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Team:Edinburgh/biology(results)
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Revision as of 21:22, 16 October 2009
Biology - Results
The OmpC promoter was characterised using procaine. Procaine acts as an antagonist to phosphorylated omp-R, the inherent activator of this promoter. The first thing to notice from the characterisation results, is 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 first test done on the YeaR promoter was the famous miller test. As you can see this test shows that pYeaR is more sensitive to Nitrates than to Nitrites, and the sensitivity increases with increased concentration. In order to get a more detailed representation to the response to nitrates, the main activator of this promoter, the Jason Kelly assay was performed.
Jason Kelly Assay
From the Jason Kelly Assay we got a higher resolution response curve to the presence of Nitrates, that will hopefully be useful for further work with pYeaR. As you see, as the concentration goes beyond 30 mM, the Fluorescence decreases, most likely due to poisoning of the cells with excess Nitrates.
Personal Note
The OmpC promoter was characterised using procaine. Procaine acts as an antagonist to phosphorylated omp-R, the inherent activator of this promoter. The first thing to notice from the characterisation results, is 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 first test done on the YeaR promoter was the famous miller test. As you can see this test shows that pYeaR is more sensitive to Nitrates than to Nitrites, and the sensitivity increases with increased concentration. In order to get a more detailed representation to the response to nitrates, the main activator of this promoter, the Jason Kelly assay was performed.
Jason Kelly Assay
From the Jason Kelly Assay we got a higher resolution response curve to the presence of Nitrates, that will hopefully be useful for further work with pYeaR. As you see, as the concentration goes beyond 30 mM, the Fluorescence decreases, most likely due to poisoning of the cells with excess Nitrates.
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
