iGEM 2022 — Team wego-taipei

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[pNP Sensor](#pnpsensor)
[Organophosphate Hydrolase (OPH)](#organophosphate)
[Antisense PhoU (AsPhoU)](#antisense)
[Polyphosphate (PolyP) Sensor](#polyphosphate)

**Engineering Success**

pNP Sensor

*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.

![](https://static.igem.wiki/teams/4271/wiki/oph-pathway.jpg)

Fig. 1. Paraoxon degradation by OPH

The degradation of paraoxon into dimethyl phosphate (DMP) and
p-nitrophenol (pNP)

![](https://static.igem.wiki/teams/4271/wiki/linear-map-of-sensor-plasmid.png)

Fig. 2. The linear map of our pNP sensor plasmid

Our sensor plasmid includes a dual-directional
*pobA/R*
promoter, pNP RBS, sfGFP,
*pobR*
operator, pNPmut1-1, and two double terminators that are composed of
RrrnB1 terminator and T7 terminator

![](https://static.igem.wiki/teams/4271/wiki/paraoxon-biosensor.png)

Fig. 3. Function of our biosensor upon IPTG induction (created by
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 Fig. 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 Result*

**Groups**
**Fluorescence**

DH5alpha

24870

DH5alpha + pNP

20650

DH5alpha-sensor

46867

DH5alpha-sensor + pNP

50783

![](https://static.igem.wiki/teams/4271/wiki/fluorescent-protein-expression-level.png)

Fig. 4. 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 (Fig.5) to further observe
if GFP expression will increase in the presence of the same amount of pNP.

![](https://static.igem.wiki/teams/4271/wiki/redesign-sensor-plasmid.png)

Fig. 5. 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.

Organophosphate Hydrolase (OPH)

*Design*

Upon iptg induction, the lacI repressor protein will be detached from the
lacI gene, leading to the transcription and translation of our target
*oph*
gene. In the process of paraoxon degradation, our target gene
*oph*
encodes for the enzyme organophosphate hydrolase (OPH), which hydrolyzes
paraoxon into dimethyl phosphate (DMP) and p-nitrophenol (pNP) (Fig.1).
Since the
*E.coli*
bacterial strain BL21 (DE3) has a high level protein expression with T7
RNA polymerase, we chose it as a host cell for our experiment. The vector
we used is pET-22b, which includes a T7 promoter (BBa\_I712074), lac
operator (BBa\_K2406019), RBS (BBa\_K2924053), OPH gene (BBa\_K4271000), and
T7 terminator (BBa\_K731721) (Fig.6). We also included a pelB signal
peptide, which plays a significant role in our experiment by directing our
target OPH enzyme to the bacterial periplasm, thereby enhancing the
enzyme’s activity at the specific location (Jain, Monika et al.).

![](https://static.igem.wiki/teams/4271/wiki/linear-map-pet22boph.jpg)

Fig. 6. The linear map of pET22b::oph

Our enzyme plasmid includes T7 promoter sequence, lac operator, RBS, pelB
signal peptide, OPH, his tag, and T7 terminator

*Build*

Synthetic
*oph*
gene we used in this study is derived from the
*opd*
(organophosphate degradation) gene in
*Agrobacterium tumefaciens*
and performed with codon usage optimization for
*E. coli*
heteroexpression. We digested the
*oph*
gene with BamHI and HindIII, subcloned it to pET22b vectors that underwent
the same restriction enzyme digestion, then transformed the recombinant
into
*E. coli*
DH5α. The transformation was conducted by plasmid extraction through
mini-prep.

We later confirmed the insertion of our
*oph*
gene into the enzyme plasmid by enzyme digestion, cutting the recombinant
DNA with BamHI and HindIII respectively, and observing the same band sizes
of 6.5 kilobases after gel electrophoresis (Fig.7). We later digested our
pET22b::OPH again with both BamHI and HindIII, two of resulting DNA bands
include the 1071 base-long
*oph*
and the 5479 base-long pET22b vector (Fig.8). Finally, the plasmid was
transformed into the competent cells
*E.coli*
BL21(DE3) via heat shock, which we later used to examine the level of
paraoxon degradation by our enzyme plasmid.

