Difference between revisions of "Part:BBa K822003"
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<partinfo>BBa_K822003 short</partinfo> | <partinfo>BBa_K822003 short</partinfo> | ||
| − | Yellow Fluorescent Protein coding sequence (partinfo>BBa_E0030</partinfo>) expressed under a constitutive promoter (<partinfo>BBa_J23100</partinfo>) for use as positive control in fluorescence measurement experiments. | + | Yellow Fluorescent Protein coding sequence (<partinfo>BBa_E0030</partinfo>) expressed under a constitutive promoter (<partinfo>BBa_J23100</partinfo>) for use as positive control in fluorescence measurement experiments. |
==Testing== | ==Testing== | ||
| − | The part was tested using a Tecan Infinite 200 Pro plate reader for fluorescence measurements. Using this instrument, using excitation and emission wavelengths of 514 and 544 nm respectively was found to be most suitable. A culture of E. coli strain DH5a containing the part was incubated for ~24 h at 37 C with shaking. Cells containing a construct with GFP (<partinfo>BBa_K082003</partinfo>) under the Jen1 promoter (<partinfo>BBa_K284002</partinfo>) were not expected to express GFP under the conditions and were used as a negative control. | + | The part was tested using a Tecan Infinite 200 Pro plate reader for fluorescence measurements. Using this instrument, using excitation and emission wavelengths of 514 and 544 nm respectively was found to be most suitable. A culture of E. coli strain DH5a containing the part was incubated for ~24 h at 37 C with shaking. Cells containing a construct with GFP (<partinfo>BBa_K082003</partinfo>) under the Jen1 promoter (<partinfo>BBa_K284002</partinfo>) were not expected to express GFP under the conditions and were used as a negative control. Fluorescence was measured using 100 uL samples in Nunclon 96-well flat bottom black polystyrol plates. |
[[image:YFP_fluorescence_1.png|center]] | [[image:YFP_fluorescence_1.png|center]] | ||
Revision as of 13:45, 26 September 2012
Constitutive YFP generator
Yellow Fluorescent Protein coding sequence (BBa_E0030) expressed under a constitutive promoter (BBa_J23100) for use as positive control in fluorescence measurement experiments.
Testing
The part was tested using a Tecan Infinite 200 Pro plate reader for fluorescence measurements. Using this instrument, using excitation and emission wavelengths of 514 and 544 nm respectively was found to be most suitable. A culture of E. coli strain DH5a containing the part was incubated for ~24 h at 37 C with shaking. Cells containing a construct with GFP (BBa_K082003) under the Jen1 promoter (BBa_K284002) were not expected to express GFP under the conditions and were used as a negative control. Fluorescence was measured using 100 uL samples in Nunclon 96-well flat bottom black polystyrol plates.
The fluorescence measurements above are not corrected for cell concentration (OD).Further experiments were made in connection with testing of oxygen-sensitive promoters, with averaging of readings and corrections made for the measured OD of the cell cultures that were sampled. These experiments failed to show activity by the oxygen-sensitive promoters, but provided further evidence for expression of YFP under the constitutive promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]