"
Team:UC Davis/Projects
From 2010.igem.org
(Difference between revisions)
Skatercomet (Talk | contribs) |
|||
| Line 44: | Line 44: | ||
var pHContent = "<br/><p class='header'>THE PROBLEM</p>Cellular machines are not isolated systems. They have an intimate relationship with their surroundings and must change with varying environmental conditions. To do this, they rely on sensory machinery to trigger internal responses based on external stimuli such as light, chemical concentrations, etc. Sensors have been engineered for the E. coli chassis but one stimulus has been neglected: pH.<br/><br/> Cells thrive in a limited pH range; the optimal range for E. coli being 6-7. If the pH is much different than this, the cell must take action to survive. This needs to be taken into account when designing cellular machines which are reactive to pH changes. A pH sensitive system from another organism would help keep the engineered response independent. This would allow the desired response to be separate from a native stress response. <br/><br/><div style='text-align:center'><img src='https://static.igem.org/mediawiki/2010/a/ac/Mes_photo.jpg'></div><br/><br/><p class='header'>APPROACH</p>In the native host, Agrobacterium tumefaciens, the pH sensing machinery is a two component system consisting of the ChvG and ChvI genes. ChvG is the membrane bound histidine kinase and ChvI is the chromosomal response regulatory gene. The pH sensing machinery is flawlessly intertwined in the vast number of other simultaneously occurring processes. Ideally, it could be transplanted into E. coli without affecting any other pathway, although the possibility of this happening is next to none. We do not know how the construct will behave in it's new environment, but we do expect some unwanted crosstalk caused by differing pH and phosphate levels within the new host.<br/><br/>Four promoters are likely to be activated by the ChvG/ChvI in A. tumefaciens: KatA, ImpA, ChvI, and AopB. We took these sequences and placed them upstream of RFP to measure the extent to which they are activated in E. coli. Another promoter, PhoA from the PhoB/PhoR two component system was also chosen to be tested with the ChvG/ChvI construct since both two-component systems are similar. We expect some activation of the PhoA promoter at high phosphate levels when the PhoB/PhoR system is deactivated.<br/><br/><p class='header'>CONSTRUCT</p><br/><br/><div style='text-align: center;'><img src='https://static.igem.org/mediawiki/2010/f/fd/Phsensorconstructs.jpg'></div><br/> The figure shows the construct for the ChvG/ChvI two component system activating 5 possible promoters.<br/><br/><p class='header'>RESULTS: KatA, ImpA, ChvI, AopB</p>Control tests of promoters KatA,Impa, ChvI, and AopB showed relatively low to no fluorescence over the background. Graphs are shown below.<br/><br/><div style='text-align:center'><img src='https://static.igem.org/mediawiki/2010/d/dc/ImpA_KatA_graph.JPG'></div><br/><br/>It is clear that there is virtually no activity of ImpA and KatA in the absence of ChvG/ChvI. This test was carried out at varying pH levels from 5.0-7.3 and at two different phosphate concentrations of 0.05mM and 5mM. All the data looks similar to this graph which means there is no activation of the promoters from anything native to E. coli.<br/><br/><div style='text-align:center'><img src='https://static.igem.org/mediawiki/2010/7/76/AopB_ChvI_graphs.JPG'></div><br/><br/>Similar curves were seen in tests of AopB and ChvI promoter constructs. There is substantially less fluorescence than the induced RFP. It may appear as if ChvI has some minimal fluorescence over background but this is merely because the tests were done on separate occasions where the gain played a part in differing the actual fluorescence. AopB and ChvI were also tested at pH levels from 5.0-7.3 and in phosphate concentrations of 0.05mM and 5mM.<br/><br/>In all tests, the induced RFP is part Bba_J04450 and was induced with 0.1-1mM IPTG in Xl1 blue cells.<br/><br/><p class='header'>RESULTS: PhoA</p><br/><br/><div style='text-align:center'><img src='https://static.igem.org/mediawiki/2010/b/b7/PhoA_graph.JPG'></div><br/><br/>We did see some fluorescence from the E. coli native promoter PhoA at low pH and low phosphate (0.05mM) which we were not expecting. We were expecting some crosstalk from the ChvG/ChvI two component system with the PhoB/PhoR system but weren't expecting the promoter construct alone to show any fluorescence. We are still looking for a possible explanation of why this is happening and have more tests planned using different reagents.<br/><br/><p class='header'>FUTURE TESTING</p>We have many more tests planned including testing the ChvG/ChvI construct with each promoter construct and further PhoA tests. We would like to find out why we see expression in the absence of the ChvG/ChvI system and hope to see and characterize expression for the other four promoters when coupled with the ChvG/ChvI system."; | var pHContent = "<br/><p class='header'>THE PROBLEM</p>Cellular machines are not isolated systems. They have an intimate relationship with their surroundings and must change with varying environmental conditions. To do this, they rely on sensory machinery to trigger internal responses based on external stimuli such as light, chemical concentrations, etc. Sensors have been engineered for the E. coli chassis but one stimulus has been neglected: pH.