![](https://static.igem.wiki/teams/4271/wiki/gel-bamhind.jpg)

Fig. 7. gel electrophoresis of pET22b::OPH after digested with BamHI and
HindIII respectively

Column 2 shows the result of pet22b::OPH digested by BamHI while column 3
shows the result of pET22b::OPH digested by HindIII. Both show 6.5
kilobases of linear DNA.

![](https://static.igem.wiki/teams/4271/wiki/gel-bam-hind.png)

Fig. 8. gel electrophoresis of pET22b::OPH after digested with both BamHI
and HindIII

The 7th column shows the result of restriction enzyme digestion by BamHI
and HindIII; the DNA bands include pET22b::OPH, OPH (1071 bases), and
pET22b vector (5479 bases).

![](https://static.igem.wiki/teams/4271/wiki/pet22b-plasmidmap.png)

Fig. 9. The plasmid map of pET22b::OPH

*Test*

To test the degradation of paraoxon by OPH, we detected the level of pNP
production with a spectrophotometer. Since pNP (yellow) reaches an
absorbance peak at 410 nm, we assume that the absorbance at 410 nm of the
colonies under different conditions will provide us with an overview of
the efficiency of paraoxon degradation by OPH. We performed two
experiments based on this assumption: the amount of pNP at various time
points (pNP conc. v.s. Time) and in the presence of different IPTG
concentrations at a fixed time (pNP conc. v.s. IPTG conc.).

*Analysis of result*

**Groups**
**(Absorbance at 410nm - background data) / OD600**
**(Supernatant Absorbance at 410nm - background data) / OD600**

1. BL2(DE3) (negative control)

0

0

![](https://static.igem.wiki/teams/4271/wiki/1.jpg )

2. BL2(DE3) +paraoxon (experimental)

0.2323266987

0.3905284832

![](https://static.igem.wiki/teams/4271/wiki/3.jpg )

3. BL2(DE3) +pNP (positive control)

8.905950096

9.966890595

![](https://static.igem.wiki/teams/4271/wiki/5.jpg )

4. PET::OPH +IPTG induction (negative control)

0

0

![](https://static.igem.wiki/teams/4271/wiki/2.jpg )

5. PET::OPH +paraoxon +IPTG induction (experimental)

6.720481928

6.916144578

![](https://static.igem.wiki/teams/4271/wiki/4.jpg )

6. PET::OPH +pNP +IPTG induction (positive control)

11.83912249

12.51005484

![](https://static.igem.wiki/teams/4271/wiki/6.jpg )

![](https://static.igem.wiki/teams/4271/wiki/time-v-s-pnp.png)

Fig. 10. The change in pNP concentration over 25 hours in culture

The results met our expectations as the pNP concentration increased over
time, showing that paraoxon is being degraded by the
*E.coli*
BL21(DE3) steadily. However, pNP concentration seems to increase rapidly
only in the first 5 hours of observation, after which it proceeds to grow
steadily, which demonstrates that the enzyme reaches optimal activity
after 5 hours of culture.

**Groups**
**Bacteria Used**
**Substrates**
**IPTG Induction (μM)**

1 (negative control)

BL21(DE3)

-

0

2 (positive control)

pNP

0

3 (experimental group)

PXN

0

4 (negative control)

BL21(DE3) engineered with OPH

-

0

5 (positive group)

pNP

0

6 (experimental group)

PXN

0

7 (experimental group)

PXN

2000

8 (experimental group)

PXN

1000

9 (experimental group)

PXN

500

10 (experimental group)

PXN

250

11 (experimental group)

PXN

125

12 (experimental group)

PXN

62.5

13 (experimental group)

PXN

31.25

14 (experimental group)

PXN

15.625

![](https://static.igem.wiki/teams/4271/wiki/iptg-v-s-pnp.png)

Fig. 11. IPTG induction (μM) vs. pNP concentration after 23 hours (μM)

We later measured the pNP concentration under exposure of different
concentrations of IPTG. We discovered that the concentration of pNP
reaches a maximum amount when around 250 μM of IPTG is introduced into
E.coli BL21(DE3) engineered with OPH. We also inferred from the data that
after pNP concentration reaches a maximum at 250μM of IPTG induction, the
amount of pNP will not increase as the concentration of IPTG increases.

