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Latest revision as of 02:27, 6 October 2011

ptsG2-GFP (ptsG2 5'UTR fused with gfp)

Background

PtsG2 is the C87G mutant of ptsG(wt) and the conjugate part of SgrS2 in our comparator device.

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). A 31-nt-long stretch in the 3’ region of SgrS is partially complementary to the translation initiation region of ptsG mRNA, and a 6 nt region overlapping the Shine-Dalgarno sequence of the target mRNA turns out to be crucial for SgrS’ function. When imperfect base-pairing interactions between ptsG and SgrS occurs and this critical region is involved, ptsG's expression is repressed.(See Fig.1).

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, and ptsG2 is one mutant of ptsG(wt) utilized in our system(See Fig.2).

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 3.

Fig. 3 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 specifity of ptsG2/SgrS interaction, 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 4 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.

The original data also provided below (Table 1).

Fig. 4 A graphical representation of the repression matrix associated with SgrS and its mutants, and ptsG and its mutants. The values represent the repression ratios, defined as the repression capacity of each ptsG/SgrS pair, denoted by the ratio of fluorescence intensity before to after the induction of SgrS, suggesting within-subgroup pairwise specificity.

Part II. The Response Curve

The sRNA-mediated gene silencing can be formulated quantitatively via a simple kinetic model. The model is cast in terms of two mass-action equations for the cellular concentrations of the sRNA (s) and its target mRNA (m):

The parameters are defined as in Table 2.

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)(see Fig.5).

Fig. 5 Predicted response curve of a target gene. (a) The red line depicts the idealized threshold-linear mode of regulation in which gene expression is completely silenced if the SgrS transcription rate exceeds a threshold set by the transcription rate of the ptsG-gfp mRNA. Under this threshold, gene expression decreases linearly with the difference between the mRNA and sRNA transcription rates. (b) The red line depicts the idealized threshold-linear mode of regulation in which gene expression is completely silenced if the ptsG-gfp mRNA transcription rate is below a threshold set by the transcription rate of the sRNA. Above this threshold, gene expression increases linearly with the difference between the mRNA and sRNA transcription rates. The idealized scenario is expected when binding between sRNA and mRNA occurs extremely rapidly. The blue line is the actual response expected using the estimated parameters of Table 2

Our data of ptsG2/SgrS2 interaction fitted into the predicted scenario quite readily, just as Fig 6 shows.

Fig. 6 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 R^2 is 0.9607.

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