<br/><br/> Cells thrive in a limited pH range; the optimal range for E. coli being 6-7. If the pH is much different than this, the cell must take action to survive. This needs to be taken into account when designing cellular machines which are reactive to pH changes. A pH sensitive system from another organism would help keep the engineered response independent. This would allow the desired response to be separate from a native stress response. <br/><br/><div style='text-align:center'><img src='https://static.igem.org/mediawiki/2010/a/ac/Mes_photo.jpg'></div><br/><br/><p class='header'>APPROACH</p>In the native host, Agrobacterium tumefaciens, the pH sensing machinery is a two component system consisting of the ChvG and ChvI genes. ChvG is the membrane bound histidine kinase and ChvI is the chromosomal response regulatory gene. The pH sensing machinery is flawlessly intertwined in the vast number of other simultaneously occurring processes. Ideally, it could be transplanted into E. coli without affecting any other pathway, although the possibility of this happening is next to none. We do not know how the construct will behave in it's new environment, but we do expect some unwanted crosstalk caused by differing pH and phosphate levels within the new host.<br/><br/>Four promoters are likely to be activated by the ChvG/ChvI in A. tumefaciens: KatA, ImpA, ChvI, and AopB. We took these sequences and placed them upstream of RFP to measure the extent to which they are activated in E. coli. Another promoter, PhoA from the PhoB/PhoR two component system was also chosen to be tested with the ChvG/ChvI construct since both two-component systems are similar. We expect some activation of the PhoA promoter at high phosphate levels when the PhoB/PhoR system is deactivated.<br/><br/><p class='header'>CONSTRUCT</p><br/><br/><div style='text-align: center;'><img src='https://static.igem.org/mediawiki/2010/f/fd/Phsensorconstructs.jpg'></div><br/> The figure shows the construct for the ChvG/ChvI two component system activating 5 possible promoters.<br/><br/><p class='header'>RESULTS: KatA, ImpA, ChvI, AopB</p>Control tests of promoters KatA,Impa, ChvI, and AopB showed relatively low to no fluorescence over the background. Graphs are shown below.<br/><br/><div style='text-align:center'><img src='https://static.igem.org/mediawiki/2010/d/dc/ImpA_KatA_graph.JPG'></div><br/><br/>It is clear that there is virtually no activity of ImpA and KatA in the absence of ChvG/ChvI. This test was carried out at varying pH levels from 5.0-7.3 and at two different phosphate concentrations of 0.05mM and 5mM. All the data looks similar to this graph which means there is no activation of the promoters from anything native to E. coli.<br/><br/><div style='text-align:center'><img src='https://static.igem.org/mediawiki/2010/7/76/AopB_ChvI_graphs.JPG'></div><br/><br/>Similar curves were seen in tests of AopB and ChvI promoter constructs. There is substantially less fluorescence than the induced RFP. It may appear as if ChvI has some minimal fluorescence over background but this is merely because the tests were done on separate occasions where the gain played a part in differing the actual fluorescence. AopB and ChvI were also tested at pH levels from 5.0-7.3 and in phosphate concentrations of 0.05mM and 5mM.<br/><br/>In all tests, the induced RFP is part Bba_J04450 and was induced with 0.1-1mM IPTG in Xl1 blue cells.<br/><br/><p class='header'>RESULTS: PhoA</p><br/><br/><div style='text-align:center'><img src='https://static.igem.org/mediawiki/2010/b/b7/PhoA_graph.JPG'></div><br/><br/>We did see some fluorescence from the E. coli native promoter PhoA at low pH and low phosphate (0.05mM) which we were not expecting. We were expecting some crosstalk from the ChvG/ChvI two component system with the PhoB/PhoR system but weren't expecting the promoter construct alone to show any fluorescence. We are still looking for a possible explanation of why this is happening and have more tests planned using different reagents.<br/><br/><p class='header'>FUTURE TESTING</p>We have many more tests planned including testing the ChvG/ChvI construct with each promoter construct and further PhoA tests. We would like to find out why we see expression in the absence of the ChvG/ChvI system and hope to see and characterize expression for the other four promoters when coupled with the ChvG/ChvI system."; | ||
