BME494 Sp2014 Tran: Difference between revisions
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'''Tetracycline Promoter'''<br> | '''Tetracycline Promoter'''<br> | ||
Biobrick: | Biobrick: [http://parts.igem.org/Part:BBa_R0040 BBa_ R0040]<br> | ||
This component will be the promoter used during the “off” state of the device. It has a constitutive transcription rate in order to ensure that the device will begin in the “off” state and will only express green fluorescence when the device is switched “on”. This promoter will also be repressed from the expression of tetracycline repressor, which is expressed in the “on” state of the device.<br><br> | This component will be the promoter used during the “off” state of the device. It has a constitutive transcription rate in order to ensure that the device will begin in the “off” state and will only express green fluorescence when the device is switched “on”. This promoter will also be repressed from the expression of tetracycline repressor, which is expressed in the “on” state of the device.<br><br> | ||
'''Lac Repressor'''<br> | '''Lac Repressor'''<br> | ||
Biobrick: | Biobrick: [http://parts.igem.org/Part:BBa_K292005 BBa_K292005]<br> | ||
This gene will be expressed when the tetracycline promoter, and will repress expression of the lac promoter utilized in the “on” state of the system. This component is essential to the bistability of the device since its expression prevents the expression of the “on” state promoter.<br><br> | This gene will be expressed when the tetracycline promoter, and will repress expression of the lac promoter utilized in the “on” state of the system. This component is essential to the bistability of the device since its expression prevents the expression of the “on” state promoter.<br><br> | ||
'''Copper Promoter'''<br> | '''Copper Promoter'''<br> | ||
Biobrick: | Biobrick: [http://parts.igem.org/Part:BBa_I760005 BBa_I760005]<br> | ||
This transcriptional unit is responsible for the expression of the “on” state genes. This promoter will be induced by excess extracellular copper concentration through the cusR / cusS cell signal pathway. In this signaling pathway, the transmembrane protein cusS reacts to excess extracellular copper concentration and phosphorylates cusR which is a transcription factor for this promoter. The promoter will then, theoretically, promote the transcription of tetracycline repressor and green fluorescence protein, indicating the “on” state. The sensitivity of this promoter is paramount to the success of the device. During testing of the device, the promoter must be sensitive to a certain concentration of copper, as related to the presence of malaria. If the promoter is unsuccessful, other copper pathways in the E. coli can be explored for different copper sensitive promoters.<br><br> | This transcriptional unit is responsible for the expression of the “on” state genes. This promoter will be induced by excess extracellular copper concentration through the cusR / cusS cell signal pathway. In this signaling pathway, the transmembrane protein cusS reacts to excess extracellular copper concentration and phosphorylates cusR which is a transcription factor for this promoter. The promoter will then, theoretically, promote the transcription of tetracycline repressor and green fluorescence protein, indicating the “on” state. The sensitivity of this promoter is paramount to the success of the device. During testing of the device, the promoter must be sensitive to a certain concentration of copper, as related to the presence of malaria. If the promoter is unsuccessful, other copper pathways in the E. coli can be explored for different copper sensitive promoters. In normal subjects, copper serum level was determined to be approximately 2.0 ppm, while in malarial patients, the copper concentration was apprximately 2.5 ppm (Baloch et al, 2011). Subsequently, the optimal promoter would have to be sensitive to concentrations of higher than 2.4 ppm. <br><br> | ||
'''Lac Promoter'''<br> | '''Lac Promoter'''<br> | ||
Biobrick: | Biobrick: [http://parts.igem.org/Part:BBa_R0010 BBa_R0010]<br> | ||
The Lac promoter is still used in this device in order to introduce a repressible element to the “on” state genes. The Lac repressor gene expressed through the tetracycline promoter in the “off” state pathway will repress this promoter, ensuring tetracycline repressor and green fluorescence protein are not expressed.<br><br> | The Lac promoter is still used in this device in order to introduce a repressible element to the “on” state genes. The Lac repressor gene expressed through the tetracycline promoter in the “off” state pathway will repress this promoter, ensuring tetracycline repressor and green fluorescence protein are not expressed.<br><br> | ||
