Part:BBa_K3059638
pBlind Curli Operon, Blue-Light Inducible
Synthetic curli operon under control of pBlind promoter, referred to as WM19_045 or "circuit 45." Within this circuit, transcription factor EL222 is constitutively expressed by J23107. Under blue light, EL222 activates pBlind and thus activates expression of curli fibers. Curli fiber expression was assessed via Congo Red spin-down assays, where any present fibers bind Congo Red dye and hold it in the cell pellet, resulting in a lighter (less red) supernatant. Congo Red results were quantified by measuring A490 of this supernatant.
Characterization of this part using Congo Red spin down assays suggests that it acts as a weak constitutive circuit rather than an optogenetic one. pBlind-EL22 had resulted in constitutive gene expression in a different circuit (circuit 46/ BBa_K3059639), which was miniprepped before circuit 45. With this in mind, we tested the function of circuit 45 when exposed to ambient light as well as blue light. Five experimental tubes were exposed to ambient light while growing in the shaking incubator overnight, while experimental tubes were covered with foil (foiled and unfoiled sample pairs were inoculated using the same colony solution, allowing for direct comparison). JS006 cultures were grown as a negative control. Some experimental tubes, both foiled and unfoiled, showed small aggregates at the bottom. However, no obvious differences in pellet color were visible to the naked eye after Congo Red staining.
Small aggregates formed for some experimental cultures, including ones that had been covered with foil during overnight growth.
The average A490 value for each group reflected expectations: average A490 was the lowest for experimental samples exposed to ambient light, followed by the average for foiled samples, followed by the average for the negative controls. However, these averages were hardly different (0.2438, 0.2440, 0.2482), and no statistical significant difference was found between ambient light and foiled samples or between ambient light and negative control samples.
A second experiment compared samples of circuit 45 grown in blue light and samples grown in darkness. A clear difference between samples with the plasmid and negative control samples was visible the next day; aggregation was observed in experimental samples whereas the turbidity within negative control samples appeared more “smooth.” This trend was apparent for both experimental cultures grown under blue light and those grown in darkness. Furthermore, experimental pellets (both those grown in darkness and those exposed to blue light) stained a darker red with Congo Red solution than negative control pellets.
For both the plate exposed to blue light (left) and the plate grown in the dark (right), experimental wells (top) showed aggregation whereas control wells (bottom) were turbid but uniformly so.
Two stained samples of circuit 45 between two negative control samples. Note the redder pellets of the middle samples. Both are similarly red, redder than the negative controls, although one was exposed to blue light and the other was grown in darkness.
No statistically significant difference was found between WM19_045 samples grown in the dark and samples exposed to blue light. Though curli expression (and thus A490) appeared to decrease from samples with the plasmid to negative control samples, it was difficult to verify this relationship statistically with only n=3. The inability of the projector to cover many samples with intense blue light prevented the use of large sample sizes. Though future experiments were designed, such experiments were unfeasible with the time constraints.
Our available data suggests that, if pBlind is truly light-sensitive, the level of induction by blue light is somewhat weak (especially compared to the pDawn system). Instead, pBlind seems to express downstream genes constitutively, even moreso than our constitutive circuit J23107-curli (WM19_045).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2730
Illegal PstI site found at 1829 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2730
Illegal NheI site found at 51
Illegal NheI site found at 74
Illegal PstI site found at 1829 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2730
Illegal XhoI site found at 607 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2730
Illegal PstI site found at 1829 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2730
Illegal PstI site found at 1829
Illegal NgoMIV site found at 177
Illegal AgeI site found at 3027 - 1000COMPATIBLE WITH RFC[1000]
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