Difference between revisions of "Part:BBa K3059632"

Latest revision as of 01:53, 22 October 2019

IPTG-Inducible Synthetic Curli Operon

Synthetic curli operon that produces curli amyloid fibers when induced by IPTG (10 uL 1M IPTG to 3 mL LB/ ~3.3 mM final concentration is sufficient). This circuit codes for the LacI repressor, which represses pLacCidar and thus the curli operon. However, addition of IPTG represses LacI, thus alleviating repression of pLac and allowing for expression of curli fibers. Curli fiber expression was assessed via Congo Red spin-down assays, where fibers bound Congo Red dye and held it in the cell pellet, resulting in a lighter (less red) supernatant. Congo Red results were quantified by measuring A490 of this supernatant, though results were often visible prior to plate reader quantification (aggregates formed at the bottom of glass culture tubes, visibly red pellets after staining with Congo Red).

Pellets formed by cultures induced with 3.3 mM IPTG.

This circuit, also called WM19_023 or "circuit 23" consistently produced aggregates at the bottom of glass culture tubes after overnight growth. Furthermore, Congo Red spin-down assays consistently suggested fiber production after IPTG induction: pellets for induced cultures were consistently redder than those for uninduced and negative control cultures. The difference between induced and uninduced samples, though visible to the naked eye, was quantified using A490 measurements of supernatants. This difference was verified by a student’s t-test as statistically significant (n=10, p=0.00298) with a confidence value of 99.7%.

Box and whisker graph comparing Congo Red spin down assay results for uninduced and induced samples. For each sample, A490 of supernatant (provided by plate reader) was subtracted from A490 of a positive control (pure 1X PBS mixed with 100 uL 0.015% Congo Red solution). Samples that produced curli fibers bound more Congo Red dye, resulting in a greater difference between pure PBS/Congo Red solution and supernatant. As seen in the graph above, induced samples bound more Congo Red dye, resulting in a greater difference between supernatant A490 and control than uninduced samples. Blue lines represent averages, while red dots represent individual samples.

Aggregates present for induced cultures (top) were not present for uninduced cultures (bottom).

As seen above, for each pair of samples, the induced/right of the two samples has a redder pellet. Uninduced samples (the left of each pair) resemble the negative control, untransformed JS006. From left to right: WM19_023 1C3 #1 uninduced, WM19_023 1C3 #1 induced, WM19_023 1C3 #2 uninduced, WM19_023 1C3 #2 induced, WM19_023 1C3 #3 uninduced, WM19_023 1C3 #3 induced, WM19_023 1C3 #4 uninduced, WM19_023 1C3 #4 induced, untransformed JS006 #1, untransformed JS006 #2, pBbB8K-csgBACEFG uninduced, pBbB8K-csgBACEFG induced. pBbB8K-csgBACEFG was used as a positive control.

To further characterize our IPTG-inducible curli operon, we compared it to a verified curli plasmid, pBbB8K-csgBACEFG from Dr. Neel S. Joshi from the Wyss Institute at Harvard. This plasmid showed dramatic curli expression when induced, compared to our IPTG-inducible circuit. However, cells with pBbB8K showed impressive curli production even when uninduced. Uninduced samples of the arabinose-inducible circuit resembled the induced samples of our IPTG-inducible circuits, rather than the negative control (untransformed JS006).

From left to right: untransformed JS006, uninduced IPTG-inducible curli circuit, induced IPTG-inducible curli circuit, uninduced pBbB8K-csgBACEFG, induced csgBACEFG. The uninduced sample of pBbB8K-csgBACEFG resembled the induced sample of our IPTG-inducible circuit, rather than the negative control. The data suggests that, though our circuit maintains weaker expression of curli fibers, expression is less leaky.

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 ~2 kb and ~5 kb.

More detailed characterization, as well as photographs, is available on our wiki.

Sequence and Features