iGEM 2017 — Team NCKU_Tainan

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# Notebook

## Construction of BBa\_K2275007

---

### 5/22

* PCR to get DNA of *nir* gene cluster from *Eschericha coli* MG1655 and analyze by DNA gel.

### 5/23

* Digest *nir* gene cluster and pSB1C3-PLacI-sfGFP with *Hind*Ⅲ and *SpeI*. Analyze by the DNA gel.
* Ligase at 16℃ overnight.

### 5/25

* Transform the ligased product into DH5α and put at 37℃ for 12 hours.

### 5/26

* Select five colonies from the plates and pre-culturing.

### 5/27

* Extract plasmid and use plasmid digestion to confirm.

## Construction of BBa\_K2275008

---

### 7/3

* PCR to get DNA of *gudB* from *Bacillus subtilis* and analyze by the DNA gel.
* Digest gene *gudB* and pSB1C3-PLacI-sfGFP with BamHI and PstI. Analyze by the DNA gel
* Ligase at 16℃ overnight.

### 7/4

* Transform the ligased product into DH5α and put at 37℃ for 12 hours.

### 7/5

* Confirmation by colony PCR.
* Select two colonies from the plates and pre-culturing.

### 7/6

* Extract plasmid and use plasmid digestion to confirm.

## Construction of BBa\_K2275009

---

<

### 8/13

* PCR to get DNA of *gudB* from *Pseudomonos putida* and analyze by the DNA gel.
* Digest gene glnA and pSB1C3-PLacI-sfGFP with *Hind*Ⅲ and PstI. Analyze by the DNA gel
* Ligase at 16℃, 4 hours and then transform into DH5α.

### 8/14

* Select two colonies from the plates and pre-culturing.
* Extract plasmid and use plasmid digestion to confirm.

## Construction of BBa\_K2275010

---

### 9/6

* PCR to get rev-*gudB*.
* Digest gene rev-*gudB* with XbaI and PstI and pSB1C3-PLacI-glnA with *Spe*I and *Pst*I. Analyze by the DNA gel.
* Ligase at 16℃ , 4 hours and then transform into DH5α

### 9/7

* Select two colonies from the plates and pre-culturing.

### 9/8

* Extract plasmid and confirm with plasmid digestion.

## Construction of BBa\_K2275011

---

### 9/13

* Add a NsrR binding site to K381001 before RBS by PCR and analyze by DNA gel.

### 9/14

* Digest linear PCR product with *Dpn*I and self-ligate by phosphorylation
* Transform the ligased product into DH5α and put at 37℃ for 12 hours.

### 9/15

* Select two colonies from the plates and pre-culturing.

### 9/16

* Extract plasmid and use plasmid digestion to confirm.

## Construction of BBa\_K2275012

---

### 9/13

* Add a NsrR binding site to K381001 after RBS by PCR and analyze by DNA gel.

### 9/14

* Digest the linear PCR product with *Dpn*I and self-ligate by phosphorylation.
* Transform the ligased product into DH5α and put at 37℃ for 12 hours

### 9/15

* Select two colonies from the plates and pre-culturing.

### 9/16

* Extract plasmid and use plasmid digestion to confirm.

## Nitrate sensing test

---

### 7/1~7/31

* Use M2 to quantitate fluorescence emitting by K381001 in lyophilized *E. coli* powder

### 8/1~8/31

* Use our device to do the nitrate sensing test by K381001 in lyophilized *E. coli* powder.

## Whole pathway test

---

### 10/7~10/10

* Use our device and modified *E. coli* to test our whole project.

BBa\_K2275007

---

BBa\_K2275008

---

BBa\_K2275009

---

BBa\_K2275010

---

BBa\_K2275011

---

BBa\_K2275012

---

Nitrate sensing test

---

Whole pathway test

---

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# Team:NCKU Tainan/Experiments

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# NCKU\_Tainan

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

##### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

##### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:NCKU Tainan/Results

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# Results

# Sensing

---

Our experiments are conducted by using our own assembling GFP sensing device. By adding 3ml of fixed concentration nitrate potassium into the cuvette, the solution will then mixed with the lyophilized *E. coli* powder. Rapidly, nitrate will activate the PyeaR promoter which lead to expression of GFP. The result is shown as below.

