Revision as of 06:44, 8 October 2022

pNP sensor (sfGFP + pNPmut1-1)

The pNP sensor plasmid was designed to provide a qualitative evaluation for our paraoxon degradation experiment. (See parts BBa_K4271001 for more information on the OPH experiment.) After the paraoxon is degraded, it produces the chemicals diethyl phosphate and p-nitrophenol (pNP). pNP then further binds to the pNPmut1-1 protein on the sensor plasmid which enhances the sfGFP production for experimental detection.

Usage and Biology

Design

The designed target gene oph, encodes for the enzyme organophosphate hydrolase (OPH), which degrades paraoxon into dimethyl phosphate (DMP) and p-nitrophenol (pNP) (Fig.1). Our oph gene is inserted in an enzyme plasmid in our pNP sensor cell, yet in order to monitor the level of paraoxon degradation by OPH in our sensor cell, we utilized the ability of pNP binding onto pNPmut1-1 to make a biosensor called pNP sensor. We referred our pNP sensor design directly to a research paper on organophosphate hydrolysis (Jha, Ramesh K., et al.). In our sensor plasmid, we included a dual-directional pobA/R promoter(BBa_K4271005), pNP RBS(BBa_K4271006), GFP sequence(BBa_I746916), pobR operator(BBa_K4271007), pNPmut 1-1 sequence for pNP binding(BBa_K4271004), and two double terminators of RrrnB1 terminator and T7 terminator(BBa_B0015) (Fig.2). Once pNP binds to pNPmut1-1, the protein complex would act as an activator to the pobR operator, enhancing the ability of RNA polymerase to bind to the pobR promoter and initiate GFP transcription and translation (Fig.3). Therefore, as the level of pNP increases, more GFP will be generated to produce strong green fluorescence.

Build

Our gene parts are synthesized by Twist Bioscience (ABreal Biotech Co., Taiwan), whose genetic synthesis is based directly on the sensor plasmid design we acquired from the paper(Jha, Ramesh K., et al.). Gene fragments that were synthesized were pNPmut1-1, dual-directional pobA//R promoter (including pobR operator and RBS), and sfGFP. Linear map of the genetic organization of the pNP sensor is shown in Figure 2, which demonstrates all parts subcloned to the pFAST vector (Cat. TTC-CA15, Tools, Taiwan).

Test

To confirm the efficiency of our pNP sensor in determining the amount of pNP produced, we measure the GFP fluorescence of E. coli BL21 (DE3) with and without pNP sensor in the presence and absence of pNP.

Analysis of Results

Sequence and Features