Revision as of 13:18, 31 October 2017

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 1.

Figure 2. Primers for VR backbone:1,3,5 ;primers for monobody: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)

Figure 3. SDS-PAGE for purification of HRP

Functional test of Horseradish Peroxidase

To prove the degradation ability of HRP, we mixed 25 µg of HRP with 1 mM H2O2 and BPA or NP in the 1 mL water. The environment of degradation is suitable for HRP to degrade the phenolic compounds(40℃ and pH=6-7). After degradation for 24 hours, we denatured HRP by briefly heating up to 80℃ and use LC-PDA (Liquid Chromatography - Photodiode Array detector) to analysis the result of degradation.(figure 4 and 5)

Figure 4. The result of LC-PDA (BPA sample)

Figure 5. The result of LC-PDA (NP sample)

From the result of LC-PDA, we found that there are two extra peaks showing after degradation and these extra peaks represent the by-products from degradation. Unfortunately, we can’t know how much EDCs is degraded by HRP because:

(1) The peak of the by-products overlap the peak of EDCs and we can’t get the information of remained EDCs. (There is a by-product have a broad peak from 2.2 min to 3.5min and its intensity also much higher than the peak of EDCs)

(2) The quality of the column from LC isn’t good enough to separate the by-products from EDCs due to their similar molecular properties. (This LC can’t distinguish between BPA and NP so we estimated that it can’t distinguish the degraded by-product from BPA or NP as well.)

Moreover, 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 6-9) 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.

Figure 6. the mass spectrum of BPA

Figure 7. the mass spectrum of BPA after degradation

Figure 8. the mass spectrum of NP

Figure 9. 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 10 and 11) After transformation, we extracted both plasmids from the cells and the vector was validated by gel electrophoresis.

Figure 10. E. coli with gene of monobody

Figure 11. 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 and Monobody (797bp).(Figure 12)

Figure 12. 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 13). 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.

Figure 13. 1 : Fragments of E. coli with gene of RFP-INP ; 2: Fragments of E. coli with gene of RFP

Characterization of detection system

To prove that our detection system 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)

Table 1. The IR signals for E. coli

In the beginning, we try to use live cells for the detection system. 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 14)

Figure 14. 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 15) This result can prove that our detection system can detect EDCs in the water.

Figure 15. 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 detection system 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 16)

Figure 16. Tthe sample under 50X microscope

We observed the density of E. coli when the samples contained different concentration of BPA and NP from 5mM to 5nM (Figure 17 and 18). This result indicates that when the concentration of EDCs decreases, the density of E. coli will decrease as well.

Figure 17. The samples with different concentration of BPA under 500X microscope

Figure 18. 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 19 and 20) From the result of observation, we can find the similar tendency as the sample without trehalose.

Figure 19. The samples with different concentration of BPA in 0.1M trehalose under 500X microscope

Figure 20. 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 21-24) 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 detection system.

Figure 21. Samples of BPA

Figure 22. Samples of NP

Figure 23. Samples of BPA in 0.1M trehalose

Figure 24. 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 25 and 26)From the results, we found that BL-21 can’t affect the outcomes of the detection system.

Figure 25. Samples of BPA

Figure 26. Sample of NP

The limitation of our detection system

To further understand the limitation of the concentration that our system 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 detection system can’t measure the concentration of EDCs less than 5µM.(Figure 25 and Table 2)

Figure 27. The background of 4 different conditions

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)

Figure 28. Expression of GFP in the E. coli for detection.

