Difference between revisions of "Part:BBa K346002:Experience"
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== Promoter Characterization ==
 
== Promoter Characterization ==
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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.
+
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 intensity could be induced by Hg (II) in a dose response manner. The PTPCAD-E0840 was then cloned into pSB3K3 backbone and BBa_J23103 (constitutive promoter)-merR into pSB1A3.
  
 
'''Protocols for promoter characterization'''
 
'''Protocols for promoter characterization'''
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1.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.  
 
1.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.  
  
−
2.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.  
+
2.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 supplied 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  measurement and another 100uL was pipetted to the transparent-96-well plates for OD600 measurement.  
  
−
3.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.
+
3.In-plate culture fluorescence and OD600 was recorded at 20min intervals from 0 to 275min and the GFP intensity of 20min before induction was also measured. Temperature was constant at 37°C.
  
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The result is shown in Fig.3.
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The result is shown in Fig.5.
  
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[[Image:od-time.jpg]]
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[[Image:od-time.jpg|center]]
  
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'''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.
+
'''Fig.5. 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.
  
 +
== Construction of ''merOP'' Library ==
  
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== Cognate with constitutive promoter+RBS+''merR'' ==
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In our project, we planned to analyze the behavior of ''merOP'' thoroughly, and it could be expected that mutation at the dyad sequence would change the Ka of MerR-DNA interactions. Thus we mutated the semiconserved sites and left the conserved sites constant.
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One biosensor construct was made by fusing PmerT and a reporting system, ''gfp'', along with a plasmid structure that a constitutive promoter was prefixed before ''merR'' coding sequence. Promoters were selected from parts-registry constitutive promoter library and differed in strength. Theses constructs reacted to Hg(II) by dose-dependent light emission. As the concentration of Hg(II) was fixed, the proportion of Hg-bound MerR dimers was decided by the expression of merR, which is controlled by the different promoter intensities. Furthermore, we used pSB1A2 and pSB3K3 as backbones because of their different copy numbers which could result in different MerR concentrations(Fig.4).
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[[Image:pc.jpg]]
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The approach was generally based upon degenerate primer PCR, with the combination of a ‘DNA shuffling’ procedure, that was performed on the target DNA sequence; the resulting library of variants was then screened for the desired feature, and selected isolates are subjected to a repeated procedure(Fig.6).
  
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'''Fig.4. The comparison of GFP data.''' Five representative lines are selected and it can be seen that the thresholds have varied apparently. The deeper the colour, the stronger the expression of MerR, leading to the higher threshold.
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[[Image:PmerT-pcr.jpg|center]]
 +
 
 +
'''Fig.6. Library construction.''' We performed a degenerate primer PCR on the merTPCAD wild-type promoter region of the alterable sites. The resulting PCR fragments, each potentially contained one or more mutation sites at a restricted location.
 +
 
 +
[[Image:mutant-PmerT.jpg|center]]
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 +
'''Fig.7. Dose-response curves of mutants.''' Among all these candidates, mutant1, 3, 25, 44, 85, 88 (also shown in Fig 3) were selected for the final careful characterization during which a higher concentration resolution was exploited. Then the dose-response curves were fitted by Hill function.
 +
 
 +
[[Image:mutant-PmerT seq.jpg|center]]
 +
 
 +
'''Fig.8 Sequence comparison of the mutants'''
 +
 
 +
It can be observed that mutations at the semiconserved region of PmerT promoter significantly influenced the response behavior of MerR/PmerT pair(Fig.7). It is probably because that the mutations alter the binding affinity between MerR and PmerT promoter. As a result of this, different sensitivity (higher or lower than the wild-type) PmerT promoters were gained by us. More details about these mutants suffixed with GFP(BBa_E0840) were described in the parts BBa_K346020 - BBa_K346025.
  
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[[Image:mutant-PmerT.jpg]]
 
 
===User Reviews===
 
===User Reviews===
 
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Latest revision as of 03:08, 28 October 2010

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K346002

Promoter Characterization

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 intensity could be induced by Hg (II) in a dose response manner. The PTPCAD-E0840 was then cloned into pSB3K3 backbone and BBa_J23103 (constitutive promoter)-merR into pSB1A3.

Protocols for promoter characterization

1.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.

2.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 supplied 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 measurement and another 100uL was pipetted to the transparent-96-well plates for OD600 measurement.

3.In-plate culture fluorescence and OD600 was recorded at 20min intervals from 0 to 275min and the GFP intensity of 20min before induction was also measured. Temperature was constant at 37°C.

The result is shown in Fig.5.

Od-time.jpg

Fig.5. 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.

Construction of merOP Library

In our project, we planned to analyze the behavior of merOP thoroughly, and it could be expected that mutation at the dyad sequence would change the Ka of MerR-DNA interactions. Thus we mutated the semiconserved sites and left the conserved sites constant.

The approach was generally based upon degenerate primer PCR, with the combination of a ‘DNA shuffling’ procedure, that was performed on the target DNA sequence; the resulting library of variants was then screened for the desired feature, and selected isolates are subjected to a repeated procedure(Fig.6).

PmerT-pcr.jpg

Fig.6. Library construction. We performed a degenerate primer PCR on the merTPCAD wild-type promoter region of the alterable sites. The resulting PCR fragments, each potentially contained one or more mutation sites at a restricted location.

Mutant-PmerT.jpg

Fig.7. Dose-response curves of mutants. Among all these candidates, mutant1, 3, 25, 44, 85, 88 (also shown in Fig 3) were selected for the final careful characterization during which a higher concentration resolution was exploited. Then the dose-response curves were fitted by Hill function.

Mutant-PmerT seq.jpg

Fig.8 Sequence comparison of the mutants

It can be observed that mutations at the semiconserved region of PmerT promoter significantly influenced the response behavior of MerR/PmerT pair(Fig.7). It is probably because that the mutations alter the binding affinity between MerR and PmerT promoter. As a result of this, different sensitivity (higher or lower than the wild-type) PmerT promoters were gained by us. More details about these mutants suffixed with GFP(BBa_E0840) were described in the parts BBa_K346020 - BBa_K346025.

User Reviews

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