Antisense PhoU (AsPhoU)

*Design*

Since the overabundance of phosphate in water bodies is one of the major
causes of eutrophication, we designed genetically engineered
*E. coli*
bacteria that are able to increase the uptake of phosphate into the cell.
Normally, the Pho regulon in
*E. coli*
bacteria is responsible for regulating the amount of phosphate entering
the bacteria to maintain the homeostasis of phosphate in the bacteria. To
allow the bacteria to consume organic phosphate from the environment
limitlessly, we engineered
*E. coli*
expressing the antisense AsPhoU (As PhoU), which binds to the phoU mRNA
and blocks the translation of
*PhoU*
protein, thereby enhancing phosphate transportation into the cell.

![](https://static.igem.wiki/teams/4271/wiki/phou.png)

Fig. 12. PhoU protein function (left) and inhibition of PhoU by AsPhoU
(right)

![](https://static.igem.wiki/teams/4271/wiki/gel-asphou.png)

Fig. 13. gel electrophoresis of pBADHisA::AsPhoU after digested with NcoI
and XhoI

The 7th column shows the result of restriction enzyme digestion by NcoI
and XhoI; the DNA bands include pBADHisA::AsPhoU (4196 bases), AsPhoU (213
bases), and pBAD vector (3983 bases).

*Build*

In order to determine the amount of phosphate entering the bacteria, we
utilized certain components of the PhoU regulon to measure the
effectiveness of phosphate transportation. To evaluate the activity of the
PstSCAB transporter, we conducted a preliminary experiment that measures
the concentration of PhoA via its coloration in low and high phosphate
environments. Since the activity of PstSCAB and PhoA are positively
correlated, an increase in PhoA concentration will indicate the activity
of the PstSCAB transporter. In this preliminary experiment, we added
solutions of 5-Bromo-4-chloro-3-indolyl phosphate (XP) because PhoA will
severe it into a phosphate ion and a 5,5′-dibromo-4,4′-dichloro-indigo,
which makes the solution blue. Arabinose also plays an important role in
our preliminary experiment, since it acts as an inducer that promotes
AsPhoU to bind on the PhoU sequence.

Another experiment we conducted to measure the effectiveness of phosphate
transportation into the cell is to measure the amount of extracellular
phosphate in the bacteria via malachite green coloration. A complex of
phosphomolybdic acid is formed when molybdate (MoO₄⁻²) interacts with
phosphate (PO₄⁻³), which would later interact with malachite and form a
green chromogenic complex.

*Test*

**Groups**
**Environmental condition**
**Resulting coloration of
*E. coli*
colonies**

E. coli DH5α

Low phosphate

Blue

![](https://static.igem.wiki/teams/4271/wiki/e-coli-low-p.png)

E. coli DH5α (withAsPhoU)

Low phosphate

Blue

![](https://static.igem.wiki/teams/4271/wiki/e-coli-asphou-low-p.png)

E. coli DH5α (with AsPhoU) + arabinose

Low phosphate

Blue

![](https://static.igem.wiki/teams/4271/wiki/e-coli-asphou-arabinose-low-p.png)

E. coli DH5α

High phosphate

Transparent

![](https://static.igem.wiki/teams/4271/wiki/e-coli-high-p.png)

E. coli DH5α (with AsPhoU)

High phosphate

Transparent

![](https://static.igem.wiki/teams/4271/wiki/e-coli-asphou-high-p.png)

E. coli DH5α (with AsPhoU) + arabinose

High phosphate

Blue

![](https://static.igem.wiki/teams/4271/wiki/e-coli-asphou-arabinose-high-p.png)

We cultured three different groups of E.coli DH5α (E.coli DH5α, E.coli
DH5α with AsPhoU, E.coli DH5α with AsPhoU and arabinose) in both low and
high phosphate concentrations. The groups cultured in low phosphate
concentration act as the positive control of our preliminary experiment,
while the groups cultured in high phosphate concentration function as the
negative control.