'''Tetracycline Repressor'''<br> | |||
Biobrick: [http://parts.igem.org/Part:BBa_S03518 BBa_S03518]<br> | |||
The tetracycline repressor is used to maintain the off state of the device by repressing the tetracycline promoter.<br><br> | |||
'''Green Fluorescence Protein'''<br> | '''Green Fluorescence Protein'''<br> | ||
Biobrick: | Biobrick: [http://parts.igem.org/Part:BBa_K823039 BBa_K823039]<br> | ||
Green fluorescence protein is used as the reporter for this device. Because the gene for transcription of green fluorescence protein is placed downstream of the copper promoter, the green fluorescence will indicate the presence of excess copper and subsequently, the presence of malaria.<br><br> | Green fluorescence protein is used as the reporter for this device. Because the gene for transcription of green fluorescence protein is placed downstream of the copper promoter, the green fluorescence will indicate the presence of excess copper and subsequently, the presence of malaria.<br><br> | ||
'''AmpR Vector Backbone'''<br> | '''AmpR Vector Backbone'''<br> | ||
Biobrick: pSB1A3<br> | Biobrick: [http://parts.igem.org/Part:pSB1A3 pSB1A3]<br> | ||
This ampicillin resistant vector will be used to host the other device components. It surrounds the input area with terminators in order to prevent transcription outside of the input area.<br><br> | This ampicillin resistant vector will be used to host the other device components. It surrounds the input area with terminators in order to prevent transcription outside of the input area.<br><br> | ||
<!-- Show a network/ circuit diagram of your device. Include a paragraph to explain how it works (e.g., how to switch the system from on to off and vice versa, and what happens to each component as the system switches between states) --> | <!-- Show a network/ circuit diagram of your device. Include a paragraph to explain how it works (e.g., how to switch the system from on to off and vice versa, and what happens to each component as the system switches between states) --> | ||
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|- | |- | ||
! Lac Promoter | ! Lac Promoter | ||
| | | [http://parts.igem.org/Part:BBa_R0010 Bba_R0010] | ||
| 200 bp | | 200 bp | ||
|- | |- | ||
! Tet Promoter | ! Tet Promoter | ||
| Bba_ R0040 | | [http://parts.igem.org/Part:BBa_R0040 Bba_ R0040] | ||
| 54 bp | | 54 bp | ||
|- | |- | ||
! RBS + Tet Repressor | ! RBS + Tet Repressor | ||
| Bba_S03518 | | [http://parts.igem.org/Part:BBa_S03518 Bba_S03518] | ||
| 703 bp | | 703 bp | ||
|- | |- | ||
! Lac Repressor | ! Lac Repressor | ||
| Bba_K292005 | | [http://parts.igem.org/Part:BBa_K292005 Bba_K292005] | ||
| 1256 bp | | 1256 bp | ||
|- | |- | ||
! Kozak RBS | ! Kozak RBS | ||
| BBa_J176012 | | [http://parts.igem.org/Part:BBa_J176012 Bba_I760005] | ||
| 18 bp | | 18 bp | ||
|- | |- | ||
! Copper Promoter | ! Copper Promoter | ||
| BBa_I760005 | | [http://parts.igem.org/Part:BBa_I760005 Bba_I760005] | ||
| 16 bp | | 16 bp | ||
|- | |- | ||
! Green Fluorescence Protein | ! Green Fluorescence Protein | ||
| Bba_K823039 | | [http://parts.igem.org/Part:BBa_K823039 Bba_K823039] | ||
| 845 bp | | 845 bp | ||
|- | |- | ||
! AmpR Vector Backbone | ! AmpR Vector Backbone | ||
| pSB1A3 | | [http://parts.igem.org/Part:pSB1A3 pSB1A3] | ||
| 2155 bp | | 2155 bp | ||
|- | |- | ||
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<!-- Briefly describe a wet lab experiment you will need to perform to compare the behavior of your device to a mathematical model (based on a type of model that is similar to the Lac operon model). What inputs will you use? How will you measure output? What type of result will confirm that the device works as expected? You do not have to describe the experimental protocol, just the overall approach. --> | <!-- Briefly describe a wet lab experiment you will need to perform to compare the behavior of your device to a mathematical model (based on a type of model that is similar to the Lac operon model). What inputs will you use? How will you measure output? What type of result will confirm that the device works as expected? You do not have to describe the experimental protocol, just the overall approach. --> | ||
