Team:William and Mary/Demonstrate
Demonstrate
Curli operon
When induced with IPTG, cultures of E. coli with the IPTG-inducible curli operon produced aggregates visible to the naked eye. Furthermore, when centrifuged and stained with Congo Red solution, cell pellets for induced cultures were visibly redder than uninduced cultures and untransformed cultures (Congo Red binds amyloids fibers, and thus ends up in the cell pellet rather than the liquid supernatant). To quantify the differences in color, absorbance at 490 nm was measured for the liquid supernatants. Induced cultures consistently presented lighter supernatants (since the dye was taken into the solid cell pellet) than uninduced and untransformed cultures. A student’s T-test verified this difference as significantly significant with a confidence interval of 95% (n=10). Though originally intended as an optogenetic system, pBlind-EL22 resulted in constitutive expression of downstream adhesin genes. The circuit pBlind + curli + AG43 produced massive AG43 aggregates, and showed a statistically significant difference from negative control (untransformed JS006) samples when subjected to Congo Red spin down assay (n=10, confidence interval of 99%).
Fap operon
We were able to isolate the fap operon from Pseudomonas aeruginosa and make it 3G compatible via Gibson site-directed mutagenesis. We then functionally confirmed and characterized the operon in BBa_K3059640, BBa_K3059634, and BBa_K3059641. The strong constitutive fap circuit BBa_K3059640 and the weak constitutive fap circuit BBa_K3059641 both produced statistically significant results (confidence interval of 95%, n = 5) in a T-test with congo red staining when compared to untransformed JS006. The strong and weak circuits were also statistically significant to one another. The IPTG-inducible circuit BBa_K3059634 also produced statistically significant results (confidence interval of 95%, n = 10) with the same tests. However, there was no significant difference between the uninduced and induced circuits.
SaSuhB
We were able to isolate the coding sequence of SaSuB from the genomic DNA of Staphylococcus Aureus and make it 3G compatible by introducing the appropriate overhangs through PCR. We then utilized this coding sequence to form several circuits. Circuit 34 (BBa_K3059766) is a pLacCidar controlled SaSuhB circuit, Circuit 40 (BBa_K3059767) is a plLac controlled SaSuhB circuit, and circuit 39 (BBa_K3059779) is a pBlind_v1 controlled SaSuhB circuit. The first two circuits are induced via addition of IPTG, and the pBlind_v1 circuit is induced when blue light activates EL222.
Through quantitative analysis via congo red staining, and qualitative analysis via microscopy, we found that our circuits behaved as expected. Induced circuit 34 has a significantly higher decrease in A490 (p = 0.0014, n=4), correlation to less congo red being present in solution, and rather being localized in the pellet. Induced (p=2.08 X 10^-06, n = 8) , and induced circuit 040(p = 0.0003, n = 8) have a significantly higher decrease in A490 as compared to uninduced circuit 033. Furthermore, while circuit 40 was a bit leaky, there was a significantly higher decrease in A490 for the induced circuits as compared to uninduced ones(p = 0.038, n = 8). For circuit 039, visible SaSuhB fibers were observed, and isolated from culture. The fibers were imaged under transmission and fluorescence microscopy.
Patterning
We sequence confirmed pLux, LuxI, and LuxR sequences, and incorporated them into several circuits. First we demonstrated functionality by inducing a circuit with constituitively expressed LuxR with the appropriate HSL molecule. The induced LuxR was able to bind to pLux and promote transcription of mScarlet. This was observed with a plate reader experiment. Next, we showed that we could have cells produce the HSL and induce the receiver cells. We co-cultured the bacteria in a microscopy dish, and conducted a time lapse experiment starting when we induced the sender cells with IPTG to produce Lux HSL. We found that the receiver cells fluoresced while the sender cells did not, as expected. We conducted several ring experiments by co transforming the cells with pDawn AG43 and forming a biofilm, and found that the sender cells were able to cause the receiver cells to fluoresce, but not in ring-like pattern.