Part:BBa_K581006

Pc+ptsG(wt)-GFP (ptsG(wt) 5' UTR fused with gfp)

Background

PtsG is a glucose permease which is subordinate to phosphotransferase system and serves as a transporter. Here,we studied this mRNA perform the conjugate part of the small RNA regulator sgrS(wt)[1]. ptsG mRNA is regulated by SgrS by short, imperfect base-pairing interactions, and its expression is thus repressed(See Fig.1).

Figure 1: Sequence alignment of wildtype ptsG/SgrS pair and its mutant complementary pairs.

Teppei Morita et.al’ s work suggests that two mutations (C85G and C87G) in ptsG mRNA could completely impair the ability of SgrS to downregulate its expression, while compensatory mutations of SgrS (G178C and G176C) restore the gene silencing ability. These results indicate that it is the base pairing of the two RNAs rather than particular nucleotides that is important for SgrS action. They have also illustrated that sequence outside this region, even though complementary, is rather dispensable for the efficient silencing (Kawamoto et al., 2006). This makes mutant ptsG/SgrS pairs orthogonal to genetic context of the host cell. Therefore we choose this couple of conjugate mRNA/sRNA as the foundation of our comparator device design.

By employing two sets of mutant ptsG mRNA as well as its complementary SgrS in the design shown in Fig 1, we set to biologically implement the comparator. In detail, ptsG1 refers to a C85G mutant of ptsG (wt) while ptsG2 is a C87G mutant. SgrS1 (G178C) and SgrS2 (G176C) are the corresponding revertants which could help restore their complementarity. And as a proof-of-concept experiment, we constructed synthetic gene circuits, in which the 5’ untranslated region of ptsG mRNA was translationally fused to the coding sequence of the reporter gfp (Levine et al., 2007), as shown in Fig 2.

Fig. 2 The modular components of the comparator. (A) Salicylate leads to the transcription of ptsG-gfp mRNA, which is the target of constitutively expressed SgrS. This is how we implemented both reporting and repressing outputs as a result of the activation of Psal. When there is more salicylate in the media, the GFP fluorescence intensity is expected to be stronger. (B) Salicylate leads to the transcription of SgrS, while the ptsG-gfp mRNA is downstream a constitutive promoter. In this scenario, as the concentration of salicylate increases, the repression effect SgrS exerts on ptsG would in turn be stronger, so the GFP fluorescence intensity is supposed to be weaker.

Furthermore, as a proof-of-concept experiment, we constructed synthetic gene circuits, in which the 5’ untranslated region of ptsG mRNA was translationally fused to the coding sequence of the reporter gfp. The fluorescence intensity of GFP could reflect the repression effect that SgrS exerts on ptsG.

Experimental Data

To qualitatively and quantitatively characterize the performance of our competitor, we conducted the following experiments.

Part I. The Orthogonal Silencing Matrix

The repression capacity of each ptsG/SgrS pair was indicated by the ratio of the average fluorescence intensity before to after the trigger of SgrS. What we expected was a significant repression within the cognate pairs (ptsG1/SgrS1, ptsG2/SgrS2, and ptsG (wt)/SgrS (wt)), and a minor repression folds among different pairs.

As Figure 3 shows, the highest ratio lie at the diagonal from the upper left to the lower right as expected, which is 5 to 6 folds. As for the ptsG (wt)/SgrS1&2, ptsG1/SgrS (wt), and ptsG2/SgrS (wt), given that these crosses differ at only one base pair, the repression efficacy is around 3 folds. By contrast, the inhibiting effect of on ptsG2 and SgrS2 on ptsG1 is rather unapparent, which can be seen as an appropriate characteristic fitting our competitor requirements.

Part II. The Response Curve

Levine et. al’s work has revealed that in the idealized scenario when binding between sRNA and mRNA occurs extremely rapidly, gene expression is completely silenced if the target transcription rate is below a threshold. Above this threshold, gene expression will increase linearly. Such threshold-linear model is based on the difference of transcription rates between sRNA and mRNA(Levine et al., 2007).

Our data fitted with the predicted scenario quite readily, just as Fig 4 shows.

Fig. 4 The dose-response curve of SgrS/ptsG-gfp interaction. (A) Salicylate-induced SgrS repressing the expression of ptsG-GFP. The promoter activity is defined as the GFP expression of the Psal+gfp strain grown in identical media. Different promoter activities were obtained by varying salicylate concentration in the media. The conjugate pairs fit into dose-response curve with variable Hill slope given as a parameter, and the R2 is 0.9974, 0.9879, and 0.9607 corresponding to ptsG(wt)/SgrS(wt), ptsG1/SgrS1 and ptsG2/SgrS2, respectively. (B) When sgrS (wt) was constitutively expressed and ptsG (wt) salicylate-induced, a dose-response curve is also fitted with an R2 of 0.9301.

Such results are in accordance with Levine et. al’s conclusion, i.e., the binding rates between mRNA and sRNA in effect are inherently limited, so the threshold-linear model couldn’t be strictly fitted (Levine et al., 2007). But the performance of SgrS/ptsG pairs is very close to the idealized threshold-linear mode of regulation.

Reference

[1] Geissmann, T.A., and Touati, D. (2004). Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator. The EMBO journal 23: 396-405

[2] Kawamoto, H., Koide, Y., Morita, T., and Aiba, H. (2006). Base-pairing requirement for RNA silencing by a bacterial small RNA and acceleration of duplex formation by Hfq. Molecular microbiology 61: 1013-1022

[3] Levine, E., Zhang, Z., Kuhlman, T., and Hwa, T. (2007). Quantitative characteristics of gene regulation by small RNA. PLoS biology 5: e229 Sequence and Features

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