This device’s performance can be tested by exposing colonies with the device to different concentrations of copper. Flow cytometry and microscopy can then be used to retrieve values of fluorescence from the exposed colonies. If the device is successful, then copper concentrations similar to those found in malaria infected patients will cause the device to fluoresce green, while lower copper concentrations will have no effect on the cells and their appearance. | This device’s performance can be tested by exposing colonies with the device to different concentrations of copper. Flow cytometry and microscopy can then be used to retrieve values of fluorescence from the exposed colonies. If the device is successful, then copper concentrations similar to those found in malaria infected patients will cause the device to fluoresce green, while lower copper concentrations will have no effect on the cells and their appearance. Further tests would also have to be done to ensure minimal crosstalk with other copper sensitive pathways within E. coli. | ||
==Human Practices - Stakeholder Assessment== | ==Human Practices - Stakeholder Assessment== | ||
<br><br> | <br><br> | ||
Line 308: | Line 312: | ||
This group does not understand the scientific basis for the malarial biosensor but also does not support further research or production of it either. Media sensationalists often use synthetic biology to instill fear and create worry about the controllability of synthetic biological systems. Many of the stories told by sensational media have no bounds in reality. People who also have ethical or moral quandaries with biological interference would also fall into this group, as they disagree with tampering with the natural state of biological systems. <br><br> | This group does not understand the scientific basis for the malarial biosensor but also does not support further research or production of it either. Media sensationalists often use synthetic biology to instill fear and create worry about the controllability of synthetic biological systems. Many of the stories told by sensational media have no bounds in reality. People who also have ethical or moral quandaries with biological interference would also fall into this group, as they disagree with tampering with the natural state of biological systems. <br><br> | ||
[[Image:BME494_HaiTran_stakeholders.PNG|520px|center]] | |||
<!--Wait until Unit 3 to fill this in. Demonstrate that you have considered the societal aspects of your project. --> | <!--Wait until Unit 3 to fill this in. Demonstrate that you have considered the societal aspects of your project. --> | ||
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<div style="color: #808080; background-color: #ffffff; width: 600px; padding: 5px"> | <div style="color: #808080; background-color: #ffffff; width: 600px; padding: 5px"> | ||
[[Image: | [[Image:BME494 HaiTran face.PNG|thumb|noframe|130px|left|'''Your Name''']] | ||
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<br> | <br> | ||
== | ==References== | ||
1. "CDC Malaria". http://www.cdc.gov/malaria/index.html<br> | 1. "CDC Malaria". http://www.cdc.gov/malaria/index.html<br> | ||
2. "WHO | Malaria". http://www.who.int/mediacentre/factsheets/fs094/en/<br> | 2. "WHO | Malaria". http://www.who.int/mediacentre/factsheets/fs094/en/<br> | ||
3. "Haynes:TypeIIS Assembly". http://openwetware.org/wiki/Haynes:TypeIIS_Assembly<br> | 3. "Haynes:TypeIIS Assembly". http://openwetware.org/wiki/Haynes:TypeIIS_Assembly<br> | ||
4. Gardner T.S., Cantor C.Reverse, Collins J.J. 2000. Nature. Vol 409: 339-342 | 4. Gardner T.S., Cantor C.Reverse, Collins J.J. 2000. Nature. Vol 409: 339-342 | ||
5. | 5. Baloch S., Gachal G.S., Memon S.A., Baloch M., 2011. Sindh University Research Journal. Vol 43(2): 147-148<br><br><br> |
Latest revision as of 08:52, 23 April 2014
My Profile Dr. Haynes OpenWetWare Previous Course Wiki Editing Help
Background & Proposed ApplicationBACKGROUND
The malarial biosensor is modeled after the classical synthetic toggle switch developed in Escherichia coli, or E. Coli (Gardner et al 2000). The precedent synthetic system was developed with two different inducible states, an “on” and “off” state. The system was created using two promoter and repressor pairs. Both repressors are inhibited by a different inducer (aTc and IPTG). The promoters were placed upstream of the repressor gene of the opposite pair. This created a bistable system where only one promoter would be expressed at any one time, since the expression of a promoter repressed expression of the other promoter. To distinguish a cell between its on and off state, a green fluorescence protein transcription gene was placed downstream of the on state promoter.
