Part:BBa_K4604029

piG_SYBS

This BioBrick (also referred to as SY sensor) consists of the LacI promoter, the AdoCbl riboswitch from Salmonella typhimurium/enterica, 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

A fluorescence measurement with different added AdoCbl concentrations was performed to test this BioBricks working range.

Figure 2: Fluorescence measurement after 12 hours and different AdoCbl concentrations for the SY sensor. Cultures were grown in 5 mL M9 medium in reaction tubes at 37°C and 200 rpm with respective AdoCbl concentrations added. Fluorescence was measured 12 hours after inoculation from the glycerol stock. PGGAselect depicts E.coli with just an empty plasmid which functions as a negative control here. Measurements were taken with the SpectraMax ID5 plate reader. The data present in these graphs are the result of at least three independent biological replicates.

When considering the graph above, one notices that the fluorescence for the SY sensor is very low compared to autofluorescence of pGGAselect. The SY sensor when induced with 200 µM IPTG, however, showed a fluorescence similar to other sensors we tested (BBa_K4604026 and BBa_K4604028). With this data, we conclude that the sensor does not work properly as expected. Possible reasons are not known at the moment and would need further testing.

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 220
  
• 1000
  COMPATIBLE WITH RFC[1000]