| - | var spatialContent = "<br/><p class='header'>MOTIVATION</p>Patterns are everywhere in biology. Some, like zebra stripes, are easy to see, while other patterns like those that appear during animal development may be more subtle. Underneath the expression of these patterns are complex genetic networks that interpret specific cues from the environment and use this data to direct cells, or even populations of cells, to self organize and act.<br/><p class='header'>THE PROJECT</p>Given the importance of pattern generation in biology, we wanted to see if we could construct a synthetic circuit that would allow us to generate patterns in a community of inter-communicating cells in response to a simple stimuli; in our case, this stimuli would be light.<br/><br/>In order to mimic the process by which groups of cells can communicate to ultimately form a pattern, we will be using a lawn of E. Coli cells as our multicellular model system. These cells would be designed to communicate with each other through quorum sensing, and based on the small signaling molecules that each cell \"patch\" produces, the subsequent cell patch will know whether to activate or remain inactive.<br/><br/>In our system, the activated cells would produce colored pigment in order to indicate that they have been activated, and the cells that remain inactive will produce no color. In this way, the lawn of E. Coli cells would produce an oscillatory pattern of active and inactive \"bands\" of color and no color.<br/><br/><p class='header'>THE CONSTRUCT</p><img src='https://static.igem.org/mediawiki/2010/a/a8/IgemConstruct.jpg' height='600px' width='675px'><br/><br/><p class='header'>HOW IT WORKS: LET THERE BE LIGHT</p>The two main components of this system are two quorum sensing constructs that detect the products Lux and Las; the Lux being the signal for a cell to turn off, and the Las being the signal for a cell to turn on.<br/><br/>The following flash animation depicts the initiation of the system; as light hits the initiating construct's light promoter, Lambda activator is produced which activates the \"On Promoter\" which promotes the production of color (the cell is now on), additional Lambda activator and Lux. However, in the presence of Lambda, the Lux activator is unable to bind to the Lux Promoter, thereby keeping the cell on. The final thing to notice about the flash is that some of the Lux activator will diffuse out of this cell into adjacent cells, where the next step of the cycle will take place.<br/><br/><object width='690' height='600'><param name='movie' value='https://static.igem.org/mediawiki/2010/6/60/InitiatorUCD.swf'><embed src='https://static.igem.org/mediawiki/2010/6/60/InitiatorUCD.swf' width='690' height='600'></embed></object><br/><br/><p class='header'>HOW IT WORKS: HOW TO TURN CELLS OFF (besides telling bad jokes...)</p>The next step in the cycle is to turn off adjacent cells with the diffused Lux activator. Upon entering a new cell, the Lux will activate the Lux Promoter which triggers the production of Delta activator, 434 repressor and TetR repressor. The Delta repressor is in charge of stopping Las activator (the on signal) from activating the Las Promoter which would otherwise ultimately turn the cell on via the 'On Promoter' construct. The 434 repressor is yet another system that will prevent transcription of the 'On Promoter'. This is a necessary component of the system because there is a very high probability that some Lambda activator will also diffuse from the neighboring cells, which could activate the 'On Promoter' to a certain degree.<br/><br/>Since the Las activator is unable to bind to the Las Promoter, it will find its way out of this cell and diffuse to neighboring cells. The following flash animation depicts this secondary process.<br/><br/><object width='690' height='600'><param name='movie' value='https://static.igem.org/mediawiki/2010/d/d7/LuxAnimation.swf'><embed src='https://static.igem.org/mediawiki/2010/d/d7/LuxAnimation.swf' width='690' height='600'></embed></object><br/><br/><p class='header'>HOW IT WORKS: THE POWER OF LAS</p>The final step in our system's cycle is activating the next adjacent batch of cells to turn on and produce color. This can be achieved by taking advantage of the diffused Las activators produced by the cells in the previous section. The following flash depicts this process.<br/><br/><object width='690' height='600'><param name='movie' value='https://static.igem.org/mediawiki/2010/1/12/FinalflashUCD.swf'><embed src='https://static.igem.org/mediawiki/2010/1/12/FinalflashUCD.swf' width='690' height='600'></embed></object><br/>First, the leftover Las activators from the previous patch of cells makes their way into the new patch of cells. This activates the Las Promoter which promotes the transcription of Lambda activator. From this point on, it is as if the Las activators are acting in place of the light stimulus from the first step: the produced Lambda will activate the 'On Promoter', and the cycle repeats from the beginning. In this way, an oscillating pattern of colored/non-colored bands can be produced on a lawn of our modified cells.<br/><br/> For more details about the construct itself and its construction, please visit the <a href='/Team:UC_Davis/ | + | var spatialContent = "<br/><p class='header'>MOTIVATION</p>Patterns are everywhere in biology. Some, like zebra stripes, are easy to see, while other patterns like those that appear during animal development may be more subtle. Underneath the expression of these patterns are complex genetic networks that interpret specific cues from the environment and use this data to direct cells, or even populations of cells, to self organize and act.