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Team:Edinburgh/biology(nitritenitratesensing)
From 2009.igem.org
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Biology - Nitrite/Nitrate-Sensing Pathway
Personal note
Before I started working on iGEM, I had never heard of “Synthetic Biology”. I understood that we were going to make genetically modified organisms but did not see how the principles of engineering can be applicable. It is my privilege to be part of the iGEM experience, to witness the shift of biology from discovery science into applied science. iGEM has inspired me to continue working in the field of synthetic biology- my final year project will involve more BioBricking. I am looking forward to the day when BioBricking and assembly of genetic networks will be as easy as building a computer in both prokaryotic and eukaryotic cells - think artificial tissue interfaces!
Evangeline
Before I started working on iGEM, I had never heard of “Synthetic Biology”. I understood that we were going to make genetically modified organisms but did not see how the principles of engineering can be applicable. It is my privilege to be part of the iGEM experience, to witness the shift of biology from discovery science into applied science. iGEM has inspired me to continue working in the field of synthetic biology- my final year project will involve more BioBricking. I am looking forward to the day when BioBricking and assembly of genetic networks will be as easy as building a computer in both prokaryotic and eukaryotic cells - think artificial tissue interfaces!
Evangeline
TNT filled landmines often leak when it contact with the soil (Jenkins et al., 2001). The chemicals leaking from landmines include 1,3-DNB, 2,4-DNT, and 2,4,6-TNT (Jenkins et al., 2001). Subsequently, these chemicals are degraded to nitrites by soil bacteria (French et al., 1998). As such, we expect a radial diffusion gradients of nitrites around a focal point (the landmine). The size of this radius would depend on both the soil structure (e.g. water content, grain size etc) and the age of the landmine.
Our system uses a promoter that is sensitive to both nitrites and nitrates. Endogenously, this promoter controls the expression of the E. coli yeaR-yoaG operon (Lin et al., 2007). We shall henceforth refer to it as PyeaR (BBa_K216005). Nitrites and nitrates bind to the NsrR repressor to relief repression and activate gene transcription (Figure 1). To learn more about PyeaR’s sensitivity to nitrites/nitrates via our characterization results, click here.
Figure 1 a) PyeaR repression b) Repression relieved by nitrites. Transcription activated.
Figure 2 PETN reduces TNT to nitrite. Along with nitrite in the soil, it binds to the NsrR repressor to activate gene transcription. LuxAB.GFP is produced and blue-green light is emitted.
The reason for creating this fusion protein is founded upon an observation by Miyawaki that led us to believe that fusing the GFP to luciferase will increase the intensity of light produced. Furthermore, the emission wavelength from the fusion protein will excite enhanced fluorescent protein (BBa_ E0430) when it is produced in response to TNT detection.We attempted to clone the genes encoding for Photobacterium phosphoreum luciferase several times, unfortunately this remained unsuccessful. As such, we decided to clone the luciferase-producing genes from Xenorhabdus luminescens (BBa_K216008). We confirmed that this part is functional.
Another essential component of the nitrate/nitrate-sensing pathway is the enzymes that will produce aldehyde, the substrate for luciferase. These were biobricked from Xenorhabdus luminescens (Bba_xxxx). The corresponding genes of P. phosphoreum were not used because they are expressed at very low quantities in E. coli. The reason for this phenomenon is not clear as yet but it could be due to the presence of repressors in E. coli or condon usage bias (by P. phosphoreum). (Lee et al., 2000 and Miyamoto et al., 1987).
After consulting our modellers, we decided that the most efficient system would have the genes coding for the aldehyde-producing enzymes under the control of a constitutive promoter. This will ensure aldehyde-on-demand. The promoter chosen was BBa_J23105.
In summary, the presence of nitrites (and nitrates) activates PyeaR, activating transcription of the luciferase-GFP fusion protein resulting in the emission of blue-green light. How intense will the light be? Check out the following .
Our system uses a promoter that is sensitive to both nitrites and nitrates. Endogenously, this promoter controls the expression of the E. coli yeaR-yoaG operon (Lin et al., 2007). We shall henceforth refer to it as PyeaR (BBa_K216005). Nitrites and nitrates bind to the NsrR repressor to relief repression and activate gene transcription (Figure 1). To learn more about PyeaR’s sensitivity to nitrites/nitrates via our characterization results, click here.
It is also noteworthy that the nitrite feeding into the nitrite-detecting pathway comes from two sources. First, nitrite that has been degraded by soil bacteria can enter the cell. Second, our E. coli features a nitroreductase (PETN reductase) that degrades TNT to nitrites within the cell (Figure 2).
In our system, PyeaR will control the expression of a fusion protein that combines Photobacterium phosphoreum luciferase (Accession #: AY341063, BBa_XXXX, hypothetical) allowing the bacteria to emit blue-green light in response to nitrates and/or nitrates in the soil.
In our system, PyeaR will control the expression of a fusion protein that combines Photobacterium phosphoreum luciferase (Accession #: AY341063, BBa_XXXX, hypothetical) allowing the bacteria to emit blue-green light in response to nitrates and/or nitrates in the soil.
The reason for creating this fusion protein is founded upon an observation by Miyawaki that led us to believe that fusing the GFP to luciferase will increase the intensity of light produced. Furthermore, the emission wavelength from the fusion protein will excite enhanced fluorescent protein (BBa_ E0430) when it is produced in response to TNT detection.We attempted to clone the genes encoding for Photobacterium phosphoreum luciferase several times, unfortunately this remained unsuccessful. As such, we decided to clone the luciferase-producing genes from Xenorhabdus luminescens (BBa_K216008). We confirmed that this part is functional.
Another essential component of the nitrate/nitrate-sensing pathway is the enzymes that will produce aldehyde, the substrate for luciferase. These were biobricked from Xenorhabdus luminescens (Bba_xxxx). The corresponding genes of P. phosphoreum were not used because they are expressed at very low quantities in E. coli. The reason for this phenomenon is not clear as yet but it could be due to the presence of repressors in E. coli or condon usage bias (by P. phosphoreum). (Lee et al., 2000 and Miyamoto et al., 1987).
After consulting our modellers, we decided that the most efficient system would have the genes coding for the aldehyde-producing enzymes under the control of a constitutive promoter. This will ensure aldehyde-on-demand. The promoter chosen was BBa_J23105.
In summary, the presence of nitrites (and nitrates) activates PyeaR, activating transcription of the luciferase-GFP fusion protein resulting in the emission of blue-green light. How intense will the light be? Check out the following .
Edinburgh University iGEM Team 2009