2. Since we don’t have time to construct the detection system to measure the change of fluorescence or surface plasmon resonance, we will construct the detection system to estimate the concentration of EDCs in the water precisely. (Figure 27)

Figure 29. Using the change of fluorescence to estimate the precise concentration of EDCs in the water

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+	To make sure the plasmid is cloned into <I>E. coli</I> BL-21 strain, we extracted the plasmid from transformed <I>E. coli</I>.(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).
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+	Figure 1.
+	</center></center></p>
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+	Figure 2. Primers for VR backbone:1,3,5 ;primers for monobody:2,4,6
+	</font></center></p>
+	<p>Expression and purification of apo-HRP and refolded-HRP</p>
-<!-- start of content -->
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+	To obtain functional horseradish peroxidase we need to purify the protein from <I>E. coli</I>, 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)
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-<h1>
+	<p><center><font size=2>
-PART I Degradation
+	Figure 3. SDS-PAGE for purification of HRP
-</h1>
+	</font></center></p>
-<br>
-<p>
-<b>
-Cloning of Horseradish Peroxidase
-</b>
-</p>
-<br>
+	<p>Functional test of Horseradish Peroxidase</p>
-<font size=4>
-<p>
-To make sure the plasmid is cloned into <I>E. coli</I> BL-21 strain, we extracted the plasmid from transformed <I>E. coli</I>.(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).
-</p>
-</font>
-<p>
-<img src="https://static.igem.org/mediawiki/2017/b/bb/T--NTHU_Taiwan--Results--hrp_ecoli.png">
-</p>
-<p>
+	<p>
-<center>
+	To prove the degradation ability of HRP, we mixed 25 µg of HRP with 1 mM H2O2 and BPA or NP in the 1 mL water. The environment of degradation is suitable for HRP to degrade the phenolic compounds(40℃ and pH=6-7). After degradation for 24 hours, we denatured HRP by briefly heating up to 80℃ and use LC-PDA (Liquid Chromatography - Photodiode Array detector) to analysis the result of degradation.(figure 4 and 5)
-<font size=2>
+	</p>
-Figure 1.
-</font>
-</center>
-</p>
-<p align="center">
-<p>
-<img src="https://static.igem.org/mediawiki/2017/c/c4/T--NTHU_Taiwan--Results--hrp_pcr.png">
-</p>
-<p>
+	<p>
-<center>
+	<img width="50%" src="https://static.igem.org/mediawiki/2017/0/03/T--NTHU_Taiwan--Results--lc_bpa.png">
-<font size=2>
+	</p>
-Figure 2. Primers for VR backbone:1,3,5 ;primers for monobody:2,4,6
-</font>
-</center>
-</p>
-<br>
+	<p><center><font size=2>
+	Figure 4. The result of LC-PDA (BPA sample)
+	</font></center></p>
-<p>
-<b>
-Expression and purification of apo-HRP and refolded-HRP
-</b>
-</p>
-<br>
+	<p>
+	<img width="50%" src="https://static.igem.org/mediawiki/2017/9/90/T--NTHU_Taiwan--Results--lc_np.png">
+	</p>
-<font size=4>
+	<p><center><font size=2>
-<p>
+	Figure 5. The result of LC-PDA (NP sample)
-To obtain functional horseradish peroxidase we need to purify the protein from <I>E. coli</I>, 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)
+	</font></center></p>
-</p>
-</font>
-<p>
-<img width="65%" src="https://static.igem.org/mediawiki/2017/3/3a/T--NTHU_Taiwan--Results--hrp_page.png">
-</p>
-<p>
-Figure 3. SDS-PAGE for purification of HRP
-</p>
-<br>
-<p>
+	<p>
-<b>
+	From the result of LC-PDA, we found that there are two extra peaks showing after degradation and these extra peaks represent the by-products from degradation.
-Functional test of Horseradish Peroxidase