![](https://static.igem.org/mediawiki/2017/1/13/T--NCKU_Tainan--result-sensing.png)

Figure 1. The graph shows difference between low and high level of nitrate.

![](https://static.igem.org/mediawiki/2017/d/db/T--NCKU_Tainan--result-sensing2.png)

Figure 2. The growing curve of electrical signals under different nitrate concentration

Figure 1 and 2 show that our sensing device has the ability to distinguish between different nitrate concentration, even if the nitrate level is lower than 10 ppm.

![](https://static.igem.org/mediawiki/2017/5/53/T--NCKU_Tainan--result-sensing3.png)

Figure 3. comparison between 40 ,60 and 100 ppm

Before 900 seconds, the growing curves of 40 ppm and 60 ppm are clearly separated, while growing curves of 60 ppm and 100 ppm are quite indistinguishable. After 1000 seconds curve of 60 ppm reached its saturation but 100 ppm kept growing, thus we are able to differentiate 60 ppm and 100 ppm.

# Regulation

---

## Nitrate Assimilation Process

---

### From nitrate to nitrite

Due to too high GC content of one part sequence of *nar*, it failed to synthesize by IDT. Thus, we turned to use original *nar* cluster existed in the wild type *E. coli* MG1655 had been used in our project. However, the activity
of *nar* in the *E. coli* MG1655 is much lower than the one we chose before. Wondering whether the activity of *nar* cluster in *E. coli* MG1655 is strong enough, we use the griess reaction to detect the nitrite concentration.
The principle and mechanism of griess reaction are shown below. When the broth contains nitrite, by adding the griess reactant which consists of naphthylethylenediamine dihydrochloride, sulphanilamide, phosphoric acid can form a diazonium salt
with nitrite. The diazonium salt will have absorbance at wavelength 545 nm. By the reaction, we can further calculate the nitrate conversion.

![](https://static.igem.org/mediawiki/2017/7/73/T--NCKU_Tainan--project-result-1.png)

Figure 1. The mechanism of griess reaction

In our experiment, we use 1-Naphthylamine to substitute naphthylethylenediamine dihydrochloride. Before starting our test, we need to do the calibration curve between the absorbance at wavelength 545 nm and different nitrite concentration. The result
was shown in Figure 2. With the calibration curve, we can deduce the nitrite concentration from absorbance in different time.

![](https://static.igem.org/mediawiki/2017/8/8d/T--NCKU_Tainan--result2.png)

Figure 2. The calibration curve for the absorbance at wavelength 545 nm of different nitrite concentration

We added 10 and 50 μM of nitrate to the broth and cultured for 4 hours. After that we add the griess reactant in order to detect the nitrite concentration. By the calibration curve, we deduced the concentration of nitrite produced, and then the conversion
of nitrate was calculated. The conversion result is shown in Figure 3.

![](https://static.igem.org/mediawiki/2017/archive/b/bc/20171129031529%21NCKU-result-fig3.png)

Figure 3. The conversion of nitrate by *nar* cluster

![](https://static.igem.org/mediawiki/2017/9/9e/T--NCKU_Tainan--result4.jpg)

Figure 4. The coloring after adding griess reactant after 5 mins. The concentration from left to right is increasing.

Figure 3 shows that after 4 hour reacted, the conversion of nitrite concentration produced about 20-30 %. Thus, the *nar* cluster in *E. coli* MG1655 has the ability to convert nitrate into nitrite.

### From nitrite to ammonium Achievement

1. Cloned the *nir* cluster(*nirB* and *nirD*) into pSB1C3 and created a new Biobrick: BBa\_K2275007
2. Characterized and qualified the function of *nir* cluster by griess reaction

After constructing the Biobrick: BBa\_K2275007 and transforming by heat shock into the DH5α, we selected several colonies from the plate and cultured in LB medium for 8 to 10 hour. Then extracted the plasmid by FAVORGEN FavorPrepTM Plasmid DNA Extraction Mini Kit and used plasmid digestion with *Eco*RΙ for confirmation. The confirmation result is shown in Figure 5.