For the experiment of malachite green coloration, we incubated E.coli DH5α
(0.1 O.D.) with AsPhoU and E.coli DH5α with AsPhoU and arabinose under
fixed high-phosphate environment (2 mM of K₂HPO₄, 0.06% glucose, and MOPS
buffer). We retrieved our E.coli colonies respectively after 1 hour, 2
hours, and 4 hours of incubation in a high-phosphate environment. We then
added molybdate and malachite into the tested groups and used a
spectrometer to detect the absorbance of phosphate at 600 and 620 nm,
since molybdate has a max absorbance rate at 600 nm while that of
malachite is at 620 nm.

*Analysis of result*

From our preliminary result, we confirmed that the Pho regulon will only
be active in a low phosphate environment as the positive control groups
all turned blue, indicating PhoA enzyme activity and indicating PstSCAB
activity. Our results also proved that arabinose has the ability to induce
AsPhoU binding to the PhoU sequence, since the
*E. coli*
DH5α cultured with both AsPhoU and arabinose appears blue even in a high
phosphate environment, showing PhoA and PstSCAB activity regardless of
PhoU inhibition.

1 hr

**Groups**
**Absorbance at 600 nm (O.D.)**
**Absorbance at 620 nm**
**Background data - absorbance at 620 nm**
**Absorbed Phosphate conc. per O.D.**

Medium

0.0439

0.7048

Medium + arabinose

0.0427

0.9448

DH5α

0.082

0.83

0.1148

1.4

DH5α + arabinose

0.0785

0.803

0.1418

1.806369427

AsPhoU

0.0836

0.7886

0.1562

**1.868421053**

AsPhoU + arabinose

0.0796

0.7392

0.2056

**2.582914573**

2 hrs

**Groups**
**Absorbance at 600 nm (O.D.)**
**Absorbance at 620 nm**
**Background data - absorbance at 620 nm**
**Absorbed Phosphate conc. per O.D.**

Medium

0.0439

0.9052

Medium + arabinose

0.0465

0.9215

DH5α

0.088

0.7818

0.1397

1.5875

DH5α + arabinose

0.0854

0.8878

0.0337

0.3946135831

AsPhoU

0.0889

0.8388

0.0827

**0.9302587177**

AsPhoU + arabinose

0.0551

0.8209

0.1006

**1.825771325**

3 hrs

**Groups**
**Absorbance at 600 nm (O.D.)**
**Absorbance at 620 nm**
**Background data - absorbance at 620 nm**
**Absorbed Phosphate conc. per O.D.**

Medium

0.0427

0.6126

DH5α

0.1043

0.5899

0.0227

0.217641

DH5α + arabinose

0.104

0.6221

-0.0095

-0.09135

AsPhoU

0.0955

0.5306

0.082

**0.858639**

AsPhoU + arabinose

0.0965

0.4867

0.1259

**1.304663**

According to the data above, we concluded that E.coli engineered with
AsPhoU and induced by arabinose has a significantly higher efficiency in
absorbing phosphate. At all three time periods, the absorbance rate of our
engineered E.coli cells was higher in the absence of arabinose. From this
result, we concluded that arabinose, along with the AsPhoU that induces,
does increase phosphate absorption through the PstSCAB transporter even
under exposure at 2 mM of phosphate. In addition, the presence of AsPhoU
is also proven effective at increasing phosphate absorption, as the two
groups of DH5α engineered with AsPhoU show levels of phosphate
significantly higher than the other two groups of normal DH5α. Both of the
conclusions we obtained from the data further prove that our engineered
bacteria has the ability to absorb phosphate from the eutrophicated water
bodies, thus reducing the concentration of phosphate in the polluted
water.

Polyphosphate (PolyP) Sensor

![](https://static.igem.wiki/teams/4271/wiki/polyp-sensor.png)

Fig. 14. Mechanism of polyP sensor (Created by BioRender)

*Design*

The design of the polyP sensor plasmid includes genes encoding for mCherry
fluorescent protein and RpoD sigma factor with a P region that easily
binds to polyphosphate, respectively. Without polyphosphate accumulation,
the sigma factor could successfully direct the RNA polymerase to the
promoter, resulting in the expression of mCherry fluorescent protein.
However, if polyphosphate is fixated, accumulated, and attached to the P
region of the sigma factor, the sigma factor loses its function and would
be unable to direct the RNA polymerase for transcription of mCherry.
Therefore, the increase in phosphate absorption and, therefore,
polyphosphate fixation results in reduced mCherry fluorescence.

