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    Cellular and Molecular Bioengineering

    (H-314) Unraveling the Q system function in prokaryotes

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row H - Poster # 314
    Introduction:: Recently the eukaryotic QF activating transcription factor from Neurospora crassa was shown to activate gene expression in prokaryotes when coupled with the T7 promoter1. The mechanisms underlying the activation of the eukaryotic Q system in prokaryotes are unknown. Understanding how these genetic parts function as activators in prokaryotes will enable the building of novel tools and approaches for activating gene expression in prokaryotes. Here, we isolate the activation domain from the QF transcription factor and fuse it to the well-characterized TetR repressor protein to investigate its behavior in Escherichia coli (E. coli). This fusion protein (TetR-QFAD) acts as an activator that is regulated with anhydrotetracycline (aTc). Our analysis of TetR-QFAD provides greater insight into QF-mediated activation and demonstrates the utility of using TetR-QFAD as a new tool to regulate gene expression in prokaryotes.

    Materials and Methods:: Traditional cloning techniques were used to construct genetic circuits in plasmids and transformed into BL21(DE3) E. coli (ThermoFisher). This bacterial strain transcribes T7 RNA Polymerase (T7RNAP) upon induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). T7RNAP is highly specific for the T7 promoter1. The TetR-QFAD fusion protein was constructed by amplifying the QFAD via PCR and inserting it on the C-terminus of TetR. A TetR operator sequence (TetO) was placed downstream of a T7 promoter driving the expression of the reporter gene, green fluorescent protein (GFP). An ssrA degradation tag was attached to the C-terminus of GFP, to allow for its rapid degradation. The TetR-QFAD protein was placed downstream of a T7 promoter in a separate circuit. Colonies were picked and grown in LB (ThermoFisher) overnight for approximately 12 hours with antibiotic selection. The overnight culture was diluted at a 1:50 ratio in LB and induced with 0.5mM IPTG at an optical density of ~ 0.2 at 600 nm (OD600) to initiate the transcription of T7RNAP. A Synergy HTX Reader (Biotek) was used to measure the OD600 of 300 uL of bacterial culture. Anhydrous tetracycline (aTc) (Alfa Aesar) was also added at the time of induction to the second induction condition. GFP expression was measured every hour using a cytoFLEX S (Beckman Coulter) flow cytometer. OD600 was also measured at each time point using 300 uL of induced culture. The measured culture was returned to the induced culture after measurement.

    Results, Conclusions, and Discussions:: Fusing the QFAD to TetR allows the TetR to function as a repressor and an activator depending on the presence of aTc (Fig. 1). The expression of GFP regulated by TetR-QFAD is significantly higher than the TetR control in the presence of aTc (Fig. 1). Attaching the well-defined eukaryotic VP16 activation domain to TetR, yielded results similar to the TetR control (Fig. 1). Furthermore, by replacing the T7 promoter with the native bacterial pTet promoter, it was demonstrated that TetR-QFAD does not interact with bacterial RNA polymerase (Fig. 2).

    These results demonstrate the uniqueness of using QFAD in bacteria to control gene expression that will enhance the genetic toolbox for prokaryotic synthetic biology. We hypothesize that QFAD strongly interacts with T7RNAP and our future work will test this hypothesis using AlphaFold modeling and experimental methods.


    Acknowledgements (Optional): :

    References (Optional): : 1. MacDonald IC, Seamons TR, Emmons JC, Javdan SB, Deans TL. Enhanced regulation of prokaryotic gene expression by a eukaryotic transcriptional activator. Nature communications 2021; 12(1): 4109.