Part:BBa_K2796041:Experience

BS-Biotin-On system module BirA, a bifunctional protein, can activate biotin and connect it with specific protein receptor in the presence of ATP, and can also bind to activated biotin to produce conformational changes, and then bind to the specific operon sequence (OBirA) located upstream of biotin biosynthesis operon to inhibit the transcription of this operon. The system utilizes the above characteristics of BirA to construct an artificial transcription activator, BirA-VP16, with the transcription activation domain of herpes simplex virus transcription activator VP16. The obtained BirA-VP16 can bind and activate the minimum CMV promoter containing three copies of OBirA in the presence of biotin, thereby activating the downstream genes.



Experimental results





• The function of biotin-inducing system was tested by HEK 293T. Firstly, we stably transfected the vector that expressed birA-VP16 gene driven by pEF1a promoter into HEK 293T cells. And then, the plasmid with the ObirA-miniCMV-mRFP element is then transferred to the engineering 293T cells.
• The result of the left figure is that the mRFP expression level of engineered HEK293T cells was measured at 4h under different biotin concentrations.
• At the same time, fluorescence intensity of mRFP was measured at different times at the concentration of 100uM biotin (right figure). From the experimental results, the mRFP fluorescence intensity gradually leveled off at when incubated for about 40 hours.
  
  

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