Part:BBa_K581003

SgrS2+Terminator (small RNA regulator, conjugate part of ptsG2)

This is the conjugate part of ptsG2-gfp.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

This BioBrick has been sequence verified.

Background

SgrS is an Hfq-binding small antisense RNA that is induced upon phosphosugar stress (Vanderpool, 2007). It forms a ribonucleoprotein complex with RNase E through Hfq to mediate silencing of the target ptsG mRNA encoding the major glucose transporter (Geissmann and Touati, 2004). 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, shown as Fig 2 (Kawamoto et al., 2006; Maki et al., 2010).

Figure 1:The complementary pair site-mutant version of ptsG2 mRNA and corresponding SgrS2.

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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 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 (Fig.1).

Fig.1A 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. Response Curves

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):

References

[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