Part:BBa_K4604027

piG_K12BSb (LacIprom_riboK12_T7RBS_lacI_trcProm_sfGFP)

piG_K12BSb (referred to as K12b) consists of the LacI promoter, the AdoCbl riboswitch from E. coli K12, the modified T7 RBS, lacI and the trc promoter. The trc promoter can be induced with IPTG. This BioBrick can be used as a sensor for AdoCbl (a bioavailable form of B12) with a variable marker gene. PiG_K12BSb is BBa_K4604026 with the addition of a stronger RBS right behind the riboswitch, decreasing background fluorescence.

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

Characterziation

Fluorescence measurement of BBA_K4604027 with different added AdoCbl concentrations was performed to test this BioBricks functionality. Fluorescence is normalized to OD_600.

Figure 2: Fluorescence measurement after 12 hours for different AdoCbl concentrations added to the medium. Cultures were grown in 5 mL M9 medium in reaction tubes at 37°C and 200 rpm. Fluorescence and OD600 were measured 12 hours after inoculation from the glycerol stock.pGGAselect depicting E.coli with just an empty plasmid and functioning 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.

We observe a rather small but still steady increase of fluorescence with increasing AdoCbl concentration until a concentration of 500 nM, followed by a stagnation in fluorescence. Interestingly, we observed a higher background fluorescence with 0 nM AdoCbl. Next, this BioBrick was compared to BBa_K4604026, referred to as K12/piG_K12BS. These contructs only differ in an additional stronger RBS on piG_K12BSb.

Figure 3: Fluorescence measurement after 12 hours for different AdoCbl concentrations added to the medium. Cultures were grown in 5 mL M9 medium in reaction tubes at 37°C and 200 rpm. Fluorescence and OD600 were measured 12 hours after inoculation from the glycerol stock. Depicted is a comparison between induced (200 µM IPTG) and non-induced states for the piG_K12BS and piG_K12BSb plasmids with pGGAselect depicting E.coli with just an empty plasmid and functioning as a negative control here. Additionally piG_04, a plasmid given to us by iGEM Freiburg 2022) (p_04) is added as a positive control. 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.

This time K12b showed reduced fluorescent values compared to the tested K12 sensor. Comparison between the K12 sensor and the K12 sensor with the new RBS (K12b) reveals a twice as high background fluorescence as well as a twice as high fluorescence when IPTG is added.

The steady increase of fluorescence can be attributed to the added AdoCbl. But still the same problem persists as with BBa_K4604026 and BBa_K4604028: the increase in fluorescence observed is very low and insignificant when compared to fluorescence values obtained by supplementation with IPTG (figure 3). It is worth noting that the K12b sensor showed much less background fluorescence than the K12 sensor. This suggests that more repressor is produced leading to a tighter regulation of the reporter gene. Therefore we conclude that placing a stronger RBS behind the riboswitch leads to higher protein expression without compromising the regulatory function of the riboswitch. This is due to the transcription regulational part of the riboswitch, since the sequestering of the RBS does not apply for the changed RBS with its different sequence.

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]