Contribution
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BBa_K1529321 (PrhlRR-GFP) and by improving plux/tet-GFP BBa_K1529311 (PluxLR-GFP) has been analyzed in vivo activity. Here, we added new data obtained by cell-free protein synthesis.
For the crosstalk experiments, we constructed two kinds of cell-free translation systems containing substantial amount of R proteins (luxR,rhlR). In those systems, we put signaling molecules (3OHSL-C4 or 3OHSL-C6) and target genes (plux/tet-GFP or prhl-RR-GFP), respectively. We measured the fluorescence of GFP expressed from plux/tet-GFP and prhl(RR)-GFP on a RT-qPCR machine.
Fig 1 shows the result of the crosstalk test in a cell-free system containing sufficient amount of LuxR protein. In the results, non-cognate Prhl(RR) promoter was highly activated by luxR-3OC6HSL complex as well as the cognate Plux/tet promoter. Fig 2 shows the result of the crosstalk test in a cell-free system containing sufficient amount of RhlR protein.
![Fig1 Prhl(RR)-GFP assay in a cell-free system containing LuxR](https://static.igem.org/mediawiki/2020/9/98/T--Waseda--crosstalk_luxR.png)
![Fig2 Prhl(RR)-GFP assay in a cell-free system containing rhlR](https://static.igem.org/mediawiki/2020/e/ec/T--Waseda--crosstalk_rhlR.png)
We have also described monotelpensynthesis by using either BBa_K3580101 (improved part) or BBa_K3580102 (new part).
![Fig. 2-2-2 GC / MS analysis results of limonene synthesis system](https://static.igem.org/mediawiki/2020/f/f7/T--Waseda--monoterpene_2-2-2_GCMS_analysis_results_of_limonene_synthesis_system.png)
We confirmed limonene synthesis GC/MS analysis with SIM. In this SIM analysis, ions with four m/z values characteristic in limonene (68 and 93) and sabinene (77 and 91, 93) were analyzed. By using authentic limonene standard, we confirmed a retention time for GC and the characteristic limonene SIM signal at the specific m/z values. At the same retention time with the standard, limonene-specific m/z value (68, 93) ions were detected in the selected ions (The upper right figure of Figure. 2-2-2). We also draw a GC chart by summation of the signals from the selected ions (The lower left figure of Figure. 2-2-2). By comparison with a negative control experiment which we omitted the extract containing GPP synthase and limonene synthase, we found clear peak from our limonene synthesis.