Team:Toronto/Proof
Proof
Comparative Functional Analysis of BBa_K2048001 and BBa_K2048002 for Device Sensitivity and Specificity
We investigated specificity and sensitivity of BBa_K1127008 against gold and cupper ion by conducting Chlorophenol red-β-D-galactopyranoside (CPRG) assay. We compared the results of our devices BBa_K2048001 and BBa_K2048002 against BBa_K1127008, which is the currently available functional device in the registry for gold sensing.
Results:
Image shown in Figure 1 was obtained after 12 hours of incubation. Lanes A to D contained copper(II) metals according to the uM concentration shown on the left hand side whereas lanes E to F contained corresponding gold(III) metal ions. Control groups: Untransformed DH10B cells containing the metal ion of interest (Lanes D and I) Experiment groups: BioBricks BBa_K2048001 labelled as GolS in lanes C and G, BBa_K2048002 labelled as P118A in lanes B and F and BBa_K1127008 labelled as York in lanes A and E.
Dark red color was observed in the presence of DH10B cells transformed with BBa_K2048001 in the presence of gold(III) metal ions. Although the expression was sensitive to the gold ions, it was not particularly specific to gold ion. Red-orange expression levels were also observed in the presence of copper(II) ions. Cells transformed with BBa_K2048002 were found to be specific to presence of gold(III) ions. Minimum to no activity was observed in the presence of copper(II) ions. However, complete color development required more than 12 hours. Cells transformed with BBa_K1127008 showed orange color development in the presence of both gold(III) and copper(II) ions. Color observed in the presence of gold was darker than the one in the presence of copper ions. Most importantly, circuit expression was observed for BBa_K1127008 and BBa_K2048001 even when no metal ion was added to the well. However this was not the case with device BBa_K2048002.
As shown in Figure 2, After >24h incubation, expression levels for BBa_K1127008 and BBa_K2048001 reached to its maximal steady state levels and expression levels for metal and gold ions were similar. BBa_K2048002 showed concentration dependent expression in the presence of gold(III) ions up to 1uM with minimum to no expression in the presence of copper(II) ions.
Conclusion:
We proved that BBa_K1127008 Biobrick expression was not specific for Gold(III) ions. Comparable expression levels were also observed in the presence of Copper(II) ions and abscence of metal ions.
Cell Free Paper Based Genee Circuit Concept
The process begins with the application of molecular biology techniques to create the plasmids holding the gene(s) of interest. Then, the paper array supplies are prepared. A hydrophobic barrier around each “well” can be made by using software to design a matrix and a printer to print wax in a given pattern onto the paper. Then transcription and translation enzymes are combined with the gene circuits, embedded into the paper, and freeze dried to form a paper disk. Upon rehydration, i.e. the addition of water, the abiotic and sterile paper will produce a functional and stable cell-free synthetic gene network. In our case, the incorporated GolB promoter is activated with the transcriptional regulator GolS, in the presence of gold ions. The LacZ reporter is then transcribed, producing the - galactosidase, which allows a dramatic color change, from yellowish-white to purple, to be observed in individual paper wells. Thus, the engineered paper can be used to test soil samples for gold. By simply isolating a soil sample, hydrating the paper based technology, placing it into the sample, and allowing it to incubate at 37 for 24 hours, the presence or absence of gold can be confirmed, by the change of colour in the paper compared to the control wells.
We have successfully proved the functionality of our circuit by performing an in vitro assay and determined the time required as 24 hours. Among the circuits available in the registry (BBa_K1127008), and devices assembled by our team ( BBa_K2048001, BBa_K2048002), we have determined that BBa_K2048002 is the best candidate for cell-free paper based reaction. However, the cell free extract and the expression levels of BBa_K2048002 need to be optimized for maximum expression differential.
Pardee, K., Green, A., Ferrante, T., Cameron, E., DaleyKeyser, A., Yin, P., & Collins, J. (2014, November 6). Paper-Based Synthetic Gene Networks. Cell, 159(4), 940–954.
