Part:BBa_K2387022

Cpx system reporter + tethered CpxP

The Cpx envelope stress reporter consists of the subunits CpxA, a membrane receptor, CpxR, the response regulator, and CpxP, an auxiliary periplasmic inhibitor of CpxA. If E. coli cells undergo stress on their envelope, the Cpx system will be activated due to the CpxA sensing stress related signals. In turn it will activate CpxR which acts as a transcription factor for genes involved in the alleviation of this stress and reinstatement of homeostasis. In non-stress conditions CpxP will inhibit activation of CpxA by direct physical interaction.

This part contains an inner membrane tethered version of CpxP (BBa_K2387019), which is able to inhibit the Cpx system in case the outer membrane of the cell is removed by spheroplasting. In the [http://2017.igem.org/Team:Wageningen_UR 2017 WageningenUR project] this part was used to verify the inhibition of the Cpx system in case the cells were spheroplasted. This procedure is necessary for the project as it allows antigens to reach the receptor system. As a reporter for activation of the Cpx system a mRFP1 coding sequence under control of CpxR promoter was used, available as BBa_K339007.

Usage and Biology

In the Wageningen iGEM 2017 project Mantis, this biobrick was used to measure the Cpx inhibitory capacity of inner membrane tethered CpxP and affinity body fusions in E. coli ΔCpxP strains that were spheroplasted, together with parts BBa_K2387023, BBa_K2387024. Specifically, this biobrick was used as an inner membrane bound native CpxP control to compare the tethered fusions to. Furthermore, BBa_K339007 was used as a positive control to show fluorescence resulting from Cpx system activation.

Figure 1 shows that the tethered fusions of CpxP with the affinity bodies were able to inhibit the Cpx system equally as well as the tethered native CpxP fusion.

For more information about this experiment see the Cpx Signal Transduction page of the 2017 Wageningen iGEM team.

Figure 1: Growth measurements of E. coli ΔCpxP strains carrying constructs of different CpxP-Aff fusions, as well as a control without CpxP. The left graph shows that the control has high activation of the Cpx system due to absence of the inhibitor. The right graph shows the same results, without the control, indicating that the membrane tethered fusions can effectively suppress the Cpx system.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1454
  Illegal BglII site found at 2372
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 636
  Illegal AgeI site found at 748
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1152