Difference between revisions of "Part:BBa K3320006:Experience"
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<div>We quantified the dynamic range of our genetic circuit (fdhF promoter. Hypoxia-inducible promoter BBa_K387003), in response to the lactate and hypoxia and how important is the architecture of the promoter in the response. Therefore, we transformed our AND gate system into E. coli Nissle 1917 and inoculated into LB broth supplemented with lactate. We used syringes to simulate the hypoxic environment. Cultures were incubated at 37º C in shaker for 16 hours at 220 rpm. We measured GFP fluorescence on the Hidex Chameleon Microplate Reader. </div> | <div>We quantified the dynamic range of our genetic circuit (fdhF promoter. Hypoxia-inducible promoter BBa_K387003), in response to the lactate and hypoxia and how important is the architecture of the promoter in the response. Therefore, we transformed our AND gate system into E. coli Nissle 1917 and inoculated into LB broth supplemented with lactate. We used syringes to simulate the hypoxic environment. Cultures were incubated at 37º C in shaker for 16 hours at 220 rpm. We measured GFP fluorescence on the Hidex Chameleon Microplate Reader. </div> | ||
<div>In this experiment, we expected to activate the expression of GFP controlled by fdhF promoter at different lactate and oxygen concentrations. Data can be analyzed below. </div> | <div>In this experiment, we expected to activate the expression of GFP controlled by fdhF promoter at different lactate and oxygen concentrations. Data can be analyzed below. </div> | ||
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| + | <img src="https://2019.igem.org/wiki/images/7/70/T--Amazonas-Brazil--gfppucpsb1c3.png"width="600"/> | ||
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<div>Figure: GFP expression under control of our AND logic gate in different hypoxic and lactate conditions.</div> | <div>Figure: GFP expression under control of our AND logic gate in different hypoxic and lactate conditions.</div> | ||
Revision as of 03:52, 22 October 2019
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Applications of BBa_K3320006
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We quantified the dynamic range of our genetic circuit (fdhF promoter. Hypoxia-inducible promoter BBa_K387003), in response to the lactate and hypoxia and how important is the architecture of the promoter in the response. Therefore, we transformed our AND gate system into E. coli Nissle 1917 and inoculated into LB broth supplemented with lactate. We used syringes to simulate the hypoxic environment. Cultures were incubated at 37º C in shaker for 16 hours at 220 rpm. We measured GFP fluorescence on the Hidex Chameleon Microplate Reader.
In this experiment, we expected to activate the expression of GFP controlled by fdhF promoter at different lactate and oxygen concentrations. Data can be analyzed below.
Figure: GFP expression under control of our AND logic gate in different hypoxic and lactate conditions.
As we can see above, we were able to successfully characterize our AND logic gate system through relative expression of GFP. There is an increase in GFP expression correlated to higher concentration of lactate, as indicated in samples with 0,1 mM and 1mM of lactate, However, we reported that in the absence of lactate - the system is ON, which indicates a leaky regulation by the hypoxia promoter. This can be solve through engineering synthetic promoters to fine-tune the interaction between the promoter and its repressor.
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UNIQ502fb401cc881192-partinfo-00000001-QINU UNIQ502fb401cc881192-partinfo-00000002-QINU