Part:BBa_K4604028

piG_PFBS (LacIprom_riboPF_lacI_trcProm_sfGFP)

This BioBrick (referred to as PF sensor) consists of the LacI promoter, the AdoCbl riboswitch from Propionibacterium freudenreichii, 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.

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_K4604028 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 OD_600 were measured 12 hours after inoculation from the glycerol stock. PGGAselect represents the negative 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.

For the PF sensor, an increase of fluorescence signal that stagnates already at 10 nM AdoCbl was visible, with no noticeable change at higher concentrations. It should be noted that a high background fluorescence was observed, as seen with the 0 nM AdoCbl (figure 2). This is higher than the controlled autofluorescence of cells expressing pGGAselect, indicating inadequate expression of the LacI repressor protein. Next, this BioBrick was compared to BBa_K4604026, referred to as K12/piG_K12BS.

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 OD_600 were measured 12 hours after inoculation from the glycerol stock. PGGAselect represents the negative control. Depicted is a comparison between sensors K12/PF non-induced and induced with 200 µM IPTG as well as the same sensor template without the riboswitches (p_04). 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 200 µM IPTG was added we saw a noticeable increase in fluorescence for both sensors with the K12 being slightly higher than the PF sensor. However, the fluorescent signal does not reach the values observed for the induced control (p_04), which is around 2-3 times higher. Since we observed an increase in fluorescence, albeit a rather small one, with rising AdoCbl concentrations, we conclude that the riboswitch used and therefore our BioBrick functions as intended, by repressing the repressor LacI expression leading to higher sfGFP expression. However, we also noticed that BBa_K4604028 does not appear to tightly regulate the repressor as even at saturating levels (>100 nM) of AdoCbl the fluorescence does not reach the fluorescence values as observed upon induction with IPTG. This could be either due to the riboswitch still allowing LacI production or LacI being produced before saturation levels inhibited its expression and staying for longer time than 12 hours, still repressing the trc promoter. We assume that the riboswitch with its own ribosome binding site (RBS) does not provide adequate repressor function for LacI expression.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]