Part:BBa_K4604026

piG_K12BSa (LacIprom_riboK12_lacI_trcProm_sfGFP)

piG_K12BSa consists of the LacI promoter, the AdoCbl riboswitch from E. coli K12, lacI and the trc promoter. The trc promoter can be induced with IPTG. This BioBrick is a sensor for AdoCbl (a bioavailable form of B12) with a variable marker gene.

Figure 1: Biosensor for the detection of AdoCbl.By placing the negative controlling riboswitches in front of the repressor gene LacI a positive signal to AdoCbl inside the cell can be achieved. Made with biorender.com

Characterization

To verify the performance of the BioBrick we added different concentrations of AdoCbl to the cultures and tested the sensitivity of the sensor by fluorescent measurement. AdoCbl is taken up by the cells, binds to the riboswitch which repressed LacI production thus enabling the expression of sfGFP.

Figure 2: Fluorescence measurement of E. coli MG1655 containing piG_K12BSa in M9 medium with different AdoCbl concentrations after 12 h incubation time. E. coli MG1655 containing pGGAselect serves as negative control. Fluorescence measurement was done using SpectraMax Plate Reader.The data present in these graphs is the result of at least three independent biological replicates.

Fluorescent measurement, as shown in figure 2, indicates that at a concentration of 100 nM AdoCbl, the riboswitch is saturated and can no longer recognize an increase of AdoCbl. This data verifies the sensibility of the sensor; it can detect AdoCbl in different concentrations and accordingly regulate the gene downstream of it. However, we also noted a high background fluorescence without the addition of AdoCbl.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 215
  
• 1000
  COMPATIBLE WITH RFC[1000]