+	Unfortunately, we can’t know how much EDCs is degraded by HRP because:
-</b>
+	</p>
-</p>
-<br>
-<font size="4">
-<p>
-To prove the degradation ability of HRP, we mixed 25 µg of HRP with 1 mM H2O2 and BPA or NP in the 1 mL water. The environment of degradation is suitable for HRP to degrade the phenolic compounds(40℃ and pH=6-7). After degradation for 24 hours, we denatured HRP by briefly heating up to 80℃ and use LC-PDA (Liquid Chromatography - Photodiode Array detector) to analysis the result of degradation.(figure 4 and 5)
-</p>
-</font>
-<p>
-<img width="50%" src="https://static.igem.org/mediawiki/2017/0/03/T--NTHU_Taiwan--Results--lc_bpa.png">
-</p>
-<p>
+	<p>
-<center>
+	(1) The peak of the by-products overlap the peak of EDCs and we can’t get the information of remained EDCs. (There is a by-product have a broad peak from 2.2 min to 3.5min and its intensity also much higher than the peak of EDCs)
-<font size=2>
+	</p>
-Figure 4. The result of LC-PDA (BPA sample)
-</font>
-</center>
-</p>
-<br>
-<p>
-<img width="50%" src="https://static.igem.org/mediawiki/2017/9/90/T--NTHU_Taiwan--Results--lc_np.png">
-</p>
-<p>
-<center>
-<font size=2>
-Figure 5. The result of LC-PDA (NP sample)
-</font>
-</center>
-</p>
-<br>
-<font size="4">
-<p>
-From the result of LC-PDA, we found that there are two extra peaks showing after degradation and these extra peaks represent the by-products from degradation.
-Unfortunately, we can’t know how much EDCs is degraded by HRP because:
-</p>
-</font>
-<br>
-<font size="4">
+	<p>
-<p>
+	(2) The quality of the column from LC isn’t good enough to separate the by-products from EDCs due to their similar molecular properties. (This LC can’t distinguish between BPA and NP so we estimated that it can’t distinguish the degraded by-product from BPA or NP as well.)
-(1) The peak of the by-products overlap the peak of EDCs and we can’t get the information of remained EDCs. (There is a by-product have a broad peak from 2.2 min to 3.5min and its intensity also much higher than the peak of EDCs)
+	</p>
-</p>
-</font>
-<br>
-<font size="4">
-<p>
-(2) The quality of the column from LC isn’t good enough to separate the by-products from EDCs due to their similar molecular properties. (This LC can’t distinguish between BPA and NP so we estimated that it can’t distinguish the degraded by-product from BPA or NP as well.)
-</p>
-</font>
-<br>
-<font size="4">
-<p>
-Moreover, 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 6-9) 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.
-</p>
-</font>
-<p>
-<img width="40%" src="https://static.igem.org/mediawiki/2017/f/fb/T--NTHU_Taiwan--Results--ms_bpa.png">
-</p>
-<p>
+	<p>
-<center>
+	Moreover, 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 6-9) 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.
-<font size=2>
+	</p>
-Figure 6. the mass spectrum of BPA
-</font>
-</center>
-</p>
-<p>
-<img width="40%" src="https://static.igem.org/mediawiki/2017/2/2f/T--NTHU_Taiwan--Results--ms_bpa_hrp.png">
-</p>
-<p>
+	<p>
-<center>
+	<img width="40%" src="https://static.igem.org/mediawiki/2017/f/fb/T--NTHU_Taiwan--Results--ms_bpa.png">
-<font size=2>
+	</p>
-Figure 7. the mass spectrum of BPA after degradation
-</font>
-</center>
-</p>
-<p>
+	<p><center><font size=2>
-<img width="40%" src="https://static.igem.org/mediawiki/2017/2/28/T--NTHU_Taiwan--Results--ms_np.png">
+	Figure 6. the mass spectrum of BPA
-</p>
+	</font></center></p>
-<p>
+	<p>
-<center>
+	<img width="40%" src="https://static.igem.org/mediawiki/2017/2/2f/T--NTHU_Taiwan--Results--ms_bpa_hrp.png">
-<font size=2>
+	</p>
