Team:TPR China/Experiment

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Results of the experiment

Demonstration:

  • √ Construction of engineering bacteria for nitrilase
  • √ Construction of 4 sensors
  • √ Nitrilase production by E. coli
  • √ Verification of nitrilase activity
  • √ The sensitivity task of 4 sensing systems by adding small molecule and PAN

1. Construction of engineering bacteria for nitrilase

We decided to use nitrilase - a protein that degrades nitriles - for degradation of PAN.
According to the literature, we selected nitrilase with high enzymatic activity and relatively complete sequences. We successfully constructed 3 NITs which are NhNIT, AdNIT, and LaNIT and tested the enzymatic activity of La & AdNITs.

pTac_NhNIT, pTac_AdNIT, pTac_LaNIT construction

The functional sequence we got from the company is constructed on pUC57 backbone. But pUC57 is not attached to all of our designs. Therefore, we need to rebuild the plasmid, and change the arrangement of sequence. Our effector system is construct on Pet28-b plasmid and bind with upstream LacI sequence.
We verified monoclones by PCR with specific primers seq-28b F/R.
pTac_NhNIT
Fig.1 The result of pTac_NhNIT construction
pTac_AdNIT
Fig.2 The result of pTac_AdNIT construction
pTac_LaNIT
Fig.3 The result of pTac_LaNIT construction
According to the design of NIT, the length of pTac_NhNIT should be 1350bp, the length of pTac_AdNIT should be 1300bp, and the length of pTac_LaNIT should be 1350bp. According to the PCR results shown in Fig.1 to Fig.3, we constructed them successfully.

2. SDS-PAGE verification of nitrilase

We performed SDS-PAGE verification on the fermentation broth of the engineered strain after induction, and the results are shown below.
Fig.4 The SDS-PAGE result of fermentation broth
Electrophoresis results show that our sample has a specific band around 31KD compared to the negative control，this proves that the engineered bacteria we constructed have expressed nitrilase protein.

3. Nitrilase Enzyme Activity

The nitrilase has PAN as substrate to degrade PAN into phenylacetic acid and ammonium. Ammonium and Nash reagents can produce reddish-brown precipitation. The shade is proportional to the concentration of NH4+ produced by PAN degradation.

Standard curve

We plotted the standard curve of the ammonium concentration.The experimental sample's NH4+ concentration can thus be calculated from the standard concentration equation.
Fig.5 Standard curve of NH4+

Nitrilase Enzyme Activity

The activity of nitrilase was verified by detecting the ammonium ions produced by the degradation of PAN by the bacterial liquid. We define U as the amount of PAN that can be degraded per microliter per hour.
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Fig.6 Experimental phenomena of enzyme activity test
Fig.7 Enzymatic activity of Nitrilase
As can be seen from Fig.7, the U value of pTac_LaNIT is 0.6115, which is 1.27 times that of the negative control;the U value of pTac_AdNIT is 0.6205, which is 1.3 times that of the negative control.These data indicate that our engineered bacteria have high activity of nitrilase.

4. Construction of engineering bacteria for sensor

We aimed to find a broad-spectrum aromatic sensor reacting to PAN molecules.In the iGEM component library, there is already an aromatic Sensor Toolbox. We selected candidates Sensor by finding the small molecule with the best response and the similarity of the chemical structure with PAN.We synthesized 3 Sensors XylS, DmpR, and NahR and designed a PaaX Sensor.

Xyls, DmpR, NahR, Paax sensor construction

Xyls sensor
To enhance Xyls-sensor's effect on the response to aromatic small molecules, we redesigned it. The BBa_K1031941 in the part library has been improved.Our new Part Xyls_sfGFP number is BBa_K3599034.
Fig.8 Biosensor circuit of BBa_K1031941
Fig.9 Biosensor circuit of Xyls_sfGFP
DmpR sensor
We constructed it based on existing part BBa_K1031222.
Fig.10 Biosensor circuit of DmpR_sfGFP
NahR Sensor
We constructed it based on existing part BBa_K1031610.
Fig.11 Biosensor circuit of NahR_sfGFP
Paax Sensor
We designed a PaaX Sensor to induction 2-Hydroxyphenylacetic Acid.
Fig.12 Biosensor circuit of Paax_sfGFP
The result of sensor construction
Fig.13 The result of Xyls_-TF construction Fig.14 The result of DmpR_-TF construction
Fig.15 The result of Paax_-TF construction Fig.16 The result of NahR_sfGFP construction
Fig.17 The result of Report construction
According to the design of sensor, the length of Xyls-report should be 1460 bp, the length of DmpR-report should be 1550 bp, the length of NahR-sfGFP should be 2640 bp, the length of Xyls-TF should be 1460 bp, the length of Dmpr-TF should be 1600 bp, the length of Paax-TF should be 1200 bp. According to the PCR results shown in Fig.13 to Fig.17, they have been constructed successfully.

5. Sensor induced by small molecules

The use of sensors can be divided into two steps. First, we used the most sensitive molecule of each sensor that was reported by the previous teams, to test whether the sensor itself is good. After we confirmed that the sensor is good, then it was tested to see whether it responds to the PAN.

The fluorescence of the sensor induced by small molecules

Fig.18 The fluorescence of the sensor induced by small molecules
Fig.19 The fluorescence of the Nahr- sensor induced by small molecules
Through the corresponding small molecule sensing experiment, except Dmpr sensor, all our sensors have great induction effects on aromatic small molecules. Among which NahR-sfGFP was the best! It is 63 times of the negative control.And here we can see that Xyls-sfGFP is 18 times of the Xyls-GPF(BBa_K1031941)

The fluorescence of the sensor induced by PAN

Fig.20 The fluorescence of the sensor induced by PAN
And here is the fluorescence of different sensors induced by PAN.This data shows the same result that NahR-sfGFP responded best.

The fluorescence of the sensor induced by guaiacol

Fig.21 The fluorescence of the sensor by guaiacol
We assisted Team SHSBNU_China to test the effect of the guaiacol response. Here we can see that Nahr-sfGFP has good effects on the guaiacol response, which can be used for their experiment. We are really happy that we have the honor to help other scholars.