Tokyo/Expression level check
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__NOTOC__ | __NOTOC__ | ||
| + | <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]] | ||
| + | <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]] | ||
| + | <br> | ||
==Expression level check on two different ''promoters + plasmid sets'' == | ==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. | 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: ==== | ||
| - | + | 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 | ||
| + | |||
<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:==== | ||
| - | Two plasmid sets, A4Δｐ+pc1-GFP and A4 hybrid+GFP PBR322TetR (+)AHL, | + | 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). |
| - | [[Image: | + | <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. |
| + | <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]] | ||
| + | [[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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