Difference between revisions of "Part:BBa K2638400"
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| − | To | + | 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. |
| − | To test the expression strength of our promoter-RBS combination | + | To test the expression strength of our promoter-RBS combination we constructed this part. |
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. | 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. | ||
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. | 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. | ||
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. | 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. | 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 full characterisation | + | For the full characterisation see [[BBa_K2638560]]. |
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Revision as of 21:28, 17 October 2018
Combination of BBa_K2638500 + BBa_K2638560
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.
To test the expression strength of our promoter-RBS combination we constructed this part.
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.
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.
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.
For the full characterisation see BBa_K2638560.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 611
Illegal AgeI site found at 723 - 1000COMPATIBLE WITH RFC[1000]