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

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<partinfo>BBa_K346001 short</partinfo>
 
<partinfo>BBa_K346001 short</partinfo>
  
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<partinfo>BBa_K346001 SequenceAndFeatures</partinfo>
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===Design Notes===
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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 teams, we prefixed an RBS part BBa_B0034 from Partsregistry.
  
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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).
  
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===Design Notes===
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[[Image:pc-merR-PmerT V3.jpg|600px|center]]
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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.
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MerR, as a regulatory protein, always binds to ''merOP'' as a homodimer and enhances the occupancy of Ptpad by RNA polymerase regardless of the presence of Hg(II), although only after Hg(II)’s binding can the dimer stop preventing the formation of the open complex by RNA polymerase. Also, MerR repress its own expression independently of Hg(II)(Fig.1).  
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'''Fig.3. The construction of bioreporter.'''
  
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[[Image:merR1.jpg]]
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We observed that cells with different MerR intensity exhibited correspondingly different sensitivity to mercury. Namely, the stronger the expression intensity of MerR is, the higher threshold will be represented(Fig.4).  
  
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'''Fig.1. The model for the interaction of MerR and Hg(II) and its role in controlling PmerT transcription. Adapted from Jon L. Hobman, John Wilkie & Nigel L. Brown,2005. A: RNA polymerase (RNAP) transcribes ''merR'' from PmerR. MerR binds to the mer promoter/operator region (''merOP'') as a homodimer, recruits RNA polymerase, and represses transcription of ''merTPAD'' from PmerT.''' B: Hg(II) enters the bacterial cell by diffusion through the outer membrane, cytoplasm and inner membrane, and binds to three cysteine residues in the apo-MerR homodimer. The Hg-bound MerR homodimer causes an underwinding of ''merOP'' DNA, allowing RNAP to proceed with transcription of the resistance genes.
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[[Image:pc.jpg|center]]
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'''Fig.4. The expression intensity of MerR significantly determines the threshold of response to mercury (II).''' Five representative lines are selected and it can be observed that the threshold has varied apparently. The letter in the bracket after each promoter name denotes the backbone (pSB3K3 or pSB1A2) where Pc-RBS-merR was cloned into. The deeper the colour represents, the stronger the expression level of MerR is, leading to a higher threshold.  
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<partinfo>BBa_K346001 SequenceAndFeatures</partinfo>
  
 
===Source===
 
===Source===
  
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The coding sequence of MerR comes from Tn, prefixed by an RBS part BBa_B0034 from Partsregistry
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The coding sequence of MerR comes from Tn21, prefixed by an RBS part BBa_B0034 from Partsregistry
  
 
===References===
 
===References===
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Brown, N. L., J. V. Stoyanov, et al. (2003). "The MerR family of transcriptional regulators." FEMS Microbiol Rev 27(2-3): 145-163.
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Hobman, J. L., J. Wilkie, et al. (2005). "A design for life: prokaryotic metal-binding MerR family regulators." Biometals 18(4): 429-436.
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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,
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Park, S. J., J. Wireman, et al. (1992). "Genetic analysis of the Tn21 mer operator-promoter." J Bacteriol 174(7): 2160-2171.

Latest revision as of 02:14, 28 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 teams, 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).

Pc-merR-PmerT V3.jpg

Fig.3. The construction of bioreporter.

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

Pc.jpg

Fig.4. The expression intensity of MerR significantly determines the threshold of response to mercury (II). Five representative lines are selected and it can be observed that the threshold has varied apparently. The letter in the bracket after each promoter name denotes the backbone (pSB3K3 or pSB1A2) where Pc-RBS-merR was cloned into. The deeper the colour represents, 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 Tn21, 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.