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Team:Peking/Project/Biosensor/PromoterCharacterization
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| - | ===Promoter Characterization=== | + | <html> |
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| + | <font size=5><font color=000><font face="Franklin Gothic Demi Cond"> Promoter Characterization</font></font></font> | ||
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| + | [[Team:Peking/Project|Project]] > [[Team:Peking/Project/Biosensor|Biosensor]] > [[Team:Peking/Project/Biosensor/PromoterCharacterization|Promoter Characterization]]<html> | ||
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| + | PmerT is a promoter from Tn21 mercury resistance (mer) operon. Transposon 21 itself is carried by plasmid NR1 (Nakaya, Nakamura et al. 1960), which was originally isolated from Shigella flexneri in Japan in the late 1950s (Liebert, Hall et al. 1999). | ||
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| + | The mer operon of Tn21 consists of two tightly overlapped, divergently oriented promoters – PR and PTPCAD.(Park, Wireman et al. 1992). PR is the promoter of the regulatory protein gene, merR, and PTPCAD is for the transcription of the structural gene – merPTCAD. They are called merOP as a whole. MerR, as a regulatory protein, always binds to merOP as a homodimer and enhances the occupancy of PTPCAD 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). 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. For the same reason, the divergent promoter PR was also removed by deletion of its -35 region (Fig 1). | ||
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| + | <html><a href="https://static.igem.org/mediawiki/2010/5/5b/Pc1.png" target="blank"><img src="https://static.igem.org/mediawiki/2010/5/5b/Pc1.png" width=650 ></a></html><br> | ||
| + | '''Fig 1 DNA sequence of the Tn21 mer operator promoter region. The MerR binding site on PmerT is marked by a box. The -35 and -10 regions for both PmerR and PmerTPAD are marked with boxes, and the dyad symmetrical DNA sequence that MerR recognizes and binds to is marked with arrows under the DNA sequence. (A) The divergently oriented promoters are marked by blue box and purple box, respectively. (B) In our project, the expression intensity of MerR should be maintained exogenously, so the divergent promoter PR (of MerR transcript) was also removed by deletion of its -35 region. The resulted promoter sequence is marked with a dark purple box Modified from (Hobman, Wilkie et al. 2005)''' | ||
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| + | As shown in Fig 1, the key sequence for MerR’s binding is a region of interrupted dyad symmetry (19bp) located between the -35 and -10 haxamers of PTPCAD (The top strand). And the structure of PR (botton strand) is similar to PTPCAD in a divergent orientation. The -10 hexamers of PTPCAD and PR actually overlap by four bases. (Park, Wireman et al. 1992). | ||
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| + | <html><a href="https://static.igem.org/mediawiki/2010/f/fc/Pc2.png"target="blank"><img src="https://static.igem.org/mediawiki/2010/f/fc/Pc2.png" width=650 ></a></html><br> | ||
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| + | '''Fig. 2. A generalized mechanism of MerR family regulator transcriptional activation. (A) The dimeric MerR regulator binds to the operator region of the promoter and recruits RNA polymerase, forming a ternary complex. Transcription is slightly repressed because the apo-MerR regulator dimer has bent the promoter DNA such that RNA polymerase does not contact it properly. (B) Upon binding the cognate metal ions (shown as cyan circles) the metallated MerR homodimer causes a realignment of the promoter such that RNA polymerase contacts the -35 and -10 sequences leading to open complex formation and transcription. Modified from Brown et al.''' | ||
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| + | The model of MerR’s action is shown in Fig 2. In (A), merR (showed as green dimer downside the DNA strand) bound to PTPCAD and recruited RNA polymerase but was unable to form an open complex. Hg (II)’s binding to the dimer changed its conformation and transcription was initiated. (Brown, Stoyanov et al. 2003) | ||
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| + | By annealing PmerT-forward (5’-3’) and PmerT-reverse (5’-3’) primers, PTPCAD carrying a sticky end of EcoRI and SpeI was cloned upstream of BBa_E0840, a GFP generator. With exogenous expression of MerR in bacteria, GFP’s expression could be induced by Hg (II) in a dose response manner. The PTPCAD-E0840 was then cloned into pSB3K3 backbone and the BBa_J23103 (constitutive promoter)-merR into pSB1A3. | ||
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| + | ===Protocols for promoter characterization=== | ||
| + | '''An overnight culture of bacteria carrying the two plasmids pSB3K3 and pSB1A3 were grown in LB broth with ampicillin and kanamycin at 37°C was reactivated by diluting the culture in a ratio of 1:100 with fresh LB. When OD600 reached 0.4-0.6, the bacteria was disposed to several EP tubes, each owning 500uL, and different dose of Mercuric chloride solution was spplied with 3 duplicates and the final concentration varied from 0 to 1.0E-6 mol/L. 100uL of the 500uL was added to the black-96-well plates for GFP intensity’s measurement and another 100uL was added to the transparent-96-well plates for OD600 measurement. In-plate culture fluorescence and OD600 was recorded at 20min intervals from 0 to 275min and the GFP intensity of 20min before inducement was also measured. Temperature was constant at 37°C. ''' | ||
