Difference between revisions of "Part:BBa K1893017"

Latest revision as of 03:28, 29 October 2016

Arabinose inducible GFP (pBAD+GFP)

This composite part is composed of a GFP reporter (BBa_E0040) downstream of a pBAD arabinose inducible promoter (BBa_K1893015). It was used to characterize the activation range of pBAD through fluorescence measurement of the GFP output.

Usage and Biology

When arabinose is absent, araC forms a homodimer that binds to the operator sites upstream of the pBAD promoter. This binding generates an inhibitory loop in the DNA that blocks access by the RNAP holoenzyme and thus prevents expression of GFP. Upon addition of L-arabinose and in the absence of glucose, the homodimer dissociates from the DNA, removing the inhibitory loop and allowing expression of GFP to occur.

Characterisation data

To determine the activation range of the pBAD promoter, we induced the pBAD+GFP device with varying concentrations of arabinose (the optimum concentration range was obtained from characterisation results from the 2010 Slovenia team), and recorded fluorescence in a BMG plate reader. TOP10 cells transformed with pBAD+GFP were grown from stocks overnight, and subcultured in the morning. After three to four hours, when the cultures were in exponential phase, the cultures were diluted to 0.05 O.D. and aliquoted into 96 well plate, before treatment with varying concentrations of arabinose (Fig. 1)

Figure 1: Characterisation of the pBAD-GFP construct with varying concentrations of L-arabinose. (BBa_K1893017).Experiments were performed in E. coli Top10 cell strain cultured at 37°C, which were diluted to 0.05 O.D. and inoculated with L-arabinose at the 0 minute timepoint. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (O.D. 600). Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation

Figure 2: Transfer function of normalised GFP fluorescence against concentration of L-arabinose

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1342
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1281
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1116
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 2017
Illegal SapI site found at 1098

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K1893017 short</partinfo>
-A GFP reporter under control of a pBAD promoter, used to characterise the activation range of the pBAD promoter
+This composite part is composed of a GFP reporter [https://parts.igem.org/Part:BBa_E0040 (BBa_E0040)] downstream of a pBAD arabinose inducible promoter [https://parts.igem.org/Part:BBa_K1893015 (BBa_K1893015)]. It was used to characterize the activation range of pBAD through fluorescence measurement of the GFP output.
 ===Usage and Biology===
+When arabinose is absent, araC forms a homodimer that binds to the operator sites upstream of the pBAD promoter. This binding generates an inhibitory loop in the DNA that blocks access by the RNAP holoenzyme and thus prevents expression of GFP. Upon addition of L-arabinose and in the absence of glucose, the homodimer dissociates from the DNA, removing the inhibitory loop and allowing expression of GFP to occur.
 ===Characterisation data===
-Without any arabinose in the cell, araC forms a homodimer, binding to operator sites upstream of the pBAD promoter, generating an inhibitory loop in the DNA that prevents access by the RNAP holoenzyme. In the absence of glucose, and the presence of L-arabinose, the dimer dissociates from the DNA, allowing transcription of GFP.
-We added varying concentrations of arabinose (optimum concentration ranges were obtained from [https://parts.igem.org/Part:BBa_I0500:Experience 2010 iGEM team Slovenia characterisation]), and recorded fluorescence in a BMG plate reader.
+To determine the activation range of the pBAD promoter, we induced the pBAD+GFP device with varying concentrations of arabinose (the optimum concentration range was obtained from characterisation results from the 2010 Slovenia team), and recorded fluorescence in a BMG plate reader. TOP10 cells transformed with pBAD+GFP were grown from stocks overnight, and subcultured in the morning. After three to four hours, when the cultures were in exponential phase, the cultures were diluted to 0.05 O.D. and aliquoted into 96 well plate, before treatment with varying concentrations of arabinose (Fig. 1)
 [[File:AraC_TOP10.png|700px|center|]]
-Figure 1: Characterisation of the pBAD-GFP construct with varying concentrations of L-arabinose. (BBa_K1893017).Experiments were performed in E. coli Top10 cell strain cultured at 37°C, which were diluted to 0.05 O.D. and inoculated with L-arabinose at the 0 minute timepoint. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (O.D. 600). Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation
+Figure 1: Characterisation of the pBAD-GFP construct with varying concentrations of L-arabinose. [https://parts.igem.org/Part:BBa_K1893017 (BBa_K1893017)].Experiments were performed in E. coli Top10 cell strain cultured at 37°C, which were diluted to 0.05 O.D. and inoculated with L-arabinose at the 0 minute timepoint. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (O.D. 600). Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation
-[[File:Transfer_curve_arac.png|700px|center|]]
+[[File:Transfer_curve_arac.png|600px|center|]]
 Figure 2: Transfer function of normalised GFP fluorescence against concentration of L-arabinose
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K1893017 SequenceAndFeatures</partinfo>