Difference between revisions of "Part:BBa K2596001"
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| + | In order to test the expression of our promoters, cpc, cpc560 (BBa_K2596006), idiA (BBa_K2596004), psbA2 (BBa_K2596003), which were incorporated into Dr. Susan Golden’s vector pAM1414 using Gibson assembly, we conducted luciferase experiments. Following Dr. Golden’s procedure, we added 5 µL of decanal to 95 µL of cyanobacteria in each well to induce expression. The decanal acted as a substrate for the bacterial luciferase enzyme, but due to the toxicity, the cells ended up dying, so the data obtained represents the end static expression. For the standard expression experiments for nighttime and daytime, we plated 95 µL of cyanobacteria into a 96 well plate, added 5 µL of decanal, parafilmed the edges and left the plate for 15 minutes before measuring the luminescence in a plate reader. For the psbA2 experiment, we plated 95 µL of cyanobacteria to half of the wells and put them under at least 500 µE of high light in our incubator for 1 hour. Then, we added 95 µL of cyanobacteria not exposed to high light to the other half of the wells and added 5 µL of decanal to all of the wells. Then, we placed the well plate in a plate reader and measured the luminescence. | ||
Revision as of 15:49, 16 October 2018
PcpcB from Synechococcus elongatus PCC7942
PcpcB is a constitutive promoter from cyanobacteria Synechococcus elongatus PCC 7942.
Usage and Biology
cpcB codes for phycocyanin, a phycobiliprotein complex, and works to harvest light energy before transmitting it to the chlorophylls. The promoter is a strong promoter but also demonstrates expression patterns following circadian oscillations. Studies have found that Pcpc can also be used in E. coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
PcpcB1A1 features two promoter sequences within the promoter, both of which were included in the BioBrick. For this promoter, derived from Synechococcus elongatus PCC 7942, we kept about 100 bp of upstream and downstream regions, the latter of which was cropped immediately before the ATG start codon. Studies have found that Pcpc can also be used in E. coli.
Luciferase Assays
In order to test the expression of our promoters, cpc, cpc560 (BBa_K2596006), idiA (BBa_K2596004), psbA2 (BBa_K2596003), which were incorporated into Dr. Susan Golden’s vector pAM1414 using Gibson assembly, we conducted luciferase experiments. Following Dr. Golden’s procedure, we added 5 µL of decanal to 95 µL of cyanobacteria in each well to induce expression. The decanal acted as a substrate for the bacterial luciferase enzyme, but due to the toxicity, the cells ended up dying, so the data obtained represents the end static expression. For the standard expression experiments for nighttime and daytime, we plated 95 µL of cyanobacteria into a 96 well plate, added 5 µL of decanal, parafilmed the edges and left the plate for 15 minutes before measuring the luminescence in a plate reader. For the psbA2 experiment, we plated 95 µL of cyanobacteria to half of the wells and put them under at least 500 µE of high light in our incubator for 1 hour. Then, we added 95 µL of cyanobacteria not exposed to high light to the other half of the wells and added 5 µL of decanal to all of the wells. Then, we placed the well plate in a plate reader and measured the luminescence.