Team:Northwestern/Experiments
What We Aimed For
With only theoretical knowledge from literature, our first ambitious goal was to be able to produce the reporter protein sfGFP (or a colorimetric enzyme) completely in vitro and at least be able to tell analyte concentration semi-quantitatively. Our other goals included high sensitivity, easy for citizens to use, precise and consistent results, generating results in less than 4 hours and holding no risk of introducing/handling/shipping living cells in environment.
Our reporter construct
Our reporter construct
We had two separate platforms on which we constructed our biosensors. The first one is the Two-Plasmid Repressor System and the other is a Two-Plasmid Activator System. The repressor plasmid constitutively produces repressor protein which is regulated by the analyte. The reporting plasmid for this system, on the other hand, contains an operator site which is regulated by the repressor protein. Therefore, it only has an output in the absence of the repressor protein.
Our metal sensors are both two plasmid repressor systems. Since neither of our construct systems have been well-characterized in literature, we decided that starting out with a more well-characterized system would be beneficial. This is why we decided to start out by testing and engineering a system to sense AHL. AHL is a signal molecule used in quorum sensing as a regulatory mechanism by which gram-negative bacteria control gene expression in response to population density. The importance and well-characterization of AHL sensing made it a prime target for us to analyze.
We started out by basing our constructs on a system characterized by the Jewett Lab at Northwestern. These were viable for a cell-free system, but they were not well optimized for such a platform. Our first aim was to optimize this system for better cell-free expression.
On the first plasmid, we replaced the endogenous pTet with a T7 promoter, because the latter is stronger, unregulated, processive polymerase, which is important to us because we want LuxR to be present in excess for accurate sensing. For the same reason we couldn’t use a processive polymerase on the second plasmid, because we didn’t want our system to leak in the off state. Additionally, we engineered in a PHP stability hairpin to our second plasmid, which helps prevent exonuclease activity, which should significantly increase the activity of our biosensor.
Experimental setup
Experimental setup
Our first step was to test our unoptimized systems in conjunction with optimized systems and compare our results and verify the functionality of our systems. Subsequently, we tested different analyte concentrations. For further information, see the Results Page.
References
1. Adam D Silverman, Nancy Kelley-Loughnane, Julius B Lucks, Michael C Jewett bioRxiv 411785; doi: https://doi.org/10.1101/411785 2. Pellinen, Teijo, et al. “A Cell-Free Biosensor for the Detection of Transcriptional Inducers Using Firefly Luciferase as a Reporter.” Analytical Biochemistry, vol. 330, no. 1, 13 May 2004, pp. 52–57., doi:10.1016/j.ab.2004.03.064.