iGEM 2017 — Team NTHU_Taiwan

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# Team:NTHU Taiwan/Notebook

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# Team:NTHU Taiwan/Experiments

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AWARDS

[Applied Design](https://2017.igem.org/Team%3ANTHU_Taiwan/Applied_Design)
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[JUDGING FORM](https://igem.org/2017_Judging_Form?team=NTHU_Taiwan)

# Experiments

---

## Integration

The first thing we should do is to transform a plasmid into *E. coli* and express the protein we want. So we get our target sequence synthesized by IDT and cloned into pSB1C3.Finally, we transform it into *E. coli* and get the target protein. In order to make the protein more effective, we purify the protein by his-tag column.

## Double Heat Shock

Transform is an important part of the experiment. It determines whether your plasmid can be amplified or not. There are two ways to transform our plasmid in our lab, mix and go and heat shock. First, mix and go is a convenient and fast way, but the success rate is low.Heat shock is more effective but it causes more time to do. However, in some experiments, even using heat shock also has a low success rate. Therefore, we change a small part of heat shock protocol, which is to incubate the competent cells at 42 degrees for two times.

![](https://static.igem.org/mediawiki/2017/a/ad/T--NTHU_Taiwan--Experiments--step.png)

## Degradation test ( HRP , [K2354012](http://parts.igem.org/Part%3ABBa_K2354012))

In order to verify whether our enzymes (HRP) can degrade EDCs or not, we added HRP, diluted H2O2, which is a cofactor for HRP to react, and BPA ( NP ) to 1 mL water totally. H2O2 and BPA (NP) will be degraded by HRP in 40°C incubator and shocked smoothly for 24 hours for the purpose of a complete reaction. After a day, we denatured and spun down HRP for fear that it may degrade BPA ( NP ) continuously. By taking 3µL supernatant of the sample and detect the compounds via LC PDA, we can determine the ability of enzymes to decompose BPA (NP). We will add HRP and various concentrations of BPA (NP) together to test the decomposition efficiency of HRP in different environments.([Results](https://2017.igem.org/Team%3ANTHU_Taiwan/Results))

## Detection test ( ER-α Part : [K2354011](http://parts.igem.org/Part%3ABBa_K2354011) and Monobody [K2354010](http://parts.igem.org/Part%3ABBa_K2354010) )

In the detection part of our experiment, we used two composite proteins to build our detector, including ERα (Estrogen Receptor alpha) which will be expressed on the surface of modified *E. coli* to capture BPA (NP) and monobody that is assembled on a gold electrode surface. Before detection test, we need to prepare modified *E. coli* with Erα and the samples of Monobody.

The following are the steps we used to produce proteins :

Erα :
We incubated 5 mL culture to reach OD=0.5 and induced with IPTG. After expressing the culture for 5 hours, the cells were centrifuged and resuspended in PBS buffer. To reduce the activity of *E. coli*, we freeze our cells in -80°C.

Monobody :
We incubated 300 mL culture to reach OD=0.5 and induced with IPTG. After expressing the culture for 12 hours, the cells were harvested by centrifugation at 4 °C. Proteins will be obtained after purification and be stored at 20°C.

As long as we mix the sample with BPA (NP) solution and *E. coli* expressing ER-alpha together, ERα will capture BPA (NP) and change their structures of complexes. On the other hand, we dropped 10µL of the solution of monobody on the gold surface and store it in 4°C for 5 hours. When the time was up, we blotted the solution of monobody gently and dropped 10µL of the sample on the place containing monobody for 30 minutes to finish our samples.