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| + | <html><a href="https://static.igem.org/mediawiki/2010/f/fa/Pc3.png"target="blank"><img src="https://static.igem.org/mediawiki/2010/f/fa/Pc3.png" width=650 ></a></html><br> | ||
| + | '''Fig. 3. Time and dose response of GFP and OD 600. The data was measured every 20 minutes since 20 minutes before supplement of different dose of Hg(II) to the wells and the plates was incubated in the shaker at 37℃during the interval of measurement. For both plates, the volume of LB medium with bacteria was 100uL per well. (a) GFP intensity was measured 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. (b) OD 600 was also measured by Tecan Microplate Reader in a transparent 96-well plate, since the depth of 100uL in the well did not reach 1 centimeter, the OD 600 value here was smaller than that was measured by a spectrophotometer.''' | ||
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| + | As shown in Fig 3, the basal level of PTPCAD is relatively low. At the concentration of 5.0E-8 mol/L, the expression of GFP was observed. With longer induction time and higher concentration of Hg (II), the fluorescent intensity dramatically increased in a narrow concentration range (Fig. 3a). After the normalization by OD 600, the trend did not change obviously (figure not shown). Around 120min later, bacteria growth reached stationary phase (Fig. 3b) while GFP still accumulated rapidly (Fig. 3a). When the concentration of Hg(II) was higher than 1.0E-6 mol/uL, GFP expression level grew slower which might be explained by the saturation of MerR by Hg(II) and the cytotoxity of mercury. | ||
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| + | <html><a href="https://static.igem.org/mediawiki/2010/2/26/Pc4.png"target="blank"><img src="https://static.igem.org/mediawiki/2010/2/26/Pc4.png" width=650 ></a></html><br> | ||
| + | '''Fig 4. Dose response curve of MerR/PmerT to mercury in vivo. Note that the GFP expression intensity was induced from 10% to the maximum across a narrow range – 7 fold change in Hg (II) concentration. The basal level is low and the activation fold is dramatic. Also the metal reorganization specificity of MerR was demonstrated by the result that lead (II) could not activate significant GFP expression in vivo. ''' | ||
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| + | Besides, it is obvious that the MerR dependent regulation worked as a hypersensitive switch which represented negligible basal level expression of GFP, but induced from 10% to 90% of maximum in response across a range of Hg (II) concentration as narrow as 7-fold change (Fig 4). When the expression was fully induced, it was even below the threshold at which Hg (II) adversely affects the growth rate of bacterial cells. Actually, the hypersensitive switch behavior is postulated to arise from the unusual metal binding mechanism of MerR. Therefore, the molecular basis of the hypersensitivity switch will be discussed thoroughly in Bioabsorbent part of our wiki. | ||
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| + | ===reference=== | ||
| + | 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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| + | Liebert, C. A., R. M. Hall, et al. (1999). "Transposon Tn21, flagship of the floating genome." Microbiol Mol Biol Rev 63(3): 507-522. | ||
| + | |||
| + | Nakaya, R., A. Nakamura, et al. (1960). "Resistance transfer agents in Shigella." Biochem Biophys Res Commun 3: 654-659. | ||
| + | |||
| + | Park, S. J., J. Wireman, et al. (1992). "Genetic analysis of the Tn21 mer operator-promoter." J Bacteriol 174(7): 2160-2171. | ||
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| + | </div> | ||
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Revision as of 06:12, 26 October 2010
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The mer operon of Tn21 consists of two tightly overlapped, divergently oriented promoters – PR and PTPCAD.(Park, Wireman et al. 1992). PR is the promoter of the regulatory protein gene, merR, and PTPCAD is for the transcription of the structural gene – merPTCAD. They are called merOP as a whole. MerR, as a regulatory protein, always binds to merOP as a homodimer and enhances the occupancy of PTPCAD 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). 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. For the same reason, the divergent promoter PR was also removed by deletion of its -35 region (Fig 1).
Fig 1 DNA sequence of the Tn21 mer operator promoter region. The MerR binding site on PmerT is marked by a box. The -35 and -10 regions for both PmerR and PmerTPAD are marked with boxes, and the dyad symmetrical DNA sequence that MerR recognizes and binds to is marked with arrows under the DNA sequence. (A) The divergently oriented promoters are marked by blue box and purple box, respectively. (B) In our project, the expression intensity of MerR should be maintained exogenously, so the divergent promoter PR (of MerR transcript) was also removed by deletion of its -35 region. The resulted promoter sequence is marked with a dark purple box Modified from (Hobman, Wilkie et al. 2005)
As shown in Fig 1, the key sequence for MerR’s binding is a region of interrupted dyad symmetry (19bp) located between the -35 and -10 haxamers of PTPCAD (The top strand). And the structure of PR (botton strand) is similar to PTPCAD in a divergent orientation. The -10 hexamers of PTPCAD and PR actually overlap by four bases. (Park, Wireman et al. 1992).