![](https://static.igem.org/mediawiki/2017/5/58/T--NCKU_Tainan--project-result-5.png)

Figure 5. The result of plasmid digestion with *Eco*RΙ. The size of BBa\_K2275007 is about 5.2k bp. The symbol (+) represents with *Eco*RΙ, (-) without.

After correct construction, we transform the plasmid into *E. coli* MG1655. In order to test the function of *E. coli* MG1655 with plasmid (Biobrick BBa\_K2275007) which contains the gene *nir* cluster. Adding the sodium nitrite
and using the method of griess reaction to detect whether the nitrite concentration was decreased with time. Furthermore, we want to make sure that nitrite won’t degrade in water by itself. We do the test by adding sodium nitrite in the water
only without any *E. coli* MG1655. The result is shown in Figure 6 & 7.

![](https://static.igem.org/mediawiki/2017/b/ba/T--NCKU_Tainan--result6.png)

Figure 6. The absorbance at 10, 20, 50 μM nitrite concentration in water without *E. coli* before and after 4 hours. The light green bar represents 0 hour, and the dark green bar represents 4 hours.

![](https://static.igem.org/mediawiki/2017/d/d1/T--NCKU_Tainan--result7.png)

Figure 7. The conversion of nitrite by *nir* culster

From Figure 6, the result indicated that nitrite concentration maintained the same before and after 4 hour. In Figure 7, the conversion of nitrite by *nir* cluster is pretty high. The result approves of that *nir* gene has the ability
to convert nitrite.

## Ammonium Assimilation Process

---

### From ammonium to glutamate Achievement

1. Successfully express the gene *gudB* from *Bacillus subtilis* in *E. coli* DH5α & MG1655.
2. Cloned the gene *gudB* into pSB1C3 and created a new Biobrick: BBa\_K2275008
3. Express the protein of *gudB* in *E. coli* MG1655.
4. Characterized and qualified the function of the gene *gudB* by Glutamate Colorimetric Assay Kit

In order to check our construction Biobrick: BBa\_K2275008. We selected several colonies from the transformed plate and cultured in LB medium for 8 to 10 hour. Then extracted the plasmid with FAVORGEN FavorPrepTM Plasmid DNA Extraction Mini Kit and used
plasmid digestion with *Bam*HΙ or *Pst*Ι for confirmation. The confirmation result is shown in Figure 8

![](https://static.igem.org/mediawiki/2017/d/d6/T--NCKU_Tainan--project-result-8.png)

Figure 8. The result of plasmid digestion. We use one cut with *Pst*Ι(left lane) and two cut with *Pst*Ι and *Bam*HΙ(medium lane). The size of BBa\_K2275008 is about 3.6k bp(left lane). The size of *gudB* is about 1.3k bp (purple arrow), and 2.3k bp for backbone (orange arrow).

Next, to ensure whether the gene *gudB* can successfully express in *E. coli* or not, we used the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the protein expression of *gudB*. The SDS-PAGE result
is shown in Figure 9.

![](https://static.igem.org/mediawiki/2017/1/15/T--NCKU_Tainan--project-result-9.png)

Figure 9. The SDS-PAGE of *gudB* in *E. coli* MG1655 with and without plasmid. The protein size of *gudB* is about 47 kDa (red arrow). Symbol “WC” is whole cell, “S” is supernatant, “P” is pellet, and “C” is control which isn’t
contain plasmid.

Figure 9 shows that the protein expression was most in pellet. Compared to the control, the expression was much more than *E. coli* MG1655 without plasmid. However, no supernatant protein can’t represent the protein is no function. Hence,
we added the ammonium and used Glutamate Colorimetric Assay Kit to detect the glutamate concentration. The glutamate Enzyme Mix recognizes glutamate as a specific substrate leading to proportional color development. The amount of glutamate can
therefore be easily quantified by spectrometer at 450 nm. At first, we need to do the calibration curve by adding different concentration of ammonium and detecting the absorbance at OD450nm . The data of calibration curve is shown in Figure
10. The next step, we do three different experiments to test if the *E. coli* MG1655 contains the Biobrick: BBa\_K2275009 can convert the ammonium into glutamate. The testing result is shown in Figure 11.