Theoretically, Erα which captured BPA (NP) can bind to monobody, causing some *E. coli* to stick on the gold surface. We can check our samples via IR test to prove the experiment.(Figure 1.)([Results](https://2017.igem.org/Team%3ANTHU_Taiwan/Results))

In order to estimate the corresponding concentration of EDCs, we washed the sample by ddH2O and dropped crystal violet solution to stain *E. coli* on the gold surface for 2 min, then we washed crystal violet solution by ddH2O and get the density of *E. coli* under a microscope.([Results](https://2017.igem.org/Team%3ANTHU_Taiwan/Results))

![](https://static.igem.org/mediawiki/2017/2/2e/T--NTHU_Taiwan--Experiments--Figure_1._20171015_IR_test.png)

Figure 1. 20171015 IR test

![](https://static.igem.org/mediawiki/2017/0/02/T--NTHU_Taiwan--Experiments--Figure_2._20171014_IR_test%28chip1-5%29_%2B_microscope_test%28chip6-11%29.png)

Figure 2. 20171014 IR test(chip1-5) + microscope test(chip6-11)

## Function of ice nucleation protein (INP)

INP is a protein which can bring other proteins to the membrane surface of *E. coli*. To check the function of INP, we transformed *E. coli* which contained RFP with INP and the other sample which contained RFP without INP. The results can be obtained by comparing the difference between RFP and RFP-IP after we spun down lysis of cells.([Results](https://2017.igem.org/Team%3ANTHU_Taiwan/Results))

## Function of gold binding protein (GBP)

In our experiment, we used GBP to bind Monobody on the surface of gold flakes. We dropped 10µL of the solution of purified monobody with GBP on the gold surface, then stored it in 4°C for 5 hours to let monoboby bind to the gold surface as far as possible.

We can prove the function of GBP by comparing the results between the gold surface and the gold surface contained Monobody via IR test.([Results](https://2017.igem.org/Team%3ANTHU_Taiwan/Results))

## Functional test of ER-alpha

To ensure the function of ER-alpha on the surface of *E. coli*, we compared the difference between BL-21 *E. coli* and *E. coli* which expressed ER-alpha in the different concentration of BPA and NP. From the [results](https://2017.igem.org/Team%3ANTHU_Taiwan/Results), we found that BL-21 can’t affect the outcomes of the biosensing chip.

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# Team:NTHU Taiwan/Results

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[Silver HP](https://2017.igem.org/Team%3ANTHU_Taiwan/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ANTHU_Taiwan/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ANTHU_Taiwan/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3ANTHU_Taiwan/Applied_Design)
[Integrated Human Practices](https://2017.igem.org/Team%3ANTHU_Taiwan/HP/Gold_Integrated)
[Hardware](https://2017.igem.org/Team%3ANTHU_Taiwan/Hardware)
[Model](https://2017.igem.org/Team%3ANTHU_Taiwan/Model)
[Software](https://2017.igem.org/Team%3ANTHU_Taiwan/Software)
[Criteria](https://2017.igem.org/Team%3ANTHU_Taiwan/Criteria)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=NTHU_Taiwan)

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# Results

---

|  |
| --- |
| Overview   1. Constructed and confirmed all of our composite parts.   2. Proved our recombinant HRP can degrade BPA and NP by the result of mass spectrum.   3. Used IR spectrum to prove the function of our detection method and we took the microscope images on the gold surface to prove the ability to estimate the concentration of EDCs in the water.   4. Found the limitation of our detection method is 5 µM (about 1ppm) of EDCs.   5. Showed comparison of our detection method and traditional ways:   ![](https://static.igem.org/mediawiki/2017/9/91/265.png) |

# PART I Degradation

## Cloning of Horseradish Peroxidase

To make sure the plasmid is cloned into *E. coli* BL-21 strain, we extracted the plasmid from transformed *E. coli*.(Figure 1) We validated the gene by PCR with specific primers and then we examined the result with Agarose gel electrophoresis. A successful cloning was verified from the results of a PCR performed with designed specific primers according to the theoretically expected length of horseradish peroxidase(927bp)(Figure 2).

![](https://static.igem.org/mediawiki/2017/b/bb/T--NTHU_Taiwan--Results--hrp_ecoli.png)

Figure 1.

