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Team:Edinburgh/biology(results)
From 2009.igem.org
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Biology - Results
We used Jason Kelly's promoter characterization kit to characterize the ompC promoter using varying concentrations of procaine. The standard promoter activity from BBa_I20260 is represented by "positive control" on the graph. Procaine acts as an agonist to phosphorylated omp-R, the inherent activator of this promoter. Observe from these results that this promoter is very leaky. Our team has found a way to overcome this problem by using an EnvZ – E. coli strain which we have constructed ourselves. Using this strain as a chassis for our construct would decrease background activation of this promoter, and thus ensure more precise expression of EYFP and onr in response to the presence of TNT.
Miller Test
The classical Miller's Assay was used to characterize the yeaR promoter. This test revealed that PyeaR is more sensitive to nitrates as compared to nitrites. The sensitivity increases with increasing concentration. In order to get a comprehensive representation of the response to nitrates and nitrites, we tested this promoter using Jason Kelly's promoter characterization kit. The result is presented below.
PyeaR characterization with fluorescence using Jason Kelly's kit
PyeaR response to nitrates
Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrates. The most sensitive time period is between 5 ½ hours (320 minutes) 7 ½ hours (440 minutes) after induction. The sensitivity decreases with time, but induction is still at a high level after the peak activity of the promoter. Only two concentrations are shown (0 mM and 10 mM), as we have demonstrated that 10 mM is the sensitivity peak of this promoter (look at last graph).
PyeaR response to nitrites
Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrites. The fluorescence levels begin to build up only after about 8 hours; therefore other irrelevant results are omitted from this graph. The sensitivity curve in this case is less steep compared to the one produced due to the presence of nitrates. This indicates that the promoter responds differently to these two nitrogenous compounds. Again, only two concentrations are shown (0 mM and 10 mM), as sensitivity peaks at 10mM.
PyeaR response to nitrates and nitrites after overnight induction
Here we can compare the activity of this promoter to different concentrations of both nitrates and nitrates. We observed that the promoter is activated more readily by nitrates compared to nitrites. Nevertheless, the activation produced by nitrites remains significant. The peak activation concentration for both compounds is 10 mM, after which activation efficiency decreases.
Personal Note
One of the best things about iGEM is that you get to learn a lot from your peers. Biologists learn about modelling, engineers learn about biology...and we all have to learn a little HTML. The learning can be intensely stressful at times but also very satisfying.
Evangeline
What is even more important, we learn from each other not just the technical knowledge, but something that is more precious to me - life views. Learning from others life views, you can extend and adjust your personal life, make it bigger, smarter and more adaptive.
JD
One of the best things about iGEM is that you get to learn a lot from your peers. Biologists learn about modelling, engineers learn about biology...and we all have to learn a little HTML. The learning can be intensely stressful at times but also very satisfying.
Evangeline
What is even more important, we learn from each other not just the technical knowledge, but something that is more precious to me - life views. Learning from others life views, you can extend and adjust your personal life, make it bigger, smarter and more adaptive.
JD
We used Jason Kelly's promoter characterization kit to characterize the ompC promoter using varying concentrations of procaine. The standard promoter activity from BBa_I20260 is represented by "positive control" on the graph. Procaine acts as an agonist to phosphorylated omp-R, the inherent activator of this promoter. Observe from these results that this promoter is very leaky. Our team has found a way to overcome this problem by using an EnvZ – E. coli strain which we have constructed ourselves. Using this strain as a chassis for our construct would decrease background activation of this promoter, and thus ensure more precise expression of EYFP and onr in response to the presence of TNT.
Miller Test
The classical Miller's Assay was used to characterize the yeaR promoter. This test revealed that PyeaR is more sensitive to nitrates as compared to nitrites. The sensitivity increases with increasing concentration. In order to get a comprehensive representation of the response to nitrates and nitrites, we tested this promoter using Jason Kelly's promoter characterization kit. The result is presented below.
PyeaR characterization with fluorescence using Jason Kelly's kit
PyeaR response to nitrates
Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrates. The most sensitive time period is between 5 ½ hours (320 minutes) 7 ½ hours (440 minutes) after induction. The sensitivity decreases with time, but induction is still at a high level after the peak activity of the promoter. Only two concentrations are shown (0 mM and 10 mM), as we have demonstrated that 10 mM is the sensitivity peak of this promoter (look at last graph).
