rrnB P1-LacI-pTrp-mCherry stress sensor: BBa_K3121005
The stringent response is a well-studied bacterial response pathway wherein the bacteria sense external stressors such as amino acid starvation, heat/pH shocks, acute nutrient deprivation, etc. and respond via signalling pathways employing guanosine tetra/penta-phosphate which primarily re-route the regular gene expression pathways. The prominent downstream effects observed are upregulation of genes implicated in survival responses, and a simultaneous decrease in those primarily employed for growth and proliferation.
The original biobrick (BBa_K639003) utilizes a clever synthesis of the Lac repressor downstream of rrnB P1 promoter. The rrnB P1 promoter is prone to quick downregulation during the stringent response. So, by placing the LacI (repressor of the Lac operon) under the rrnB-P1 promoter, along with the mCherry reporter construct under the control of a Plac promoter (a hybrid of P(L) of phage lambda with cI binding sites replaced with lacO1), a system amenable to governance by stress responses may be generated. During regular growth conditions, the rrnB-P1 promoter would be active, which ensures LacI expression is maintained, thereby repressing the Plac promoter, and by extension, any mCherry expression. In contrast, during acute stress conditions, the bacterial stringent response pathway would shut down the rrnB P1 promoter, thereby lowering the LacI expression, and relieving the repression on the Lac operator, hence allowing for abundant mCherry expression - which may be conveniently adjudged by measuring the fluorescence intensity levels.
While sound in principle, the experimental results with this biobrick give mixed results as a high fluorescence is observed in both non-stress, as well as stress conditions. This is evidenced in the results reported by the Trondheim team, wherein we observe largely comparable levels of mCherry expression under all conditions. While the fluorescence readouts under the stress conditions are undeniably higher, the difference between both states is not significant enough.
This anomaly may be attributed to the significant amount of leaky expression of the mCherry even under non-stress conditions which may be contributing towards the high readouts. This is quite possibly due to the fact that the Plac promoter used herein shows leaky expression. Although the Plac promoter is IPTG regulated, it gives upto even two-thirds of the maximum expression without IPTG induction (as indicated by existent data from the iGEM repository). Also, the plasmid used for the constructs herein is pSB1A2, which has a high copy number of around 100-300. A brief analysis of the literature helps pinpoint why this may be a strong contributing factor. In the study by Magnusson, L. U., A. Farewell, et al. (2005), it has been reported that the rrnB P-1 promoter is regulated by cis-acting UP elements, as well as a trans-acting FIS transcription factor. These play an important role, as the study implicates them in driving forward most of the rrnB P-1 activity - in fact, the core promoter has been reported to contribute only upto <1% of the overall promoter activity.
