Part:BBa_K2387023
Cpx system reporter + tethered CpxP-Affinity molecule
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-Affinity molecule fusion (BBa_K2387020), 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 whether the CpxP-Affinity molecule fusion was able to inhibit activation of the Cpx system in case the bacterial 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_K2387022, BBa_K2387024. Specifically, this biobrick was expected to inhibit the Cpx system in a spheroplasted situation in a similar way as the tethered CpxP gene could. 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.
Furthermore, this MalE24-1-CpxP-Aff fusion containing biobrick was used in an experiment to test the systems response to place-holder antigen IgG. By addition of IgG to spheroplasted cells we hypothesized activation of the Cpx system due to binding and dissociation of the tethered CpxP-Aff fusion. After dissociation the CpxA membrane sensor could be activated leading to fluorescence production.
Figure 2 shows that the presence of IgG (0.1 mg/mL, highest concentration measured) did in fact not induce the system in a significant way. Based on the experiment performed we could not identify what the leading cause was for this result. Due to time constraints no troubleshooting could be done to fix this system.
For more information about these experiments see the Cpx Signal Transduction page of the 2017 Wageningen iGEM team.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1454
Illegal BglII site found at 2372 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 636
Illegal AgeI site found at 748 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1152
None |