Part:BBa_K581004

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

PtsG2 is the C85G 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)[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.

A GFP reporter with C-terminus fused ssrA degredation tag was used to report the transcriptional activity at the promoters. The rhl-box promoter-GFP ssrA was then cloned into pSB4A5 backbone and the BBa_J23106 (constitutive promoter)-rhlR- BBa_B0015 (terminator) into pSB1K3.

Figure 3: The genetic construction of rhl repressible promoter-rhlR system used for characterization.

To evaluate the properties of rhl repressible promoter, we applied dose response assay and time dependence assay. In these assays GFP intensity was measured to quatitatively evaluate the promoter acitivity by Tecan Microplate Reader with excitation wavelength at 470nm and emission wavelength at 509nm. A black 96-well plate was used to minimize the interference of different well. OD 600 was also measured by Tecan Microplate Reader in a transparent 96-well plate.

Dose response assay

An overnight culture of bacteria grown in LB with ampicillin and kanamycin at 37°C was reactivated by diluting the culture in a ratio of 1:1000 with fresh LB. The LB we used was pre-mixed with different dose of C4-HSL and its final concentration varied from 0 to 1mM/1μM. When OD600 reached 0.6-0.8, pellet bacterial cells by 4 min centrifugation at 4000 rpm, discard the supernatant. Resuspend the pelleted cells in 500 μl of PBS, and then pippete 200uL of bacterial resuspention into each well of 96-well plate.

Figure 4: Repression effect of previous transcription activators at rhl repressible promoter under a gradient of C4-HSL concentrations.

Figure 4 shows repression of GFP expression in circuits depicted in Fig 2 under gradient concentrations of C4-HSL, which indicates that the activity of rhl repressible promoter was C4-HSL dependent, with strongest repression at 5*10^-4 M of C4-HSL.

Quantitative analysis was also conducted. As shown in the following figure and table, the dose response curve represents as a hill function and the decrease of transcription activity at promoter was approximate 20-fold.

Figure 5: Fold repression of GFP/OD expressed by rhl repressible promoter on C4-HSL signal concentrations. In this experiment, GFP expression in E. coli containing pSB1K3 and pSB4A5 was measured at a series of concentrations of C4-HSL. The fold induction of GFP/OD is expressed as the percentage of expression in the absence of C4-HSL. Error bars correspond to the standard deviation from multiple measurements.

Table 1: The parameters of Hill function fitting

Therefore, we have demonstrated that the AHL repressible promoter with rhl-box owes the ability to convert transcriptional activator rhlR into repressor.

Time dependence assay

An overnight culture of grown in LB with ampicillin and kanamycin at 37°C was reactivated by diluting the culture in a ratio of 1:1000 with fresh LB. When OD600 reached 0.4, the bacteria was disposed to several EP tubes, each owning 500uL, and C4-HSL was supplied with 3 duplicates and the final concentration was 1mM. We cultured the fluorescence in EP tubes with 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8 hours at 37°C. Then pellet bacterial cells by 4 min centrifugation at 4000 rpm, discard the supernatant. Resuspend the pelleted cells in 500 μl of PBS, and then pippete 200uL of bacterial resuspention into each well of 96-well plate.

The results of the assay are displayed below.

Figure 6: Time dependence of AHL mediated repression at rhl repressible promoter under 1mM of C4-HSL . (A) GFP/OD; (B) fold repression of GFP/OD. Error bars correspond to the standard deviation from multiple measurements.

Noting that the GFP expression intensity was reduced from 100% to 10% in merely 2-3 hours, we were delighted by the dramatic repression performance of quorum sensing repressors. The time dependence assay provides further promising clues towards the expansion of our quorum sensing repressor design to more QS systems.

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 Sequence and Features

Assembly Compatibility:

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• 1000
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  Illegal BsaI.rc site found at 747