By measuring the mCherry fluorescence, we could monitor the accumulation
of polyP in the cell. The method is employed in the design of our
implementation. To ensure that our AsPhoU cells perform at their highest
efficiency, we designed the hardware to detect reduced fluorescence levels
to the minimum, which signals that the cell has reached maximum phosphate
fixation. A notification would then be sent through the designed software
to remind the users to replace the filter in time, thus maintaining the
effectiveness of our device and further ensuring biosafety. For further
information on the implementation design, please visit our
[Implementation page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D)
.

![](https://static.igem.wiki/teams/4271/wiki/polyp-sensor-without-tag.png)

Fig. 15. The linear map of the polyP sensor (without the degradation tag)

*Partnership*

During our partnership with NYCU\_Taipei, we adopted their advice of
flanking the mCherry gene with a degradation tag in order to increase the
specificity of the sensor. The degradation tag enhances the rate of
fluorescent protein degradation so that the protein would not accumulate
and generate false signals for detection. For more information on our
team’s partnership with NYCU\_Taipei, please visit our
[Partnership page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27partnership%27%29%20%7D%7D)
.

![](https://static.igem.wiki/teams/4271/wiki/polyp-sensor-with-tag.png)

Fig. 16. The linear map of the polyP sensor (with the degradation tag)

*Build*

We committed to Twist Bioscience (ABreal Biotech Co., Taiwan) for
synthetic
*ropD*
gene (BBa\_K4271010), comprising N-terminal 69 amino acid for polyP binding
(Yang et al., 2010),
*E. coli*
RpoD (1-555 a.a), C-terminal 4.2 region for UreA promoter recognition
(BBa\_K4271011) (Beier et al., 1998). We conducted reverse PCR to amplify
the plasmids, consisting of mCherry (BBa\_J18932), and transcriptional
terminators (BBa\_B0015). Two fragments, Tac promoter (BBa\_K4271009) and
transcriptional terminators (BBa\_B0015) were prepared by PCR. We also
amplified the genetic synthetic UreA promoter (containing RBS,
BBa\_K4271012) by PCR, respectively.

Due to time constraints, we weren’t able to complete the engineering cycle
of this particular construct. Yet we have drawn up a future plan for the
building of the polyP sensor. Tac promoter (189 bps), synthetic ropD gene
(1986 bps), transcriptional terminator (173 bps), and plasmid (2885 bps)
will be connected by Gibson assembly to generate the polyP sensor (Fig.
14). The assembly product will then be transformed to
*E. coli*
DH5 alpha via the heat shock method. Colony PCR with two primers, PTac
\_forward and Tt\_reverse, will be used to confirm and generate a 3.7 bps
fragment if the assembly succeeded.

References

Beier, D., Spohn, G., Rappuoli, R., & Scarlato, V. (1998, October).
Functional analysis of theHelicobacter pyloriprincipal sigma subunit of
RNA polymerase reveals that the spacer region is important for efficient
transcription. Molecular Microbiology, 30(1), 121–134.
https://doi.org/10.1046/j.1365-2958.1998.01043.x

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.

Jain, Monika et al. “Recombinant organophosphorus hydrolase (OPH)
expression in
*E. coli*
for the effective detection of organophosphate pesticides.” Protein
Expression and Purification, Volume 186, 2021, 105929, ISSN 1046-5928,
https://doi.org/10.1016/j.pep.2021.105929

Yang, Z. X., Zhou, Y. N., Yang, Y., & Jin, D. J. (2010, June 11).
Polyphosphate binds to the principal sigma factor of RNA polymerase during
starvation response in Helicobacter pylori. Molecular Microbiology, 77(3),
618–627. https://doi.org/10.1111/j.1365-2958.2010.07233.x

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