-Figure 8. the mass spectrum of NP
-</font>
-</center>
-</p>
-<p>
+	<p><center><font size=2>
-<img width="40%" src="https://static.igem.org/mediawiki/2017/b/b3/T--NTHU_Taiwan--Results--ms_np_hrp.png">
+	Figure 7. the mass spectrum of BPA after degradation
-</p>
+	</font></center></p>
-<p>
+	<p>
-<center>
+	<img width="40%" src="https://static.igem.org/mediawiki/2017/2/28/T--NTHU_Taiwan--Results--ms_np.png">
-<font size=2>
+	</p>
-Figure 9. the mass spectrum of NP after degradation
-</font>
-</center>
-</p>
-<br>
-<h1>
+	<p><center><font size=2>
-PART II   Detection
+	Figure 8. the mass spectrum of NP
-</h1>
+	</font></center></p>
-<br>
+	<p>
+	<img width="40%" src="https://static.igem.org/mediawiki/2017/b/b3/T--NTHU_Taiwan--Results--ms_np_hrp.png">
+	</p>
-<p>
+	<p><center><font size=2>
-<b>
+	Figure 9. the mass spectrum of NP after degradation
-Cloning of ER-alpha and monobody
+	</font></center></p>
-</b>
-</p>
-<br>
-<font size="4">
-<p>
-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 10 and 11)
-After transformation, we extracted both plasmids from the cells and the vector was validated by gel electrophoresis.
-</p>
-</font>
-<p>
+	<center><h1>
-<img width="27%" src="https://static.igem.org/mediawiki/2017/c/ce/T--NTHU_Taiwan--Results--mono_ecoli.png">
+	PART II   Detection
-</p>
+	</center></h1>
-<p>
-<center>
-<font size=2>
-Figure 10. <I>E. coli</I> with gene of monobody
-</font>
-</center>
-</p>
-<br>
-<p>
-<img width="27%" src="https://static.igem.org/mediawiki/2017/9/98/T--NTHU_Taiwan--Results--er_ecoli.png">
-</p>
-<p>
+	<p>
-<center>
+	Cloning of ER-alpha and monobody
-<font size=2>
+	</p>
-Figure 11. <I>E. coli</I> with gene of ER-alpha
-</font>
-</center>
-</p>
-<br>
-<font size="4">
-<p>
-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 and Monobody (797bp).(Figure 12)
-</p>
-</font>
-<p>
-<img width="50%" src="https://static.igem.org/mediawiki/2017/b/b0/T--NTHU_Taiwan--Results--er_mono_pcr.png">
-</p>
-<p><blockquote><blockquote><blockquote>
+	<p>
-<center>
+	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 10 and 11)
-<font size=2>
+	After transformation, we extracted both plasmids from the cells and the vector was validated by gel electrophoresis.
-Figure 12.   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 )
+	</p>
-</font>
-</center></blockquote></blockquote></blockquote>
-</p>
-<br>
-<p>
-<b>
-Function of ice nucleation protein (INP)
-</b>
-</p>
-<br>
-<font size="4">
+	<p>
-<p>
+	<img width="27%" src="https://static.igem.org/mediawiki/2017/c/ce/T--NTHU_Taiwan--Results--mono_ecoli.png">
-We compared the difference between <I>E. coli</I> expressed RFP and RFP-INP(figure 13). 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 <I>E. coli</I> plasma membrane when we centrifuged the cell lysate, RFP-INP is spun down with the plasma membrane, and we see a red pellet.
+	</p>
-</p>
-</font>
-<br>
-<font size="4">
+	<p><center><font size=2>
-<p>
+	Figure 10. <I>E. coli</I> with gene of monobody
-On contract, after cell lysis of RFP expression <I>E. coli</I>, 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.
+	</font></center></p>
-</p>
-</font>
-<br>
-<font size="4">
-<p>
-This experiment showed that INP can bring RFP to the membrane of <I>E. coli</I>, and this result proved the function of INP.
-</p>
-</font>
-<p>
+	<p>
-<img width="30%" src="https://static.igem.org/mediawiki/2017/d/d7/T--NTHU_Taiwan--Results--inp.png">
+	<img width="27%" src="https://static.igem.org/mediawiki/2017/9/98/T--NTHU_Taiwan--Results--er_ecoli.png">
-</p>
+	</p>
-<p>
+	<p><center><font size=2>
-<center>