![](https://static.igem.org/mediawiki/2017/7/70/T--NTHU_Taiwan--Results--hrp_pcr3.png)

Figure 2. Primers for VR backbone:1,3,5 ;primers for HRP:2,4,6

## Expression and purification of apo-HRP and refolded-HRP

To obtain functional horseradish peroxidase we need to purify the protein from *E. coli*, but this kind of protein does not have any function and it is called apoprotein. After the first time purification, we refolded the protein to construct the correct structure and then we activated the apo-HRP with hemin to produce the functional HRP. We examined the existence of this protein by SDS-PAGE after purification. (Figure 3)

![](https://static.igem.org/mediawiki/2017/3/3a/T--NTHU_Taiwan--Results--hrp_page.png)

Figure 3. SDS-PAGE for purification of HRP

## Functional test of Horseradish Peroxidase

We use the mass spectrum to prove HRP can degrade BPA and NP by the signals of the large molecular weight of by-products.(Figure 4-7) We found that there are lots of additional peaks show up after degraded by HRP, and this result can prove our HRP can degrade BPA and NP.

![](https://static.igem.org/mediawiki/2017/f/fb/T--NTHU_Taiwan--Results--ms_bpa.png)

Figure 4. the mass spectrum of BPA

![](https://static.igem.org/mediawiki/2017/2/2f/T--NTHU_Taiwan--Results--ms_bpa_hrp.png)

Figure 5. the mass spectrum of BPA after degradation

![](https://static.igem.org/mediawiki/2017/2/28/T--NTHU_Taiwan--Results--ms_np.png)

Figure 6. the mass spectrum of NP

![](https://static.igem.org/mediawiki/2017/b/b3/T--NTHU_Taiwan--Results--ms_np_hrp.png)

Figure 7. the mass spectrum of NP after degradation

# PART II Detection

## Cloning of ER-alpha and monobody

We cloned the sequence of ER-alpha and Monobody into the vector, and then we transformed the plasmid into BL-21 competent cells, respectively.(Figure 8 and 9)
After transformation, we extracted both plasmids from the cells and the vector was validated by gel electrophoresis.

![](https://static.igem.org/mediawiki/2017/c/ce/T--NTHU_Taiwan--Results--mono_ecoli.png)

Figure 8. *E. coli* with gene of monobody

![](https://static.igem.org/mediawiki/2017/9/98/T--NTHU_Taiwan--Results--er_ecoli.png)

Figure 9. *E. coli* with gene of ER-alpha

A successful cloning was verified from the results of a PCR performed with specifically designed primers according to the theoretically expected length of ER-alpha\_INP fusion gene (2199bp) and Monobody (797bp).(Figure 10)

![](https://static.igem.org/mediawiki/2017/2/28/T--NTHU_Taiwan--Results--PCR2.png)

Figure 10. 1 : RFP ( mRFP F-primer and mRFP R-primer ); 2 : IPTG (promoter) + INP + RFP2 (ER F-primer and ER R-primer ); 3 : IPTG (promoter) + INP + RFP 1 (ER F-primer and ER R-primer ) ; 4 : IPTG (promoter) + ER with red spot (ER F-primer and ER R-primer ); 5 : IPTG (promoter) + ER2 (ER F-primer and ER R-primer ); 6 : IPTG (promoter) + ER1 (ER F-primer and ER R-primer ); 7 : IPTG (promoter) + Monobody with red spot (Monobody F-primer and Monobody R-primer ); 8 : IPTG (promoter) + Monobody 2 (Monobody F-primer and Monobody R-primer ) ; 9 : IPTG (promoter) + Monobody 1 (Monobody F-primer and Monobody R-primer ); 10 : RFP ( VR primer and VF2 primer ); 11 : IPTG (promoter) + INP + RFP 2 ( VR primer and VF2 primer ); 12 : IPTG (promoter) + INP + RFP 1 ( VR primer and VF2 primer ); 13 : IPTG (promoter) + ER with red spot ( VR primer and VF2 primer ); 14 : IPTG (promoter) + ER2 ( VR primer and VF2 primer ); 15 : IPTG (promoter) + ER1 ( VR primer and VF2 primer ); 16 : IPTG (promoter) + Monobody with red spot ( VR primer and VF2 primer ); 17 : IPTG (promoter) + Monobody 2 ( VR primer and VF2 primer ); 18 : IPTG (promoter) + Monobody 1 ( VR primer and VF2 primer )

