pmom. We chose Pmom because it is the most characterized. It is activated by the C protein. The final bio-bricks were: Pm (BBa_K427003), Pmom (BBa_K427004)
The Promoters
These devices were designed with two purposes in mind: increase the PoPS output of the pBad promoter (any promoter would work) and change its almost linear production into an almost Boolean one. These parts have the following structure: activator protein, transcriptional stop and
The Sensitivity Tuners
We characterized both sensitivity tuners using Hill equations. In order to do this, we had to build another two parts which were only used to characterize the sensitivity tuners: BBa_K427007 for the MuC sensitivity tuner and BBa_K427008 for the MuMor sensitivity tuner; such parts consisted of a bidirectional terminator (BBa_B0014), PBad Weak (BBa_K206001), the sensitivity tuner and GFP reporter (BBa_E0840). Graphs for the characterization can be seen in Figures 6 and 7.
Characterization
Pmom
MuC MuMor
Pm
To develop a genetic frame, compatible with the BioBrick standard, for the creation of a concentration-sensitive bacterial sensor. In the process we also developed and characterized BioBricks for two new families of phage
Biosensors are intact, microbial organisms capable of producing a measurable signal when there is a physical or chemical agent present in their environment. Fluorescent proteins were chosen to be the measurable signals since they offer the
There are several advantages to using a bacterial bioreporter instead of a traditional physical or chemical sensor, for example, bacteria can offer the same specificity and sensitivity that traditional sensors offer, but they are much more portable and grow in
Further research and development in the areas of genetic engineering and synthetic biology have resulted in many more applications such as detection of contaminants (Willardson, et. Al., 1998) and sugar and amino acid availability in soils (Jaeger, et.
Even though the reporters have gotten more sophisticated and sensitive, we realized that there isn’t much mention of a single bacterial bioreporter capable of detecting different concentrations of a substance and reacting differently depending on the concentration. Therefore, we decided to pave way for the
Figure 4: MuC sensitivity tuner (BBa_K427005) Figure 5: MuMor sensitivity tuner (BBa_K427006)
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. 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.
friends and teachers.
synthetic biology in Mexico.
also delays the progress of science in our country.
involving enzymatic chain reactions can be improved. Reporters.
seen in Figure 2.
indicated in Figure 3.
phage promoter. The final BioBricks are shown in Figures 4 and 5.
utility when making whole cell bacterial reporters.
because they were both working as expected.
activators.
advantage of being read on a real-time basis.
al., 1999).
inexpensive media.
creation of a concentration-sensitive bacterial sensor.
to come up with novel implementations of this system.
The development of this project wouldn’t have been possible without the backing of our university and the guidance of our instructors: Dr. Mario Moisés Álvarez and M. Sc. Sergio García Echauri. We also appreciate the support given to us by our sponsors, and the people that surrounded us during our work, specially our families,
that we could use. We found two projects that were of interest to us:
compound and reacting differently depending on the concentration.
The final bio-bricks were: Mor (BBa_K427002) and C (BBa_K427001).
iGEMTEC DE MONTERREY
250 pb
1000 pb
2500 pb
5000 pb
4300 pb
9500 pb9400 pb
1600 pb
10000 pb
500 pb
300 pb
900 pb
G EM
Listen to
them!
