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.

Paraoxon degradation by OPH, creating dimethyl phosphate (DMP) and p-nitrophenol (pNP)

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). 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_K4271015). 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. Therefore, as the level of pNP increases, more GFP will be generated to produce strong green fluorescence.

Function of our biosensor upon IPTG induction (created with BioRender).Our biosensor contains an enzyme plasmid and a sensor plasmid that would enhance GFP expression, thereby indicating the amount of paraoxon detoxified by OPH.

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

Groups	Fluorescence
Dh5alpha	24970
Dh5alpha + pNP	20650
DH5alpha-sensor	46867
Dh5alpha-sensor + pNP	50783

GFP fluorescence of DH5 alpha and DH5 alpha with biosensor in the absence/presence of pNP

The result we acquired from the experiment is not consistent with the data previously published (Jha, Ramesh K., et al.). The difference in the level of GFP fluorescence with and without adding 125 µM of pNP is not significant enough to prove the effectiveness of our pNP sensor (Jha, Ramesh K., et al.). Given that the genetic organization and sequence of our pNP sensor is identical to the plasmid design in the research paper, we went back to further examine and check the pNP sensor design. As a result, we discovered the lack of commonly used RBS sequence in front of pNPmut1-1 in the sensor plasmid, from which we inferred that the poor transcription of pNPmut1-1 might be the reason behind the relatively weak and undetectable green fluorescence signals. In Redesign, we are planning to insert RBS by flanking 4 bases apart from the start codon of pNPmut1-1 in the pNP sensor backbone to further observe if GFP expression will increase in the presence of the same amount of pNP.

Linear map of pNP sensor plasmid after redesign. We plan on inserting an additional RBS in front of pNPmut1-1 to enhance the transcription of pNPmut1-1

Contribution

The genetic organization and sequence of our pNP sensor plasmid is directly acquired from the published data in section 1G of supplemental data(Jha, Ramesh K., et al.). Observely, we did not acquire data that was consistent with results in the research paper. We have redesigned the plasmid sequences by inserting RBS in the sensor plasmid, which contributes to future research related to pNP sensor design.

References

Jha, Ramesh K., et al. “A Microbial Sensor for Organophosphate Hydrolysis Exploiting an Engineered Specificity Switch in a Transcription Factor.” Nucleic Acids Research, vol. 44, no. 17, 2016, pp. 8490–500. Crossref, https://doi.org/10.1093/nar/gkw687.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 862
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1666
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 148