## Function of ice nucleation protein (INP)

We compared the difference between *E. coli* expressed RFP and RFP-INP(Figure 11). After the lysis of culture expressing RFP-INP, most of RFP was on the fragments of the membrane. Due to the larger structure of plasma membrane, it is easily spin down. If RFP-INP is on the *E. coli* plasma membrane when we centrifuged the cell lysate, RFP-INP is spun down with the plasma membrane, and we see a red pellet.

On contract, after cell lysis of RFP expression *E. coli*, most of RFP suspended in the solution. Since the centrifugation force, we set in this experiment is not enough to spin down particles as small as RFP. We see RFP suspended in the supernatant even after centrifugation.

This experiment showed that INP can bring RFP to the membrane of *E. coli*, and this result proved the function of INP.

![](https://static.igem.org/mediawiki/2017/d/d7/T--NTHU_Taiwan--Results--inp.png)

Figure 11. 1 : Fragments of *E. coli* with gene of RFP-INP ; 2: Fragments of *E. coli* with gene of RFP

## Characterization of biosensing chip

To prove that our biosensing chip can distinguish the sample has EDCs or not, we used IR spectrum to measure specific bonds on *E. coli* and verify the detection function.(Table 1)

![](https://static.igem.org/mediawiki/2017/7/71/T--NTHU_Taiwan--Results--table_1.png)

Table 1. The IR signals for *E. coli*

In the beginning, we try to use live cells for the biosensing chip. However, we found that all the samples have the information of *E. coli*. Showing that there is no difference between the sample with 5mM EDCs and the sample with no EDCs. (Figure 12)

![](https://static.igem.org/mediawiki/2017/1/1f/T--NTHU_Taiwan--Results--ir.png)

Figure 12. The IR spectrum of samples in different conditions (live *E. coli*)

To decrease the activity of *E. coli*, we tried to freeze *E. coli* in -80℃ for 24 hours and use it after thawing on ice immediately. We found that there is a significant difference between the sample with 5mM EDCs and EDCs-free and the intensity of IR signals also decrease due to the reduction of mobility of *E. coli*.(Figure 13) This result can prove that our biosensing chip can detect EDCs in the water.

![](https://static.igem.org/mediawiki/2017/6/6c/T--NTHU_Taiwan--Results--ir2.png)

Figure 13. The IR spectrum of samples in different conditions (freezed *E. coli*)

## Proof of concept: quantifying a number of EDCs in the water

To prove our biosensing chip can measure the concentration of EDCs, we use crystal violet to stain *E. coli* on the surface of gold and then observe the density of stained *E. coli* under microscope. Due to the surface tension force of water, there are lots of *E. coli* remained around the drop edge of sample, and we only observed the area in the middle of the sample to reduce the error. (Figure 14)

![](https://static.igem.org/mediawiki/2017/2/29/T--NTHU_Taiwan--Results--demon.png)

Figure 14. The sample under 50X microscope

We observed the density of E. coli when the samples contained a different concentration of BPA and NP from 5mM to 5nM (Figure 15 and 16). This result indicates that when the concentration of EDCs decreases, the density of E. coli will decrease as well.

![](https://static.igem.org/mediawiki/2017/3/32/T--NTHU_Taiwan--Results--bpa_500.png)

Figure 15. The samples with different concentration of BPA under 500X microscope

![](https://static.igem.org/mediawiki/2017/8/81/T--NTHU_Taiwan--Results--np_500.png)

Figure 16. The samples with different concentration of NP under 500X microscope

For the practical propose, we added trehalose (0.1M) to prevent ER-alpha from denaturing and increase the time of conservation. We also observed the density of *E. coli* with samples containing the different concentration of BPA and NP from 5mM to 5nM.(Figure 17 and 18) From the result of observation, we can find the similar tendency as the sample without trehalose.

