Part:BBa_K3059639
pBlind Curli + AG43
This circuit, referred to as WM19_046 or "circuit 46," include AG43 and a synthetic curli operon under control of pBlind promoters. Within this circuit, transcription factor EL222 is constitutively expressed by J23107. Under blue light, EL222 activates pBlind and thus activates expression of both curli and AG43 (this circuit contains two transcriptional units with pBlind; the first is pBlind-curli and the second is pBlind-AG43). 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. The presence of AG43 was assessed visually.
As with circuit 45/ pBlind-curli (BBa_K3059638), pBlind-EL222 results in constitutive, uninduced expression. Inoculations for miniprep, though covered completely in foil and grown in a shaking incubator, exhibited massive aggregates at the bottom of glass culture tubes and minimal cells in the LB supernatant.
Inoculations for miniprep with large aggregates, even though glass culture tubes were completely covered with foil.
This behavior persisted in JS006 samples inoculated for experiment-- both foiled and unfoiled cultures of circuit 46 produced massive aggregates and clearer LB supernatants.
Top photo: cultures of JS006 with circuit 46 that had been exposed to ambient light during overnight growth in the shaking incubator. Bottom photo: culture of JS006 with circuit 46 that had been foiled during overnight growth in the shaking incubator.
Though Congo Red spin down assays found no statistically significant difference between foiled and unfoiled experimental samples, a statistically significant difference was found between unfoiled experimental samples and the negative control (n=5, calculated t=-3.8069, 3.8069 > 3.747) with a confidence interval of 98%. If one assumes that light plays little to no role in adhesin production and combines foiled and unfoiled groups, the confidence interval rises to 99% (n=10, calculated t=-4.1175, 4.1175 > 3.250).
Box and whisker graph comparing circuit 46 samples to untransformed JS006, the negative control. Samples expressing curli fibers bound more Congo Red dye, resulting in lighter supernatants and lower A490 values. As shown in the graph above, circuit 46 samples had generally lighter supernatants than JS006 samples. Blue lines represent averages while red circles represent individual samples.
An additional experiment was conducted to compare AG43 expression of samples exposed to blue light and those grown in darkness. Before crystal violet staining, aggregation was visible in experimental wells (as opposed to uniformly turbid negative control wells). Unfortunately, aspirating the media and subsequently washing with 1X PBS removed most of the aggregation produced by experimental samples. This suggests that AG43 and curli fibers were expressed constitutively by cells floating freely within the liquid media. Had the cells expressed AG43 as a result of adsorbing onto the region of the plate exposed to blue light (as proposed Jin and Riedel-Kruse in their 2018 PNAS paper on Biofilm Lithography), a purple film would have been observed after aspiration and washing. Instead, it appears that most of the adhesin was suspended in the liquid media.
Though optogenetics was the intended use for circuits 45 and 46, we now have constitutive circuits to replace the ineffective circuit 24 (J23107-curli, BBa_K3059635).
More information (as well as photographs) are available on our wiki.
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 NgoMIV site found at 4999
Illegal NgoMIV site found at 6223
Illegal NgoMIV site found at 6733
Illegal NgoMIV site found at 6754
Illegal AgeI site found at 3027
Illegal AgeI site found at 5749
Illegal AgeI site found at 6493
Illegal AgeI site found at 6907
Illegal AgeI site found at 7149 - 1000COMPATIBLE WITH RFC[1000]
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