Difference between revisions of "Part:BBa K2660010"

Latest revision as of 02:32, 18 October 2018

glucose-responsive regulatory circuit

A complete glucose-responsive regulatory circuit (pGRS) for gram-positive bacteria. This device was built in Bacillus subtilis and tested with glucose, fructose, and lactose. It activates the gene expression in presence of glucose or fructose and deactivates in lactose.

Experiments
We grew B. subtilis carrying plasmid pGRS in LB media supplemented with 2% fructose and lactose overnight. The cultures were centrifuged, the cell pellet was resuspended in NaCl 0,9%, and we measured the absorbance at 600 nm and fluorescence of mCherry. Figure 2 shows results for the positive control (B. subtilis producing mCherry constitutively) and the test strain (B. subtilis carrying the synthetic circuit). B. subtilis 168 was used as negative control and its intrinsic cell fluorescence was considered as blank.

Figure 1. Relative fluorescence of positive control and test strain in NaCl 0,9%. The test strain exhibits strong fluorescence at the same intensity as the positive control when grew in fructose and no fluorescence when grew in lactose, confirming that fructose activates the gene expression in our system and lactose deactivates it. The experiment was made in duplicate. (*): error bars not shown due experimental problems in one of the replicates. pGRS: Plasmid Glucose-Responsive System.

Our Glucose Responsive System works as expected. Gene expression is ON when glucose/fructose is present, and it is completely OFF in the absence of it. This is a very important result to show the specificity of our circuit and to demonstrate that we accomplished our goal of avoiding leakages with this design.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 We grew B. subtilis carrying plasmid pGRS in LB media supplemented with 2% fructose and lactose overnight. The cultures were centrifuged, the cell pellet was resuspended in NaCl 0,9%, and we measured the absorbance at 600 nm and fluorescence of mCherry. Figure 2 shows results for the positive control (B. subtilis producing mCherry constitutively) and the test strain (B. subtilis carrying the synthetic circuit). B. subtilis 168 was used as negative control and its intrinsic cell fluorescence was considered as blank.
-[[File:T--Unesp Brazil--graficonathan.png | alt="Figure 1. Relative fluorescence of positive control and test strain in NaCl 0,9%. The test strain exhibits strong fluorescence at the same intensity as the positive control when grew in fructose and no fluorescence when grew in lactose, confirming that fructose activates the gene expression in our system and lactose deactivates it. The experiment was made in duplicate. (*): error bars not shown due experimental problems in one of the replicates. pGRS: Plasmid Glucose-Responsive System."]]
+[[File:T--Unesp Brazil--graficonathan.png]]
+<br>
+Figure 1. Relative fluorescence of positive control and test strain in NaCl 0,9%. The test strain exhibits strong fluorescence at the same intensity as the positive control when grew in fructose and no fluorescence when grew in lactose, confirming that fructose activates the gene expression in our system and lactose deactivates it. The experiment was made in duplicate. (*): error bars not shown due experimental problems in one of the replicates. pGRS: Plasmid Glucose-Responsive System.
+<br><br>
 Our Glucose Responsive System works as expected. Gene expression is ON when glucose/fructose is present, and it is completely OFF in the absence of it. This is a very important result to show the specificity of our circuit and to demonstrate that we accomplished our goal of avoiding leakages with this design.