![](https://static.igem.org/mediawiki/2017/a/ae/T--NCKU_Tainan--result10.png)

Figure 10. The calibration curve by adding different concentration of ammonium and detecting the absorbance at OD450nm.

![](https://static.igem.org/mediawiki/2017/5/50/T--NCKU_Tainan--result11.png)

Figure 11. The glutamate concentration in situation with or without ammonium and plasmid

Figure 10 shows that *E. coli*, which contains the plasmid, can convert ammonia into glutamate. The higher concentration of ammonia, the higher glutamate produced by *E. coli*. Seen in Figure 11, when both ammonium and plasmid were added,
the glutamate produced was more than the either plasmid or ammonium was added. This feature explains that the *gudB* can successfully express by *E. coli* MG1655 and convert ammonium into glutamate.

### From glutamate to glutamine Achievement

1. Successfully express the gene *glnA* from *Pseudomonas putida* in *E. coli* DH5α & MG1655.
2. Cloned the gene *glnA* into pSB1C3 and created a new Biobrick: BBa\_K2275009
3. Express the protein of *glnA* in *E. coli* MG1655.
4. Characterized and qualified the function of the gene *glnA* by Glutamine Colorimetric Assay Kit

After ligation and transformation into DH5α, we chose colonies culturing for 8 to 10 hour in LB medium at 37℃ and then extracted the plasmid. Digest with *Hind*ΙΙΙ and *Pst*Ι to examine if the construction
BBa\_K2275009 is correct. The confirmation is shown in Figure 12.

![](https://static.igem.org/mediawiki/2017/8/80/T--NCKU_Tainan--project-result-12.png)

Figure 12. The result of plasmid digestion. Plasmid is digested into two fragment of 1.4 kbp and 2.3 kbp indicating our construction was succeed.

Following, to confirm whether the *glnA* can successfully express in *E. coli* or not, we used SDS-PAGE to see the protein expression of *glnA*. The SDS-PAGE result was shown in Figure 13.

![](https://static.igem.org/mediawiki/2017/a/ad/T--NCKU_Tainan--project-result-13.png)

Figure 13. The SDS-PAGE of *glnA* expressed in *E. coli* MG1655. The protein size of *glnA* is about 52 kDa (red arrow). Symbol “WC” is whole cell, “S” is supernatant, “P” is pellet, and “C” is control which isn’t contained
plasmid.

Opposite to protein expression of *gudB*, the expression of *glnA* was most in supernatant. To test if the biobrick can work in the *E. coli*, we added the glutamate and use Glutamine Colorimetric Assay Kit to detect the glutamine
concentration. The assay is based on the hydrolysis of Glutamine to Glutamate producing a stable signal, which is directly in proportion to the amount of Gln in the sample. The process of the assay is shown below.

![](https://static.igem.org/mediawiki/2017/7/7d/NCKU-result-formular.png)

At first, we also need to do the calibration curve by adding different concentration of glutamate and detecting the absorbance at OD450nm . The data of calibration curve is shown in Figure 14. Following, we also do three different experiments to test
if the *E. coli* MG1655 contain the Biobrick: BBa\_K2275009 is better to produce more glutamine. The assay result is shown in Figure 15.

![](https://static.igem.org/mediawiki/2017/0/0e/T--NCKU_Tainan--result14.png)

Figure 14. The calibration curve by adding different concentration of glutamate and detecting the absorbance at OD450nm.

![](https://static.igem.org/mediawiki/2017/d/dd/T--NCKU_Tainan--result15.png)

Figure 15. The glutamine concentration in situation with or without plasmid and glutamate

Figure 14 shows that *E. coli*, which contains the plasmid, can convert glutamate into glutamine. The higher concentration of glutamate, the higher glutamine produced by *E. coli*. Shown in Figure 15 when both glutamate and plasmid were
added, the glutamine produced was more than the either plasmid or glutamate was added. This feature explains that the *glnA* can successfully express by *E. coli* MG1655 and convert glutamate into glutamine.