<br/><p class='header'>THE PROJECT</p>Given the importance of pattern generation in biology, we wanted to see if we could construct a synthetic circuit that would allow us to generate patterns in a community of inter-communicating cells in response to a simple stimuli; in our case, this stimuli would be light.<br/><br/>In order to mimic the process by which groups of cells can communicate to ultimately form a pattern, we will be using a lawn of E. Coli cells as our multicellular model system. These cells would be designed to communicate with each other through quorum sensing, and based on the small signaling molecules that each cell \"patch\" produces, the subsequent cell patch will know whether to activate or remain inactive.<br/><br/>In our system, the activated cells would produce colored pigment in order to indicate that they have been activated, and the cells that remain inactive will produce no color. In this way, the lawn of E. Coli cells would produce an oscillatory pattern of active and inactive \"bands\" of color and no color.<br/><br/><p class='header'>THE CONSTRUCT</p><img src='https://static.igem.org/mediawiki/2010/a/a8/IgemConstruct.jpg' height='600px' width='675px'><br/><br/><p class='header'>HOW IT WORKS: LET THERE BE LIGHT</p>The two main components of this system are two quorum sensing constructs that detect the products Lux and Las; the Lux being the signal for a cell to turn off, and the Las being the signal for a cell to turn on.<br/><br/>The following flash animation depicts the initiation of the system; as light hits the initiating construct's light promoter, Lambda activator is produced which activates the \"On Promoter\" which promotes the production of color (the cell is now on), additional Lambda activator and Lux. However, in the presence of Lambda, the Lux activator is unable to bind to the Lux Promoter, thereby keeping the cell on. The final thing to notice about the flash is that some of the Lux activator will diffuse out of this cell into adjacent cells, where the next step of the cycle will take place.<br/><br/><object width='690' height='600'><param name='movie' value='https://static.igem.org/mediawiki/2010/6/60/InitiatorUCD.swf'><embed src='https://static.igem.org/mediawiki/2010/6/60/InitiatorUCD.swf' width='690' height='600'></embed></object><br/><br/><p class='header'>HOW IT WORKS: HOW TO TURN CELLS OFF (besides telling bad jokes...)</p>The next step in the cycle is to turn off adjacent cells with the diffused Lux activator. Upon entering a new cell, the Lux will activate the Lux Promoter which triggers the production of Delta activator, 434 repressor and TetR repressor. The Delta repressor is in charge of stopping Las activator (the on signal) from activating the Las Promoter which would otherwise ultimately turn the cell on via the 'On Promoter' construct. The 434 repressor is yet another system that will prevent transcription of the 'On Promoter'. This is a necessary component of the system because there is a very high probability that some Lambda activator will also diffuse from the neighboring cells, which could activate the 'On Promoter' to a certain degree.<br/><br/>Since the Las activator is unable to bind to the Las Promoter, it will find its way out of this cell and diffuse to neighboring cells. The following flash animation depicts this secondary process.<br/><br/><object width='690' height='600'><param name='movie' value='https://static.igem.org/mediawiki/2010/d/d7/LuxAnimation.swf'><embed src='https://static.igem.org/mediawiki/2010/d/d7/LuxAnimation.swf' width='690' height='600'></embed></object><br/><br/><p class='header'>HOW IT WORKS: THE POWER OF LAS</p>The final step in our system's cycle is activating the next adjacent batch of cells to turn on and produce color. This can be achieved by taking advantage of the diffused Las activators produced by the cells in the previous section. The following flash depicts this process.<br/><br/><object width='690' height='600'><param name='movie' value='https://static.igem.org/mediawiki/2010/1/12/FinalflashUCD.swf'><embed src='https://static.igem.org/mediawiki/2010/1/12/FinalflashUCD.swf' width='690' height='600'></embed></object><br/>First, the leftover Las activators from the previous patch of cells makes their way into the new patch of cells. This activates the Las Promoter which promotes the transcription of Lambda activator. From this point on, it is as if the Las activators are acting in place of the light stimulus from the first step: the produced Lambda will activate the 'On Promoter', and the cycle repeats from the beginning. In this way, an oscillating pattern of colored/non-colored bands can be produced on a lawn of our modified cells.<br/><br/> For more details about the construct itself and its construction, please visit the <a href='/Team:UC_Davis/Notebook'>Notebook</a> section."; |
switch(tab) | switch(tab) | ||
Revision as of 11:58, 27 October 2010
|
|
|
