Difference between revisions of "Part:BBa K2638400"
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To identify the optimal strength of the gene knock-down through our RNAi / siRNA vectors, we tested different promoters and RBS combinations for the expression of the RNAi/siRNA system.  
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In synthetic biology the control of transcription and translation is of enormous importance. Therefore, promoters and ribosome binding sites (RBS) play a central role in each iGEM project. The selection of a suitable promoter/RBS combination is absolutely necessary to achieve the appropriate expression level for the respective purpose. We tested different combinations of known promoters and RBS, which are often used by the iGEM community. To test the expression strength of these promoter-RBS combinations we constructed a modified pSB1C3 backbone. An eCFP (BBa_E0022) under the control of a promoter from the Anderson collection, a synthetic RBS and two terminators were integrated into the pSB1C3 backbone. This expression unit is used as a reference. To test the expression strength of other promoter-RBS combinations a second reporter gene mRFP (BBa_E1010) under control of the respective target promoter-RBS combination was integrated between the pSB1C3 prefix and suffix. With the combination of the reference reporter gene in the backbone and the mRFP integrated between the pSB1C3 prefix and suffix, the expression strength of the different promoter-RBS combinations can be normalized to the certain expression of the reporter gene in the backbone. The detection of the differential expression strength of the different promoter-RBS combinations can be calculated through the ration of the fluorescence signals at 608 nm and 485 nm. For the detailed characterization see [[BBa_K2638560]].
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To test the expression strength of our promoter-RBS combination we constructed this part.
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We integrated an eCFP (BBa_E0022) under the control of a promoter from the Anderson collection, a synthetic RBS and two terminators in the pSB1C3 backbone. This expression unit is used as a reference.  
+
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To test the expression strength of our promoter-RBS combination a second reporter gene (mRFP BBa_E1010) under control of the target promoter-RBS combination was integrated between the pSB1C3 prefix and suffix.
+
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With the combination of the reporter gene in the backbone and the gene of interest integrated between the pSB1C3 prefix and suffix, the expression strength of the different promoter-RBS combinations can be normalized to the certain expression of the reporter gene in the backbone.  
+
−
The detection of the differential expression strength of the different promoter-RBS combinations can be calculated through the ration of the fluorescence signals at 608 nm and 485 nm.  
+
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For the full characterisation see [[BBa_K2638560]].
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Revision as of 22:32, 17 October 2018


Combination of BBa_K2638500 + BBa_K2638560


In synthetic biology the control of transcription and translation is of enormous importance. Therefore, promoters and ribosome binding sites (RBS) play a central role in each iGEM project. The selection of a suitable promoter/RBS combination is absolutely necessary to achieve the appropriate expression level for the respective purpose. We tested different combinations of known promoters and RBS, which are often used by the iGEM community. To test the expression strength of these promoter-RBS combinations we constructed a modified pSB1C3 backbone. An eCFP (BBa_E0022) under the control of a promoter from the Anderson collection, a synthetic RBS and two terminators were integrated into the pSB1C3 backbone. This expression unit is used as a reference. To test the expression strength of other promoter-RBS combinations a second reporter gene mRFP (BBa_E1010) under control of the respective target promoter-RBS combination was integrated between the pSB1C3 prefix and suffix. With the combination of the reference reporter gene in the backbone and the mRFP integrated between the pSB1C3 prefix and suffix, the expression strength of the different promoter-RBS combinations can be normalized to the certain expression of the reporter gene in the backbone. The detection of the differential expression strength of the different promoter-RBS combinations can be calculated through the ration of the fluorescence signals at 608 nm and 485 nm. For the detailed characterization see BBa_K2638560.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 611
    Illegal AgeI site found at 723
  • 1000
    COMPATIBLE WITH RFC[1000]