Seeking advice from a knowledgable member concerning a whole-cell biosensor
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antomicblitz
Oct 5, 2015, 7:12:44 PM10/5/15
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Hello guys,
I had a research idea that I wanted to pursue, but I need some help refining my question and designing the experiment. I was inspired by a journal article published in Applied and Environmental Microbiology by a Stanford/Berkeley group describing the successful creation of a whole-cell uranium biosensor that fluoresces in the presence of micromolar concentrations of uranium (http://www.ncbi.nlm.nih.gov/pubmed/17905881). The coolest part was that the researchers managed to get the signal strong enough to be seen with the naked eye after 4 hours of exposure and with only a standard UV flashlight (shown in the figure I attached).
So now, my question is whether such a system could be used with a different promoter and the sensitivity of the system increased by incorporating a membrane-integrating protein to help localize the inducible promoter and/or the reporter construct to the periphery of the cell membrane. While searching through some protein databases, I found an interesting membrane integrating protein called Mistic that can apparently integrate fusion proteins into the membrane independent of the cell's translocon machinery, and can do so at relatively high concentrations, so I want to see if it could be used to make a better whole-cell biosensor. Is this idea worth pursuing? Or do you think there are better ways to create a whole-cell bacterial biosensor? So far some points that a friend of mine has brought up that will need to be addressed are:
1) What concentrations of protein do I need to make GFPuv visible to the naked eye.
2) What concentration of the molecules will I be able to detect with the promoter.
3.) How will the choice of the promoter affect expression? How will the system be optimized?
So, the basic idea is to create and test something like this, but with a lead-inducible promoter:
I would like to try to crowdfund the cost of this experiment, which I estimate will be around $4000-$6000 to pay for oligo synthesis and various reagents/supplies. Any advice from the community is much appreciated ^_^
Warmest Wishes,
Antonio
I had a research idea that I wanted to pursue, but I need some help refining my question and designing the experiment. I was inspired by a journal article published in Applied and Environmental Microbiology by a Stanford/Berkeley group describing the successful creation of a whole-cell uranium biosensor that fluoresces in the presence of micromolar concentrations of uranium (http://www.ncbi.nlm.nih.gov/pubmed/17905881). The coolest part was that the researchers managed to get the signal strong enough to be seen with the naked eye after 4 hours of exposure and with only a standard UV flashlight (shown in the figure I attached).
So now, my question is whether such a system could be used with a different promoter and the sensitivity of the system increased by incorporating a membrane-integrating protein to help localize the inducible promoter and/or the reporter construct to the periphery of the cell membrane. While searching through some protein databases, I found an interesting membrane integrating protein called Mistic that can apparently integrate fusion proteins into the membrane independent of the cell's translocon machinery, and can do so at relatively high concentrations, so I want to see if it could be used to make a better whole-cell biosensor. Is this idea worth pursuing? Or do you think there are better ways to create a whole-cell bacterial biosensor? So far some points that a friend of mine has brought up that will need to be addressed are:
1) What concentrations of protein do I need to make GFPuv visible to the naked eye.
2) What concentration of the molecules will I be able to detect with the promoter.
3.) How will the choice of the promoter affect expression? How will the system be optimized?
So, the basic idea is to create and test something like this, but with a lead-inducible promoter:
I would like to try to crowdfund the cost of this experiment, which I estimate will be around $4000-$6000 to pay for oligo synthesis and various reagents/supplies. Any advice from the community is much appreciated ^_^
Warmest Wishes,
Antonio
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