Team:Macquarie Australia/Contribution
CHARACTERISATION
Full-length stationary phase osmY promoter [BBa_J45992]
For our project, we utilised the full-length stationary phase osmY promoter to control the transcription of our riboswitch + eGFP reporters, R ON [BBa_K3151011] & R OFF [BBa_K3151002]. This part was particularly relevant for our project as the secondary messenger which we utilise to communicate between our sensor and reporter, cyclic-di-GMP, is primarily synthesised as the cells enter into their stationary phase.
In order to characterise the mode of action of the riboswitch/promoter combinations, we collaborated with Team Sydney Australia to perform a GFP fluorescence assay[1]. These assays were used to quantify the production of eGFP between our reporter constructs, as well as comparing the function of the stationary phase promoter against two inducible promoters, Lac [BBa_R0010] and Tac [BBa_J45992]. Samples were measured with the BMG Pherastar plate reader to measure eGFP (Ex485 nm Em520 nm) and OD (600 nm).
This results demonstrate that while the production of eGFP increases at 6-8 hours for all the promoters (i.e. roughly the point at which samples would be entering the stationary phase), the Lac (Figures 1 & 4) and Tac (Figures 2 & 5) inducible promoters show a steady, somewhat linear accumulation of eGFP over time, the stationary phase promoter (Figure 3) shows a sharp increase and beings to level out after ~20 hours.
These results are consistent with previous characterisations of the promoter, which observed significant increases in β-galactosidase concentration after entry into the stationary phase at 5 hours.[2]
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| Figure 1: eGFP produced by cells transformed with the Lac riboswitch constructs over 60 hours. | Figure 2: eGFP produced by cells transformed with the Tac riboswitch constructs over 60 hours. | Figure 3: eGFP produced by cells transformed with the Stationary phase riboswitch constructs over 60 hours. |
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| Figure 4: eGFP produced by cells transformed with the Lac riboswitch constructs over 30 hours. Data provided by Usyd iGEM. | Figure 5: eGFP produced by cells transformed with the Tac riboswitch constructs over 30 hours. Data provided by Usyd iGEM. |
References
- Dell EJ. Bottom reading of cell-based assays: direct optic approach enhances fluorescent protein and other microplate analyses. Genetic Engineering & Biotechnology News. 2012 May 1;32(9):22-3.
- Hengge-Aronis R, Lange R, Henneberg N, Fischer D. Osmotic regulation of rpoS-dependent genes in Escherichia coli. Journal of Bacteriology. 1993 Jan 1;175(1):259-65.