Part:BBa_K1442100:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1442100

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.

Our first experiment was performed with both the RNA dependent RNA polymerase (RdRp) and mutant RdRp with various different 3’ UTRs (Fig 6. S3, R5 and C2/HP). Our results indicate, as shown in Fig 6 – S3 showed higher fluorescence output relative to R5 and and C2/HP in the S3-RdRp construct (Fig 6. Black square). Fluorescence activity for the mutant RdRp (Fig 6. Red circle) had lower fluorescence. This suggests RdRp works better with the S3 3’ UTR than the mutant RdRp. However, in comparison to our positive controls (Fig 6. IPTG + RBS ~4-100µl) it appears that positive controls had higher relative fluorescence in comparison to constructs with RdRp and the 3’ UTRs. This suggests a number of possibilities: contamination of control wells, leading to higher relative fluorescence output (as bacteria naturally emit some fluorescence).

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.

Experiments were repeated; in this case induction at 0.2 and 0.4 OD was taken into account to omit any contaminants altering our results. 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.

To determine if this was the case, experiments were repeated as before, with the same conditions and same tests performed. Time course activity over fluorescence over optical density is displayed in Fig 7. As shown, mutant RdRp induction in this case showed higher fluorescence/OD than RdRp that was not induced. As this seemed contradictory to results obtained in Table 1. Data points which describe the best informative range (during exponential phase) were taken and linear regression was used to determine the rate of RdRp activity. As seen in Figure 8, RdRp following induction showed a higher rate of reaction (Fluorescence output) in comparison to non-induced RdRp. Similarly, the same situation in which mutant RdRp (designated mRdRp in Fig 8) showed higher rate in comparison to non-induced mRdRp.

User Reviews

UNIQ3d18340e638f6713-partinfo-00000001-QINU UNIQ3d18340e638f6713-partinfo-00000002-QINU