Investigating RpoE-Dependent EspPΔ5 Assembly

AD202 transformed with pJH208 and either a reconstructed version of pLC45 (68 (link)) or pTrc99A were grown in M9 medium as described above. When cultures reached an OD₅₅₀ of ∼0.2, they were treated with 10 μM IPTG for 25 min to induce rpoE expression and then 0.2% rhamnose for 5 min to induce EspPΔ5^{Y1298A, F1300A} synthesis. EspPΔ5^{Y1298A, F1300A} assembly was analyzed by pulse-chase labeling as described above, and samples were collected for qRT-PCR and Western blotting. For qRT-PCR, cells (1 OD₅₅₀ equivalent) were collected, and RNA was prepared by using an RNeasy minikit (Qiagen). RNA preps were treated with DNase I (NEB) and used as the templates to synthesize cDNA using the SuperScript III first-strand synthesis system (Thermal Fisher). qPCR was performed using Power-Up SYBR Green Master Mix (Thermal Fisher) and Bio-Rad CFX-96 real-time PCR detection system to determine the expression of rpoE and the housekeeping gene rssA as a normalization reference. For Western blots, proteins were TCA precipitated.