PyeaR response to nitrites
Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrites. The fluorescence levels begin to build up only after about 8 hours; therefore other irrelevant results are omitted from this graph. The sensitivity curve in this case is less steep compared to the one produced due to the presence of nitrates. This indicates that the promoter responds differently to these two nitrogenous compounds. Again, only two concentrations are shown (0 mM and 10 mM), as sensitivity peaks at 10mM.
PyeaR response to nitrates and nitrites after overnight induction
Here we can compare the activity of this promoter to different concentrations of both nitrates and nitrates. We observed that the promoter is activated more readily by nitrates compared to nitrites. Nevertheless, the activation produced by nitrites remains significant. The peak activation concentration for both compounds is 10 mM, after which activation efficiency decreases.
After 10 weeks, we have managed to make a significant number of basic BioBrick parts and a few composite BioBrick parts. However, the project is far from being practically perfect! Here are three plans we have installed!
Testing TNT receptors
First, we are determined to test the TNT receptors, TNT.R1 and TNT.R3. To accomplish this, we have inserted tnt.r1 downstream of the constitutive promoter BBa_J23105. We expect bacteria expressing tnt.r1 to move towards TNT as TNT.R1 has been derived from ribose binding protein that naturally induces chemotaxis towards ribose. Additionally, TNT binding to the receptor is going to be tested as part of an undergraduate research project (by team member Rachael). A variety of techniques will be employed, including spectroscopy to ensure that conformational change is induced when TNT binds. The signalling pathway from TNT.R1-Trz will also be tested.
Environmental aspect
For our system to be used in the environment, it is imperative that we include a self-destruct mechanism within the host. With such a mechanism in place, we can be confident that the bacteria will not live longer than required. This will alleviate fears of harm to the environment and undesirable bacteria gene transfer between our host and naturally occurring soil bacteria. Once our bacteria is release, the visual output can be recorded using aerial photography before the system self-destructs.
Field test
Finally, the system has to be tested in the field to ensure that it can accurately detect both TNT and nitrite found in the soil. This can be done by a number of methods including lab based soil experiments or in outside in restricted areas (once permission has been granted). It has been suggested that the light emitted would not be strong enough to be seen by the naked eye in the field. We propose two ways to overcome this problem. The first is a physical method where standard military night vision goggles can be used to intensify the signal. The biological solution to this is to construct a protein scaffold that tethers the luciferase.gfp fusion protein to lumazine protein and enhanced yellow protein so that the emission-excitation mechanism is efficient.
Testing TNT receptors
First, we are determined to test the TNT receptors, TNT.R1 and TNT.R3. To accomplish this, we have inserted tnt.r1 downstream of the constitutive promoter BBa_J23105. We expect bacteria expressing tnt.r1 to move towards TNT as TNT.R1 has been derived from ribose binding protein that naturally induces chemotaxis towards ribose. Additionally, TNT binding to the receptor is going to be tested as part of an undergraduate research project (by team member Rachael). A variety of techniques will be employed, including spectroscopy to ensure that conformational change is induced when TNT binds. The signalling pathway from TNT.R1-Trz will also be tested.
Environmental aspect
For our system to be used in the environment, it is imperative that we include a self-destruct mechanism within the host. With such a mechanism in place, we can be confident that the bacteria will not live longer than required. This will alleviate fears of harm to the environment and undesirable bacteria gene transfer between our host and naturally occurring soil bacteria. Once our bacteria is release, the visual output can be recorded using aerial photography before the system self-destructs.
Field test
Finally, the system has to be tested in the field to ensure that it can accurately detect both TNT and nitrite found in the soil. This can be done by a number of methods including lab based soil experiments or in outside in restricted areas (once permission has been granted). It has been suggested that the light emitted would not be strong enough to be seen by the naked eye in the field. We propose two ways to overcome this problem. The first is a physical method where standard military night vision goggles can be used to intensify the signal. The biological solution to this is to construct a protein scaffold that tethers the luciferase.gfp fusion protein to lumazine protein and enhanced yellow protein so that the emission-excitation mechanism is efficient.
Edinburgh University iGEM Team 2009