![](https://static.igem.org/mediawiki/2017/7/70/T--NTHU_Taiwan--Results--tbpa_500.png)

Figure 17. The samples with different concentration of BPA in 0.1M trehalose under 500X microscope

![](https://static.igem.org/mediawiki/2017/c/c9/T--NTHU_Taiwan--Results--tnp_500.png)

Figure 18. the samples with different concentration of NP in 0.1M trehalose under 500X microscope

To clarify the relationship between the concentration of EDCs and the density of *E. coli*, we use image J to count the amount of *E. coli* on the surface. (Figure 19-22) From the results of imageJ, we can get the similar tendency as the result from the pictures of the microscope. However, the density of *E. coli* decreases in the presence of 0.1 M trehalose, and this result suggested that although trehalose can improve the time to store lyophilized *E. coli*, it can decrease the precision of our biosensing chip.

![](https://static.igem.org/mediawiki/2017/7/71/21.png)

Figure 19. Samples of BPA

![](https://static.igem.org/mediawiki/2017/2/2f/T--NTHU_Taiwan--Results--np-22.png)

Figure 20. Samples of NP

![](https://static.igem.org/mediawiki/2017/5/5e/T--NTHU_Taiwan--Results--tbpa-23.png)

Figure 21. Samples of BPA in 0.1M trehalose

![](https://static.igem.org/mediawiki/2017/5/55/T--NTHU_Taiwan--Results--tnp-24.png)

Figure 22. Samples of NP in 0.1M trehalose

## Functional test of ER-alpha

To ensure the function of ER-alpha on the surface of *E. coli*, we compared the difference between the BL-21 *E. coli* and ER-alpha expressed *E. coli* in the different concentration of BPA and NP. (Figure 23 and 24)From the results, we found that BL-21 can’t affect the outcomes of the biosensing chip beyond 5µM of EDCs.

![](https://static.igem.org/mediawiki/2017/f/f8/T--NTHU_Taiwan--Result--BPA.png)

Figure 23. Samples of BPA

![](https://static.igem.org/mediawiki/2017/c/c8/T--NTHU_Taiwan--Result--NP.png)

Figure 24. Sample of NP

## The limitation of our biosensing chip

To further understand the limitation of the concentration that our biosensing chip can achieve, we observed the density of *E. coli* in four different conditions of background. We find that when there are no EDCs in the sample, the density of *E. coli* is close to the concentration of EDCs below 5µM. Showing that our biosensing chip can’t measure the concentration of EDCs less than 5µM.(Figure 25 and Table 2)

![](https://static.igem.org/mediawiki/2017/b/be/T--NTHU_Taiwan--Results--4%2A25.png)

Figure 25. The background of 4 different conditions

![](https://static.igem.org/mediawiki/2017/6/6f/T--NTHU_Taiwan--Results---t-26.png)

Table 2. The amounts of *E. coli* in the 4 different backgrounds

# Part III Future Work

1. Since we failed to express GFP and ER-alpha at the same time, we will try to construct the gene containing GFP and ER-alpha and express them together for the purpose of detection. (Figure 26)

![](https://static.igem.org/mediawiki/2017/b/be/T--NTHU_Taiwan--Results--f-1.png)

Figure 26. Expression of GFP in the *E. coli* for detection.

2. Since we don’t have time to construct the biosensing chip to measure the change of fluorescence, we will construct the biosensing chip to estimate the concentration of EDCs in the water precisely. (Figure 27)

![](https://static.igem.org/mediawiki/2017/f/f7/T--NTHU_Taiwan--Results--f-2.png)

Figure 27. Using the change of fluorescence to estimate the precise concentration of EDCs in the water

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