Tokyo/Expression level check

From 2007.igem.org

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__NOTOC__
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<br>[[Tokyo/Works|Works top]]　　0.[[Tokyo/Works/Hybrid promoter|Hybrid promoter]]　　1.[[Tokyo/Works/Formulation |Formulation]]　　2.[[Tokyo/Works/Assay |Assay1]]　　3.[[Tokyo/Works/Simulation |Simulation]]　　'''4.[[Tokyo/Works/Assay2 |Assay2]]'''　　5.[[Tokyo/Works/Future works |Future works]]
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<br>[[Tokyo/Works|Works top]]&nbsp;&nbsp;&nbsp;0.[[Tokyo/Works/Hybrid promoter|Hybrid promoter]]&nbsp;&nbsp;&nbsp;1.[[Tokyo/Works/Formulation |Formulation]]&nbsp;&nbsp;&nbsp;2.[[Tokyo/Works/Assay |Assay1]]&nbsp;&nbsp;&nbsp;3.[[Tokyo/Works/Simulation |Simulation]]&nbsp;&nbsp;&nbsp;'''4.[[Tokyo/Works/Assay2 |Assay2]]'''&nbsp;&nbsp;&nbsp;5.[[Tokyo/Works/Future works |Future works]]
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<br><br>[[Tokyo/Activation check by cell-produced AHL |Activation check by cell-produced AHL ]]　　[[Tokyo/Expression level check|Expression level check on ''promoters + plasmid sets'' of A and B sides]]
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<br><br>[[Tokyo/Activation check by cell-produced AHL |Activation check by cell-produced AHL ]]&nbsp;&nbsp;&nbsp;[[Tokyo/Expression level check|Expression level check on ''promoters + plasmid sets'' of A and B sides]]
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<br>
==Expression level check on two different ''promoters + plasmid sets'' ==
==Expression level check on two different ''promoters + plasmid sets'' ==
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==== Purpose: ====  
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==== Objective: ====  
To measure and compare the activities of two different ''promoters + plasmid sets'' by fluorescence of GFP downstream of each promoter.
To measure and compare the activities of two different ''promoters + plasmid sets'' by fluorescence of GFP downstream of each promoter.
Lambda cI-regulated promoter and the lux lac hybrid promoter were tested.
Lambda cI-regulated promoter and the lux lac hybrid promoter were tested.
==== Samples: ====
==== Samples: ====
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・A4Δｐ　pc1-GFP
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Each sample cells has two plasmids.
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<br>・A4 hybrid GFP PBR322TetR (+)AHL
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<br>・A4 hybrid GFP PBR322TetR (-)AHL
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*A4ΔP([http://partsregistry.org/Part:BBa_I751100 BBa_I751100]) / pc1-GFP ([http://partsregistry.org/Part:BBa_I751311 BBa_I751311])
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*A4 hybrid promoter([http://partsregistry.org/Part:BBa_I751101 BBa_I751101]) / pBR322TetR (+)AHL
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*A4 hybrid promoter([http://partsregistry.org/Part:BBa_I751101 BBa_I751101]) / pBR322TetR (-)AHL
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<br>*The promoter pcI sequence was designed to contain OR1, -35, and -10 regions, but not R2 or OR3.
<br>*The promoter pcI sequence was designed to contain OR1, -35, and -10 regions, but not R2 or OR3.
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====Result & Conclusion:====
====Result & Conclusion:====
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Two plasmid sets, A4Δｐ+pc1-GFP and A4 hybrid+GFP PBR322TetR (+)AHL, shows almost the same fluorescence of GFP, indicating that expression levels of both sets are almost the same though the latter is a bit smaller.
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Two plasmid sets, A4Δｐ+pc1-GFP and A4 hybrid+GFP PBR322TetR (+)AHL, show almost the same fluorescence of GFP, indicating that expression levels of both sets are almost the same though the latter is a bit smaller (Fig. 1).
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<br>From the results the ranges of bistability and those of mono-stability are calculated as shown in Fig. 1.
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<br>In Fig2, this expression balance indicated by red line are compared to the ranges of bistability and those of mono-stability shown by the simulations. In the next construction, RBS and/ or promoter modifications are required to adjust the expression balance of the repressors.  
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<br>.[[Image:pC1GFP.jpg|thumb|250px| '''Fig.1''' Genes downstream of the promoters on the both sides were expressed to almost the same degrees in terms of GFP fluorescence.|left]]
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<br>.[[Image:pC1GFP.jpg|thumb|350px| '''Fig.1''' Genes downstream of the promoters on the both sides were expressed to almost the same degrees in terms of GFP fluorescence.|left]]
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[[Image:Parameter set.jpg|thumb|500px|'''Fig.2 Calculated coexistance stable region''']]
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[[Image:Balanced.jpg|thumb|500px|'''Fig.2 Calculated coexistance stable region'''|center|left]]

Latest revision as of 04:28, 27 October 2007


Works top   0.Hybrid promoter   1.Formulation   2.Assay1   3.Simulation   4.Assay2   5.Future works

Activation check by cell-produced AHL    Expression level check on promoters + plasmid sets of A and B sides

Expression level check on two different promoters + plasmid sets

Objective:

To measure and compare the activities of two different promoters + plasmid sets by fluorescence of GFP downstream of each promoter. Lambda cI-regulated promoter and the lux lac hybrid promoter were tested.

Samples:

Each sample cells has two plasmids.

  • A4ΔP([http://partsregistry.org/Part:BBa_I751100 BBa_I751100]) / pc1-GFP ([http://partsregistry.org/Part:BBa_I751311 BBa_I751311])
  • A4 hybrid promoter([http://partsregistry.org/Part:BBa_I751101 BBa_I751101]) / pBR322TetR (+)AHL
  • A4 hybrid promoter([http://partsregistry.org/Part:BBa_I751101 BBa_I751101]) / pBR322TetR (-)AHL


*The promoter pcI sequence was designed to contain OR1, -35, and -10 regions, but not R2 or OR3.

Procedure:

AHL assay Standard protocol
Wash
OD and fluorescence were measured 0, 3, and 6 hours after the fresh culture incubation started.

Result & Conclusion:

Two plasmid sets, A4Δｐ+pc1-GFP and A4 hybrid+GFP PBR322TetR (+)AHL, show almost the same fluorescence of GFP, indicating that expression levels of both sets are almost the same though the latter is a bit smaller (Fig. 1).
In Fig2, this expression balance indicated by red line are compared to the ranges of bistability and those of mono-stability shown by the simulations. In the next construction, RBS and/ or promoter modifications are required to adjust the expression balance of the repressors.


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Fig.1 Genes downstream of the promoters on the both sides were expressed to almost the same degrees in terms of GFP fluorescence.
Fig.2 Calculated coexistance stable region
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