Difference between revisions of "Part:BBa K239000"

Latest revision as of 00:36, 30 October 2010

DegP promoter, activated by Phosporylated CpxR and SigmaE

Sequence contains 3 phosphorylated CpxR (Conjugative Plasmid eXpression) binding sites and transcription is initiated by sigma factor E(σ24).

Function: Damage to E.coli cell envelope --> PoPS output.

Application: Can detect various periplasmic stresses such as Copper (4mM CuCl2), Indole (4mM) and Ethanol (5%). The DegP protein is expressed by the cell to repair damage caused in the periplasm. The DegP promoter biobrick (BBaK239000) is designed to monitor the state of health for the E.coli cell envelope and periplasmic space. Possibly it can also be used to detect presence of heterogeneous unfolded proteins in the periplasmic space (e.g. target pharmaceuticals) or to detect high levels of shear stress during cultivation in a bioreactor.

Usage and Biology

The DegP gene and promoter

The gene degP encodes a protease and chaperon designated to the periplasm. (Miyadai et al. 2004) It is positively regulated by the CxpAR system and Sigma-E (Ruiz and Silhavy 2005). When misfolded proteins are accumulating in the periplasm these two mechanisms are inducing the transcription of DegP to “clean up” in the periplasm. Over expression of some native lipoproteins (also with their destination in the cell envelope) in E.coli is leading to uncontrolled expression of DegP. (Miyadai et al. 2004)

Extracytoplasmic function sigma, Sigma-E (24)

Sigma-E was first discovered as an alternative sigma factor recognised by one of the promoters for RpoH. However, it is mostly involved in transcription of genes with function in the periplasm, many of which are also part of the CpxA-CpxR regulon. The pathway of Sigma-E is activated when the periplasmic sensor protease DegS cleaves the anti-sigma factor RseA. This occurs after exposure of DegS to misfolded outer membrane proteins. Following the cleavage of RseA in the periplasm another inner-membrane bound protease YaeL (also named RseP) degrades the trans-membrane domain of RseA. The latter event releases the Sigma-E transcription factor in the cytoplasm and activates its large regulon.(Rhodius et al., 2005) However, sigma E can also be activated independently of misfolded OMPs by cytoplasmic alarmone ppGpp, which is dependent on nutrient availability. (Costanzo and Ades 2006)

CpxA-CpxR envelope stress response

The CpxA-CpxR (Conjugative Plasmid eXpression) response functions as a two-component signal transduction system. The CpxA is the system’s sensor kinase which auto-phosphorylates when proteins designated for the outer membrane or secretion (e.g. pili or curli fibers) are mounting in the periplams. The concentration of proteins can be due to problems in transport or damage to the outer membrane. (Snyder and Champess 2007) The phosphorylated CpxA transfers its phosphate to CpxR which becomes activated as a DNA binding protein activating or increasing the transcription of more than 100 different genes. The genes activated are mostly chaperones and proteases involved in the refolding or degradation of proteins in the periplasm. DiGuiseppe and Silhavy (2003) have showed that of all the genes being induced by CpxR the cpxP (part of the cpx operon and involved in feedback inhibition) promoter is the strongest induced of them all. The CpxA-CpxR system also regulates the pore size in the membrane by increasing the transcription of ompC and repressing the transcription ompF with the effect that fewer toxins can get in through the smaller porin OmpC. OmpC and OmpF are the two major porin proteins in E.coli and their main function is to balance the osmotic pressure of the cell. They are otherwise regulated due to changes in the osmolarity via EnvZ and OmpR. (Snyder and Champess 2007) Yamamoto and Ishihama (2005) have demonstrated that CpxAR is being activated as a response to external copper. An example of an additional protein regulating the activity of CpxA is NlpE (an outer membrane lipoprotein), which increases activity following adhesion. (Bury-Moné et al., 2009) GTAAANNNNNGTAAA has been proposed as the CpxR binding site. (Wulf et al. 2002) However, an examination of a large amount of CpxR induced operons by Price and Ravio (2009) indicates that the level of consensus with the proposed sequence or its orientation seems to play a minor role when determining the strength of induction by CpxR for a particular promoter. Of larger importance seems to be the location of the binding site.

Note: The DegP promoter is dependent of Sigma E (and hence also the growth phase) and can not be induced only by over expression of e.g. CpxR. In comparison, the related Spy promoter (BBa_K239001), which is dependent on sigma factor 70, can be induced up to 40 fold by over expression of CpxR. (Bury-Moné et al. 2009) Experiments by the The UCL_London_2009 team have also showed that the activity of the DegP promoter (BBa_K239000), compared to the spy promoter (BBa_K239001), is significantly dependent on the growth phase of the bacteria.

Sequence and Features

Functional Parameters

The graph above shows the RPU strength of the DegP and Spy promoter at different OD values while growing in LB media.

The Relative Promoter Unit strength of the DegP promoter (BBa_K239000) is 0.3-0.4. However, this is only true for the exponential phase. RPU strength for DegP is increasing as the bacteria are approaching their stationary phase (Probably due to ppGpp activating Sigma-E).

(Due to problems with the transformation, experiments were carried out in comparison the TetR promoter (BBa_R0040) instead of BBa_J23101. TetR is measured to have an RPU activity of ca 1.4)

Characterization of the degP promoter's response to folding and misfolding proteins- University of Calgary 2010

Protocol

Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pDegP coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then subcultured in 20 ml LB broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose. This was incubated in the shaker for 12-14 hours and RFP output was measured using 555/632 nm.

"http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/DegPinduction.png"

This figure demonstrates that the DegP promoter activated with 15 different concentrations of arabinose. This diagram shows that the DegP promoter (K239000) is not particularly sensitive to misfolding proteins.

Discussion and conclusions

The figure shows that MalE and MalE31 express the DegP promoter in a similar fashion. This is slightly contradictory compared to the literature. The literature claims that the DegP promoter is upregulated in the case of protein misfolding, graph shown . Since MalE and MalE31 have been tested using other experiments described in this page, it is reasonable to conclude that K230009 is not very responsive to protein folding stress, that is , the DNA might not be consistent.