Difference between revisions of "Part:BBa J23106"

Revision as of 02:09, 2 November 2017

constitutive promoter family member

 Variant RFP (au)
 J23112           1
 J23103           17
 J23113           21
 J23109           106
 J23117           162
 J23114           256
 J23115           387
 J23116           396
 J23105           623
 J23110           844
 J23107           908
 J23106           1185
 J23108           1303
 J23118           1429
 J23111           1487
 J23101           1791
 J23104           1831
 J23102           2179
 J23100           2547

Constitutive promoter family
Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. J23119 is the "consensus" promoter sequence and the strongest member of the family. All parts except J23119 are present in plasmid J61002. Part J23119 is present in pSB1A2. This places the RFP downstream of the promoter. Reported activities of the promoters are given as the relative fluorescence of these plasmids in strain TG1 grown in LB media to saturation. See part BBa_J61002 for details on their use.

These promoter parts can be used to tune the expression level of constitutively expressed parts. The NheI and AvrII restriction sites present within these promoter parts make them a scaffold for further modification. JCAraw

Jasonk:I suspect that J23102 is in this well rather than J23106, I'd sequence it before using.

Usage and Biology

Sheffield 2016's Characterisation

Measured strength

Sheffield 2016 has improved the characterisation of both BBa_J23100 and BBa_J23106. These parts are a strong and medium promoter respectively, that we have used to design our iron detecting device. We have experimentally validated through fluorimetry that there is indeed a significant difference between expression levels of GFP coupled to the strong and medium promoters. Comparative analysis of promoter strengths can be directly interpreted from the data we obtained. This data can be found both on the original part experience pages of BBa_J23100 and BBa_J23106, as well as on our website.

Fluorescence of JC28 mutants or W3110 wild types transformed with RyhB-GFP constructs under the control of medium (MedGFP) or strong promoters (StrGFP).

Determination of Noise Levels in Constitutive Promoter Family Members

Fluorescence microscopy and flow cytometry revealed decrease in fluorescence over time for members of the constitutive promoter family.
The expression levels and the noise of four different members of the <a href="https://parts.igem.org/Promoters/Catalog/Anderson" target="_blank">Anderson promoter collection</a> and their RFP reporter systems, were studied by fluorescence microscopy. These were, in increasing promoter strength, <a href="https://parts.igem.org/Part:BBa_J23114" target="_blank">BBa_J23114</a>, <a href="https://parts.igem.org/Part:BBa_J23110" target="_blank">BBa_J23110</a>, <a href="https://parts.igem.org/Part:BBa_J23106" target="_blank">BBa_J23106</a>, and <a href="https://parts.igem.org/Part:BBa_J23102" target="_blank">BBa_J23102</a>.
Additionally, the change in RFP expression levels and noise during growth were tested for the promoters with the highest and lowest relative promoter strength by flow cytometry and qualitative analysis by fluorescence microscopy. Combining these two techniques, the expression and noise levels for the promoters were determined as follows:
- The weak promoter, <a href="https://parts.igem.org/Part:BBa_J23114" target="_blank">BBa_J23114</a>, exhibited a relatively low expression of RFP, indicating low gene expression and an increasing high level of noise throughout growth.
- Both medium strength promoters, <a href="https://parts.igem.org/Part:BBa_J23110" target="_blank">BBa_J23110</a> and <a href="https://parts.igem.org/Part:BBa_J23106" target="_blank">BBa_J23106</a>, displayed a moderate level of both noise and protein expression of the RFP reporter.
- The strong promoter, <a href="https://parts.igem.org/Part:BBa_J23102" target="_blank">BBa_J23102</a>, exhibited a comparatively high expression of the reporter RFP and an increasing high level of noise throughout growth.

Sequence and Features

Functional Parameters

Relative promoter strength estimates (see [http://2009.igem.org/Team:Groningen/Promoters this page] from Groningen 2009):