Part:BBa_K3259002
Contructs for characterization of pYcgZ-Rhl hybrid promoter
This construct was designed to quantify BBa_K3259000. BBa_K3259000 is expected to be induced when both blue light and RhlR /PAI2 complex are present.
This construct was created by combining parts with GFP downstream of BBa_K3259000 and parts that constitutively produce RhlR.
[Purpose]
This part helps quantify the activity of pYcgZ-Rhl hybrid promoter by measuring the fluorescence intensity of downstream reporter protein.
[Description of this part]
About pYcgZ: pYcg is repressed by the BluR repressor, but is induced by the binding of BluR to BluF protein when exposed to blue light at 470 nm. [1] About Rhl system: RhlO is derived from BBa_R0071 and is induced by the binding of RhlR / C4-HSL onto it. [1]
[Method]
The characterization was done as follows: 1. Transformation of finished parts (DH5α) 2. Colony pickup 3. Shake culture in LB medium (wrap in aluminum foil so that room lights do not hit) 4. Add PAI2 and place under blue light conditions. 5. Compare the fluorescence intensity from before the reaction under each condition of PAI2 and blue light.
[Result]
In this experiment, GFP was expressed in the presence of PAI2 with or without blue light as the following graph shows
Graph
[Analysis]
We successfully quantified the activity of pYcgZ-Rhl hybrid promoter. From the experiment, it can be considered that the repression by RhlO is successful in this part, but the control by blue light is not so good. This is probably due to destabilization of the BluR binding site by the addition of RhlO. However, as a result of examination, it was found that the DH5α we used is susceptible to growth inhibition by blue light among E. coli. [1] [2] In other words, while DH5α is subject to growth suppression by blue light, gene expression may also weaken. As a result of the experiment, the fluorescence intensity per unit E. coli is equal between the blue light and the one cultured in the dark. The fact that gene expression is weakened by irradiating blue light and that there is no change between those that are not suppressed can be considered to be the activity of gene expression. We think this indicates that this part is working as intended.
[Reference]
[1] C. M. Abana et al., Microbiologyopen., 6, 4, e00466 (2017) [2] iGEM Parts Page of iGEM12_ETH_Zurich “YcgZ promoter with multiple BluR (YcgE) operator sites” https://parts.igem.org/Part:BBa_K909010
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 899
Illegal NheI site found at 922 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1193
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1668
Illegal BsaI.rc site found at 808
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