Difference between revisions of "Part:BBa K3121005"

Latest revision as of 16:17, 21 October 2019

rrnB P1-LacI-pTrp-mCherry stress sensor

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 standard Lac promoter, 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 Lac 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 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.

This helps develop a perspective as to why leaky expression is a pertinent issue in the aforementioned construct. While the plasmid has a high copy number and carried the rrnB P-1 promoter on all of its copies, the FIS transcription factor is still produced only by its endogenous genomic copies, which is optimally efficient only for few of the endogenous promoters. The excessive extra demands placed by the 100-300 additional copies of rrnB P-1 promoters would most probably render the overall activation of these very poor, simply because of insufficient FIS levels. This, in turn, implies that the LacI protein produced is woefully scarce to effectively repress all the 100-300 Lac operator sites. This ineffective repression allows for heavy leaky expression, thereby contributing towards the high readouts even under non-stress conditions.

We aim to ameliorate this situation by exploring two modifications:

Employing a low copy number plasmid, for instance, pET28a Shifting from a Lac promoter to a Ptrc promoter

The first is an obvious strategic choice, since the most straightforward method to ensure LacI is sufficient is to ensure it has lesser number of Lac operator sites to bind to, and, the roughly constant levels of endogenous FIS has only a limited number of rrnB P-1 sites it's required to bind and activate. The pET28a helps achieve this, as it has a significantly lower copy number of just around 40.

The catch here is that while we would probably be able to ensure LacI is produced in sufficient quantities to temper the leaky expression under non-stress conditions, we would be significantly hampering the system's potential to generate sufficient mCherry under actual stress conditions, as we now have a significantly reduced number of reporter construct copies. This may lead to poorer experimental results, as insufficient reporter expression would make detection difficult due to low readout intensities.

As a counter to this, we have included the second modification. The PTrc (a hybrid of the Lac and Trp operators) is a much stronger promoter than the traditional PLac itself and is also regulated by Lac repressor, and thereby, even with a limited number of copies, we may be able to force a high enough expression of the reporter protein under stress conditions. Thereby, we would be able to compensate for the low copy number plasmid used, while improving the extent of repressive control during non-stress conditions.

The experimental strategies to generate this modified biobrick may be seen in the flowchart below. We have used overlapping extension PCR to generate these modified constructs

Sequence and Features