Difference between revisions of "Part:BBa K3059635"

Revision as of 23:34, 20 October 2019

Constitutive Curli Operon: J23107

Synthetic curli operon under medium-weak constitutive promoter J23107. 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 are quantified by measuring A490 of this supernatant.

For this circuit (WM19_024 or "circuit 24"), so visible pellets formed in the glass culture tubes during overnight growth, and no difference in redness was observed between control and experimental pellets after Congo Red staining. Furthermore, the average A490 measurement for experimental samples’ supernatants was slightly higher than the average for control circuits (if curli fibers were expressed, the supernatant should be lighter, resulting in a lower A490 measurement). Future constitutive circuits may require a stronger promoter such as J23100.

Though this circuit contains non-Biobrick EcoRI and PstI cutsites, it is type IIS compatible (no BsaI or SapI) and is thus legal. The additional EcoRI cutsite within csgE allowed us to perform a diagnostic restriction digest where two EcoRI cuts (and thus two fragments) were expected, one Biobrick and one internal. Both fragments were present at the expected lengths of ~1.9 kb and ~3.5 kb.

More detailed characterization (as well as photos) is available on our wiki.

Sequence and Features