Malaria is a disease caused by any one of five different species of plasmodium, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowles. The life cycle of these plasmodium consist three different phase. During one phase, the plasmodium reproduces within a female mosquito. When the mosquito takes a blood meal from a human, the saliva of the mosquito, abundant with the pathogen, transfers the disease into the new human host. From there, the parasite infects and multiplies inside of liver and blood cells. The World Health Organization estimated that malaria was responsible for approximately 207 million clinical episodes and more than 600,000 deaths. More than 90% of fatal cases of malaria originate in Africa. Malaria presents itself with fever, sweats, chills, and other symptoms commonly associated with a common cold. When discovered early, malaria has a low mortality rate and can be readily treated. Unfortunately, because malarial symptoms can be difficult to recognize, diagnosing malaria can often be an arduous task. Currently, the most widely and accepted standard for diagnosis consists of microsopy. Microscopy techniques consist of taking a drop of a patient’s blood and staining it with a dye that gives plasmodium a distinct appearance. The drop is then examined under microscope for the presence of these dyed plasmodium. Microscopy is the most reliable method of diagnosis but requires quality reagents, microscopes, and an experienced lab technician. Generally, microscopy requires travel to an apt-equipped facility with the proper equipment which is not always a possibility. Another widely used method of diagnoses is Rapid Diagnostic Tests (RDTs). RDTs test a patient's blood for the presence of malaria related antigens. The fallback to RDTs though are they still need to be improved for accuracy, often require the use of a clinician, and can be costly. The proposed device hopes to remedy the problem of diagnosing malaria in remote areas and rural communities which do not have access to advanced tools such as microscopes. If successful, the device will be placed into small kits which would be used to test a drop of the patient’s blood and results would be readable within the day. The kit would designed to be easy to use so rural communities without experienced clinicians could still have a preliminary means of diagnosing malaria.
Design of a New DeviceThe device will be designed as a diagnostic tool for malaria. Studies have shown that malaria often presents itself with a higher concentration of copper in the bloodstream (Iqbal, 2000). The proposed device will capitalize on this correlation and use copper as an input. Similar to Gardner’s classical device, will use two promoter-repressor pairs in order to establish a bistable system. The key difference between the proposed device and Gardner’s device is that the “on” state of the system in which green fluorescence is expressed will be induced by the presence of excess copper. Topological diagram of device. "On" state induced by presence of copper. "Off" state on constitutively and reinduced by aTc. Tetracycline Promoter Lac Repressor Copper Promoter Lac Promoter Tetracycline Repressor Green Fluorescence Protein AmpR Vector Backbone
Building the New DeviceThe malarial biosensor will be constructed with a series of Biobricks which have already been created and can be found in the iGEM registry. All of the components of the system will be assembled together in a specific order through use of the Type IIS Assembly process, which utilizes BsmBI enzymes and cleavage sites. The device consist of a lac promoter-repressor pair and a tetracycline promoter-repressor pair. The lac repressor gene will be placed downstream of the tetracycline promoter. Next to the tetracycline promoter will be a copper induced promoter followed by the lac promoter, tetracycline repressor, and green fluorescence protein genes, all of which will have a reversed transcriptionally to the lac repressor and tetracycline promoter genes. These genes will be placed into a a pSB1A3 vector, which carries ampicillin resistance and surrounds the input area of the gene with terminators to prevent transcription out of the desired area.
TYPE IIS ASSEMBLY PCR Digestion Ligation Reaction
Primers Reagents and Procedures
The reagents will be mixed and then thermal cycled as follows: For the digestion-ligation reaction, these reagents will be used:
These reagents will then be mixed and thermal cycled as follows: Testing the New DeviceLAC OPERON MODEL SIMULATION
Similarities:
The Lac operon model is useful because it relates to the Tet operon and can be used to infer how aTc affects the transcription rate of the tet promoter. Furthermore, the model can be used to better understand how the lac promoter and lac repressor genes will react with one another inside the biosensor system.
Differences:
Unfortunately, the lac operon model cannot be used to gleam exact tet promoter reactions to different concentrations of aTc. Furthermore, the Lac operon model does not explore the most critical component of the malarial biosensor, which is the effect of copper concentration on the copper promoter. This is a paramount parameter to explore since the reaction of the copper promoter is paramount to the viability of the device.
This device’s performance can be tested by exposing colonies with the device to different concentrations of copper. Flow cytometry and microscopy can then be used to retrieve values of fluorescence from the exposed colonies. If the device is successful, then copper concentrations similar to those found in malaria infected patients will cause the device to fluoresce green, while lower copper concentrations will have no effect on the cells and their appearance. Further tests would also have to be done to ensure minimal crosstalk with other copper sensitive pathways within E. coli. Human Practices - Stakeholder Assessment
DOES NOT SUPPORT BUT UNDERSTANDS SUPPORTS BUT DOES NOT UNDERSTAND DOES NOT SUPPORT NOR UNDERSTANDS
About the Designer
References1. "CDC Malaria". http://www.cdc.gov/malaria/index.html |