### From ammonium to glutamine Achievement

1. Cloned the gene *gudB* into BBa\_K2275009 and created a new Biobrick: BBa\_K2275010.
2. Express the protein of *gudB* and *glnA* in *E. coli* MG1655.
3. Characterized and qualified the function of the gene *gudB* and *glnA* by Glutamate Colorimetric Assay Kit and Glutamine Colorimetric Assay Kit

We also constructed gene *gudB* and *glnA* into the same backbone. After constructing and transforming into DH5α, we selected several colonies from the plates to confirm if the construction is correct or not and extracted the
plasmid by kit. Using plasmid digestion with *Xba*Ι and *Pst*Ι to confirm. The result is shown in Figure 16.

![](https://static.igem.org/mediawiki/2017/0/01/T--NCKU_Tainan--project-result-16.png)

Figure 16. Confirmation the construction of BBa\_K2275010 by restriction enzyme digestion. We use one cut with *Xba*Ι (left lane) and two cut with *Pst*Ι and *Xba*Ι (right lane). The size of BBa\_K2275008 is about
5.4 kbp, while the digested fragment is about 3.2 kbp and 2.1 kbp (right lane).

Next, we use the SDS-PAGE to analyze if the two proteins can express in *E. coli* at the same time. The SDS-PAGE result is shown in Figure 17.

![](https://static.igem.org/mediawiki/2017/0/04/T--NCKU_Tainan--project-result-17.png)

Figure 17. The SDS-PAGE of Biobrick : BBa\_K2275010 expressed in *E. coli* MG1655. The protein size of *glnA* is about 52 kDa (red arrow). The protein size of *gudB* is about 42 kDa (Brown arrow). Symbol “WC” is whole
cell, “S” is supernatant, “P” is pellet, and “C” is control which isn’t contained plasmid.

We can see that the *glnA* and *gudB* both expressed in *E. coli* at the same time. Next, we want to test if the Biobrick can function to convert ammonium to glutamine, so we add different ammonium concentration and use kit to detect
the glutamine concentration. The result is in Figure 18.

![](https://static.igem.org/mediawiki/2017/5/52/T--NCKU_Tainan--result18.png)

Figure 18. The glutamine concentration in situation with or without plasmid and ammonium

In Figure 18, the lane 2 and lane 3 can show the function of our biobrick. When only adding ammonium, the concentration of glutamine was very low. However, both adding the plasmid and ammonium, the concentration of glutamine is higher. The result indicated
that our biobrick has the function which can convert ammonium to glutamine.

### Function test of whole pathway

Before applying our Biobrick on the device, we in advance co-culture *E. coli* MG1655 which contains two kinds of plasmid, one is the BBa\_K2275007 and the other is the BBa\_K2275010, with nitrite adding under 37℃ for 12 hours. Afterward,
we took out some broth and analyzed the glutamine concentration by kit.

![](https://static.igem.org/mediawiki/2017/5/56/NCKU-result-fig19.png)

Figure 19. The glutamine concentration in situation with or without plasmid and ammonium

In Figure 19, with adding plasmid, the glutamine concentration of lane 3 is much more than lane 2. It indicated that the overall pathway of converting nitrite into ammonium was functional.

### Combined with device

In order to test our Biobricks can work in the device. We co-culture *E. coli* MG1655 which contains two kinds of plasmid and one is the BBa\_K2275007 and the other is the BBa\_K2275010, for 12 hours. After that we added 100 ppm sodium nitrate
into the broth. Using sponge to absorb the broth and putting into the device. Take 2 mL broth from the device every two hours and analyze nitrite concentration with method of griess reaction.

![](https://static.igem.org/mediawiki/2017/2/2c/NCKU-result-fig20.png)

Figure 20. The conversion of nitrite every two hours

According to the data, the result was consistent with our prediction. The conversion of nitrite increased with time. This indicated that nitrite concentration was decreased. Therefore, our biobrick can function in the device that converted the nitrite
into glutamine.

## Conclusion

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Based on our functional test results, all of our constructed biobricks worked. Though the *E. coli* MG1655 already had the *nir* gene, our biobricks further improved the efficiency of the conversion. In conclusion, our biobricks can change nitrate waste into valuable substance.

Sensing

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Nitrate Assimilation Process

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Ammonium Assimilation Process

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Conclusion

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