Part:BBa_K1442100:Experience

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Figure 5. 3’ UTR and Reverse GFP general construct depiction. Plasmid containing reverse GFP and 3’ UTR is depicted below. This was used to characterize RdRp and mRdRp activity in E. coli. 3’ UTRs were interchangeable and are referred to as S3, R5 and C2/HP. 3’ UTRs and Reverse GFP are biobrick compatible. For clarity, biobrick prefix and suffix sequences not shown.

To characterize RdRp and mRdRp activity, co-transformation of the two plasmids with the relevant constructs (Figures 4 & 5) was performed. Table 1 summarizes the experiments and relevant 3’ UTRs tested. Data was obtained using the Tecan micro plate reader

Figure 6. Characterization of mRdRp and RdRp activity with S3, R5 and C2/HP 3’ UTRs. Data was collected at different time points using Tecan Microplate reader. Controls also present: (IPTG + RBS + GFP) and NEB only.

Table 1. RNA dependent RNA polymerase activity following induction at 0.2 and 0.4 OD, relative to control wells (not induced at 0.2 and 0.4 OD). Error bars indicate standard error.

Horizontal axis depicts various different points (1-9: due to the nature of the analysis time in seconds is not displayed). RdRp induction at 0.2 and 0.4 OD at points 1-3 indicates initial activity and relative increase in fluorescence output. Initial RdRp activity then decays following induction, with decrease in fluorescence activity at both OD 0.2 and 0.4. Conclusively, results suggest initial induction of RdRp activity causes a relative increase in fluorescence with a stabilization/decrease thereafter.

Figure 7. RNA dependent RNA polymerase activity following induction at 0.2 and 0.4 OD, relative to control wells (not induced at 0.2 and 0.4 OD).

Figure 8. RNA dependent RNA polymerase activity following induction at 0.2 and 0.4 OD. Rates were extracted by linear regression and is displayed. Error bars indicate standard error of the mean. Rdrp Ni: Non induced, Rdrp I: Induced, mRdRp follows the same conventions.

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