Fig. 2. A generalized mechanism of MerR family regulator transcriptional activation. (A) The dimeric MerR regulator binds to the operator region of the promoter and recruits RNA polymerase, forming a ternary complex. Transcription is slightly repressed because the apo-MerR regulator dimer has bent the promoter DNA such that RNA polymerase does not contact it properly. (B) Upon binding the cognate metal ions (shown as cyan circles) the metallated MerR homodimer causes a realignment of the promoter such that RNA polymerase contacts the -35 and -10 sequences leading to open complex formation and transcription. Modified from Brown et al.
The model of MerR’s action is shown in Fig 2. In (A), merR (showed as green dimer downside the DNA strand) bound to PTPCAD and recruited RNA polymerase but was unable to form an open complex. Hg (II)’s binding to the dimer changed its conformation and transcription was initiated. (Brown, Stoyanov et al. 2003)
By annealing PmerT-forward (5’-3’) and PmerT-reverse (5’-3’) primers, PTPCAD carrying a sticky end of EcoRI and SpeI was cloned upstream of BBa_E0840, a GFP generator. With exogenous expression of MerR in bacteria, GFP’s expression could be induced by Hg (II) in a dose response manner. The PTPCAD-E0840 was then cloned into pSB3K3 backbone and the BBa_J23103 (constitutive promoter)-merR into pSB1A3.
Protocols for promoter characterization
An overnight culture of bacteria carrying the two plasmids pSB3K3 and pSB1A3 were grown in LB broth with ampicillin and kanamycin at 37°C was reactivated by diluting the culture in a ratio of 1:100 with fresh LB. When OD600 reached 0.4-0.6, the bacteria was disposed to several EP tubes, each owning 500uL, and different dose of Mercuric chloride solution was spplied with 3 duplicates and the final concentration varied from 0 to 1.0E-6 mol/L. 100uL of the 500uL was added to the black-96-well plates for GFP intensity’s measurement and another 100uL was added to the transparent-96-well plates for OD600 measurement. In-plate culture fluorescence and OD600 was recorded at 20min intervals from 0 to 275min and the GFP intensity of 20min before inducement was also measured. Temperature was constant at 37°C.
Fig. 3. Time and dose response of GFP and OD 600. The data was measured every 20 minutes since 20 minutes before supplement of different dose of Hg(II) to the wells and the plates was incubated in the shaker at 37℃during the interval of measurement. For both plates, the volume of LB medium with bacteria was 100uL per well. (a) GFP intensity was measured 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. (b) OD 600 was also measured by Tecan Microplate Reader in a transparent 96-well plate, since the depth of 100uL in the well did not reach 1 centimeter, the OD 600 value here was smaller than that was measured by a spectrophotometer.
As shown in Fig 3, the basal level of PTPCAD is relatively low. At the concentration of 5.0E-8 mol/L, the expression of GFP was observed. With longer induction time and higher concentration of Hg (II), the fluorescent intensity dramatically increased in a narrow concentration range (Fig. 3a). After the normalization by OD 600, the trend did not change obviously (figure not shown). Around 120min later, bacteria growth reached stationary phase (Fig. 3b) while GFP still accumulated rapidly (Fig. 3a). When the concentration of Hg(II) was higher than 1.0E-6 mol/uL, GFP expression level grew slower which might be explained by the saturation of MerR by Hg(II) and the cytotoxity of mercury.
Fig 4. Dose response curve of MerR/PmerT to mercury in vivo. Note that the GFP expression intensity was induced from 10% to the maximum across a narrow range – 7 fold change in Hg (II) concentration. The basal level is low and the activation fold is dramatic. Also the metal reorganization specificity of MerR was demonstrated by the result that lead (II) could not activate significant GFP expression in vivo.
Besides, it is obvious that the MerR dependent regulation worked as a hypersensitive switch which represented negligible basal level expression of GFP, but induced from 10% to 90% of maximum in response across a range of Hg (II) concentration as narrow as 7-fold change (Fig 4). When the expression was fully induced, it was even below the threshold at which Hg (II) adversely affects the growth rate of bacterial cells. Actually, the hypersensitive switch behavior is postulated to arise from the unusual metal binding mechanism of MerR. Therefore, the molecular basis of the hypersensitivity switch will be discussed thoroughly in Bioabsorbent part of our wiki.
reference
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.
Liebert, C. A., R. M. Hall, et al. (1999). "Transposon Tn21, flagship of the floating genome." Microbiol Mol Biol Rev 63(3): 507-522.
Nakaya, R., A. Nakamura, et al. (1960). "Resistance transfer agents in Shigella." Biochem Biophys Res Commun 3: 654-659.
Park, S. J., J. Wireman, et al. (1992). "Genetic analysis of the Tn21 mer operator-promoter." J Bacteriol 174(7): 2160-2171.
