Difference between revisions of "Part:BBa K346001:Design"

Revision as of 17:10, 26 October 2010

RBS (B0034) + MerR (mercury-responsive transcription factor)

Design Notes

The native RBS of MerR is not strong enough and its intensity can not be predicted as there is no such a corresponding RBS part in Registry. In order to facilitate the use and further characterization of MerR for future team, we prefixed an RBS part BBa_B0034 from Partsregistry.

The model of MerR controlling PmerT tracscription indicates that the apo-merR and Hg-bound merR have a competition. We speculate that the threshold of MerR response can be also manipulated by controlling the concentration of MerR in cytosol. As with the bacteria in natural environment, the concentration of MerR is stabilized at a certain level. In our design, merR was isolated from the operon and assembled with constitutive promoters of certain strength to maintain its expression intensity at certain level, then the sensitivity of PmerT under different MerR concentrations can be denoted by mercury threshold concentration at which reporter (GFP) expression emerges. (Fig.3).

Fig.3. The construction of bioreporter. This biosensor construct was made by fusing PmerT and a reporting system, gfp, along with a plasmid structure that constitutive promoters prefixed before merR coding sequence.

We observed that cells with different MerR intensity exhibited correspondingly different sensitivity to mercury, indicating that the stronger the expression level of MerR is, a higher threshold is represented(Fig.4).

Fig.4. The expression intensity of MerR significantly determines the threshold of sensitivity to mercury (II). Five representative lines are selected and it can be seen that the thresholds have varied apparently. The letter in the bracket after the promoter name denotes the backbone (pSB3K3 or pSB1A2) where Pc-RBS-merR was cloned. The deeper the colour, the stronger the expression level of MerR is, leading to a higher threshold.

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]

Source

The coding sequence of MerR comes from Tn, prefixed by an RBS part BBa_B0034 from Partsregistry

References

Brown, N. L., J. V. Stoyanov, et al. (2003). "The MerR family of transcriptional regulators." FEMS Microbiol Rev 27(2-3): 145-163.

Hobman, J. L., J. Wilkie, et al. (2005). "A design for life: prokaryotic metal-binding MerR family regulators." Biometals 18(4): 429-436.

Diana M. Ralston, Tomas V. O'Halloran, et al. (1990). “Ultrasensitivity and heavy-metal selectivity of the allostericalyl modulated MerR transcription complex” Proc. Natl. Acad. Sci. USA, Vol. 87, pp. 3846-3850,

Park, S. J., J. Wireman, et al. (1992). "Genetic analysis of the Tn21 mer operator-promoter." J Bacteriol 174(7): 2160-2171.

@@ Line 5: / Line 5: @@
 The native RBS of MerR is not strong enough and its intensity can not be predicted as there is no such a corresponding RBS part in Registry. In order to facilitate the use and further characterization of MerR for future team, we prefixed an RBS part  BBa_B0034 from Partsregistry.
-The model of MerR controlling PmerT tracscription indicates that the apo-merR and Hg-bound merR have a competition. We speculate that the threshold of MerR response can be also manipulated by controlling the concentration of MerR in cytosol. As with the bacteria in natural environment, the concentration of MerR is stabilized at a certain level. In our design, merR was isolated from the operon and assembled with constitutive promoters of certain strength to maintain its expression intensity at certain level(Fig.3).
+The model of MerR controlling PmerT tracscription indicates that the apo-merR and Hg-bound merR have a competition. We speculate that the threshold of MerR response can be also manipulated by controlling the concentration of MerR in cytosol. As with the bacteria in natural environment, the concentration of MerR is stabilized at a certain level. In our design, merR was isolated from the operon and assembled with constitutive promoters of certain strength to maintain its expression intensity at certain level, then the sensitivity of PmerT under different MerR concentrations can be denoted by mercury threshold concentration at which reporter (GFP) expression emerges. (Fig.3).
 [[Image:pc-merR-PmerT.jpg]]
-Fig.3. The construction of bioreporter. This biosensor construct was made by fusing PmerT and a reporting system, gfp, along with a plasmid structure that constitutive promoters prefixed before merR coding sequence.
+'''Fig.3. The construction of bioreporter.''' This biosensor construct was made by fusing PmerT and a reporting system, ''gfp'', along with a plasmid structure that constitutive promoters prefixed before merR coding sequence.
+We observed that cells with different MerR intensity exhibited correspondingly different sensitivity to mercury, indicating that the stronger the expression level of MerR is, a higher threshold is represented(Fig.4).
+[[Image:pc.jpg]]
+'''Fig.4. The expression intensity of MerR significantly determines the threshold of sensitivity to mercury (II).''' Five representative lines are selected and it can be seen that the thresholds have varied apparently. The letter in the bracket after the promoter name denotes the backbone (pSB3K3 or pSB1A2) where Pc-RBS-merR was cloned. The deeper the colour, the stronger the expression level of MerR is, leading to a higher threshold.
 ----