Fig. 4: Intracellular asymmetry arises when DivIVA is used as a reporter.
From: Construction of intracellular asymmetry and asymmetric division in Escherichia coli
a Localization of DivIVA-sfGFP when the expression was induced from the Ptac promoter with 50 μM IPTG for 1 h. Scale bars: 1 μm. b Localization of DivIVA-sfGFP and mRFP-PopZ when the two were co-expressed. DivIVA-sfGFP expression was induced from the Ptac promoter with 50 μM IPTG for 1 h. mRFP-PopZ expression was induced from the PBAD promoter with 2% arabinose for 2 h. Scale bars: 1 μm. c Graphic comparing freely diffusing GFP and limited diffusion via DivIVA. d Circuit diagram for asymmetric gene expression when DivIVA-sfGFP was used as the reporter. pNSSCP and pNSSC denote plasmids containing SpmXΔC-fused RNAP halves expressed with or without mRFP-PopZ, respectively. eT7pN-spmXN denotes the SpmXΔC-fused PACE-evolved RNAP-N; spmXN-eT7pC denotes the SpmXΔC-fused PACE-evolved RNAP-C. Both SpmXΔC-fused T7 RNAP halves were expressed from the IPTG-inducible promoter, Ptac. divIVA-sfgfp is the reporter gene under the expression of T7 promoter. mrfp-popZ gene was expressed from the Anderson promoter J23116. e Asymmetric gene expression when DivIVA-sfGFP was used as a reporter. NSSCP and NSSC denote SpmXΔC-fused RNAP halves expressed with or without mRFP-PopZ, respectively, with DivIVA-sfGFP as a reporter. SpmXΔC-fused RNAP halves were induced from the Ptac promoter with 50 μM IPTG for 1 h. Scale bars: 1 μm. f Quantification of DivIVA-sfGFP asymmetry in a, b, and e using the ratio between opposite poles (“Methods”). NSSCP and NSSC denote SpmXΔC-fused RNAP halves expressed with or without mRFP-PopZ, respectively. Div denotes DivIVA-sfGFP expressed alone. DivRP denotes DivIVA-sfGFP co-expressed with mRFP-PopZ. The gray dashed line indicates one (i.e. no asymmetry); n = 89, 78, 99, and 91 cells for Div, DivRP, NSSC, and NSSCP, respectively. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. Statistical difference was determined by two-tailed Student’s t-test; **** denotes P < 0.0001. g Fluorescence intensity (sfGFP) profiles along the long axis of the cell when NSSC circuits were expressed alone (gray lines) or co-expressed with mRFP-PopZ (red lines); x = 0 marks the PopZ pole. Fluorescence intensity was normalized by the maximal mean intensity in NSSCP. Solid lines indicate averages; colored belts indicate standard deviations; n = 99 and 91 cells for NSSC and NSSCP, respectively. Source data are provided as a Source Data file.
