Part:BBa_K1343012
Plux-amilCP
blue reporter protein amilCP under LuxR-AHL regulated promoterDetermine the qualitative expression of AmilCP under the promoter Plux
Objective
We aspire to verify the expression of the reporter pigment protein AmilCP under the promoter Plux.
Description
In this experiment, a culture of E. coli K-12 Top 10 expressing AmilCP (a dark blue pigment protein) under the promoter Plux was grown overnight. The bacteria were engineered to contain a plasmid with the gate Plux-AmilCP. We originally aspired to determine the activity of the promoter Plux using the reporter gene AmilCP, by cloning and testing bacteria containing the gate Pcat-luxR-Plux-AmilCP. Pcat is a constitutive promoter- therefore, luxR is expressed in excess in the bacteria, creating a dimer with AHL. This dimer binds to the Plux promoter, resulting in expression of AmilCP. However, we ran out of time, so we decided to at least show that the construct Plux-AmilCP functions properly while relying on basal levels of transcription. Our next step would have been a scar ligation, similar to the one conducted to create Pcat-luxR-Plux-mcherry-luxI (visit our wiki page for more details)
Protocol
A starter was prepared by growing the cells in LB medium + appropriate antibiotics at 37°C for 19 hours. The bacteria were then centrifuged for 10 minutes, and a picture of the pellet was taken.
Results and conclusions
The pellet was dark blue, it is highly probable that the bacteria contain the gate Plux-AmilCP.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 64
Illegal XhoI site found at 57 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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