![](https://parts.igem.org/images/partbypart/icon_composite.png)
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.
Characterziation
Fluorescence measurement of BBA_K4604028 with different added AdoCbl concentrations was performed to test this BioBricks functionality. Fluorescence is normalized to OD_600.
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
None |