How does a native E. coli cell combat protein related stress in the Cytoplasm?

There are several heat shock pathways in E. coli which are actively transcribed in response to cellular stress. There are housekeeping genes called sigma factors that are responsible for maintaining homeostasis in the cell and helping with protein folding. In the cytoplasm, stress, in particular misfolded protein, is largely regulated through the sigma32 pathway. Normally, sigma factor 32 is bound to heat shock proteins such as GroE and DnaK. In the presence of misfolding protein however, these heat shock proteins bind to the misfolded proteins, levaing sigma 32 free to form a complex with RNA Polymerase. This allows for transcription from various sigma-32 dependent promoters, driving the expression of anything downstream,. Many studies have found sigma-32 dependent promoters to be very effective at measuring levels of cytoplasm protein misfolding in E. Coli. One such promoter is the ibpAB promoter, which controls a heat shock operon in E. Coli.

iGEM Calgary cytoplasmic stress detection circuit

The cytoplasmic stress detector has a fusion of sigma 32 activated heat shock promoter which allows a higher output compared to the ibpAB promoter and FxsA promoter

The ibpAB Promoter

The ibpAB promoter contorls the trasncription of two small proteins: ibpA and ibpB. These are small heat shock proteins called inclusion body binding proteins. In the presence of inclusion bodies within the cytoplasm, they are thought to form mixed complexes, ibpA allowing ibpB to bind to the inclusoon body at higher temperatures. The binding of these proteins to the misfolded protein lowers its hydrofobicity, previngin firtyher binding of exposed peptide chains, thus stabilizing the protein and mediating its refolding by the DnaK/DnaJ/GrpE chaperone protein system (Matuszewska at al., 2005). Transcription levels from this promoter have been found to increase the most upon heat shock as compared to other heat shock promoters (Chuang et al., 1993). .

B: MalE31 induction with IPTG; C: MalE31 induction and reporter reading with just ibpAB promoter; D: MalE31 induction and reporter reading with just fxsA promoter; E: MalE31 induction and reporter reading with ibpAB/FxsA fusion promoter (Kraft et al, 2006)

How are we utilizing this promoter?

This fusion promoter will be connected to the registry part I13504 which is RBS-GFP-B0015. The ibpAB/fxsA circuit will be activated in the presence of aggregation in the cell. We will be using MalE31 with a signal sequence deletion (MalE31∆SS) which was designed by Betton et al. The native E. coli protein MalE generally exported into the periplasmic space but this mutated protein does not get exported to the periplasmic space due to the signal sequence deletion. Also Betton et al designed MalE31such that there are two amino acid changes in the protein and it misfolds. The MalE31∆SS protein coding region will be used in order to induce cytoplasmic protein stress in E. coli.

Ideally, this misfolded MalE31∆SS should activate the plasmid system containing ibpAB/fxsA-I13504 which will produce GFP alerting the researcher that their protein is not being expressed in the cell because it is misfolding and as a result getting degraded. Our circuit should also be activated much faster than the native stress system because the ibpAB/fxsA promoter is much more sensitive to the presence of aggregate bodies in the cell. The promoter also gives a much higher output compared to the promoters individually, which is the case in the E. coli genome which should allow us to detect the fluorescence level much faster.