+	Figure 11. <I>E. coli</I> with gene of ER-alpha
-<font size=2>
+	</font></center></p>
-Figure 13. 1 : Fragments of <I>E. coli</I> with gene of RFP-INP ; 2:  Fragments of <I>E. coli</I> with gene of RFP
-</font>
-</center>
-</p>
-<br>
-<p>
+	<p>
-<b>
+	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 and Monobody (797bp).(Figure 12)
-Characterization of detection system
+	</p>
-</b>
-</p>
-<br>
-<font size="4">
-<p>
-To prove that our detection system can distinguish the sample has EDCs or not, we used IR spectrum to measure specific bonds on <I>E. coli</I> and verify the detection function.(Table 1)
-</p>
-</font>
-<p>
+	<p>
-<img width="50%" src="https://static.igem.org/mediawiki/2017/7/71/T--NTHU_Taiwan--Results--table_1.png">
+	<img width="50%" src="https://static.igem.org/mediawiki/2017/b/b0/T--NTHU_Taiwan--Results--er_mono_pcr.png">
-</p>
+	</p>
-<p>
+	<p><center><font size=2>
-<center>
+	Figure 12.   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 )
-<font size=2>
+	</font></center></p>
-Table 1. The IR signals for <I>E. coli</I>
-</font>
-</center>
-</p>
-<br>
-<font size="4">
-<p>
-In the beginning, we try to use live cells for the detection system. However, we found that all the samples have the information of <I>E. coli</I>. Showing that there is no difference between the sample with 5mM EDCs and the sample with no EDCs. (Figure 14)
-</p>
-</font>
-<p>
+	<p>Function of ice nucleation protein (INP)</p>
-<img width="75%"src="https://static.igem.org/mediawiki/2017/1/1f/T--NTHU_Taiwan--Results--ir.png">
-</p>
-<p>
-<center>
-<font size=2>
-Figure 14. The IR spectrum of samples in different conditions (live <I>E. coli</I>)
-</font>
-</center>
-</p>
-<br>
-<font size="4">
+	<p>
-<p>
+	We compared the difference between <I>E. coli</I> expressed RFP and RFP-INP(figure 13). 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 <I>E. coli</I> plasma membrane when we centrifuged the cell lysate, RFP-INP is spun down with the plasma membrane, and we see a red pellet.
-To decrease the activity of <I>E. coli</I>, we tried to freeze <I>E. coli</I> 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 <I>E. coli</I>.(Figure 15) This result can prove that our detection system can detect EDCs in the water.
+	</p>
-</p>
-</font>
-<p>
-<img width="75%" src="https://static.igem.org/mediawiki/2017/6/6c/T--NTHU_Taiwan--Results--ir2.png">
-</p>
-<p>
-<center>
-<font size=2>
-Figure 15. The IR spectrum of samples in different conditions (freezed <I>E. coli</I>)
-</font>
-</center>
-</p>
-<br>
-<p>
+	<p>
-<b>
+	On contract, after cell lysis of RFP expression <I>E. coli</I>, 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.
-Proof of concept: quantifying a number of EDCs in the water
+	</p>
-</b>
-</p>
-<br>
-<font size="4">
-<p>
-To prove our detection system can measure the concentration of EDCs, we use crystal violet to stain <I>E. coli</I> on the surface of gold and then observe the density of stained <I>E. coli</I> under microscope. Due to the surface tension force of water, there are lots of <I>E. coli</I> remained around the drop edge of sample, and we only observed the area in the middle of the sample to reduce the error. (Figure 16)
-</p>
-</font>
-<p>
-<img src="https://static.igem.org/mediawiki/2017/2/29/T--NTHU_Taiwan--Results--demon.png">
-</p>
-<p>
+	<p>
-<center>
+	This experiment showed that INP can bring RFP to the membrane of <I>E. coli</I>, and this result proved the function of INP.
-<font size=2>
+	</p>
-Figure 16. Tthe sample under 50X microscope
-</font>
-</center>
-</p>
-<br>
-<font size="4">
-<p>
-We observed the density of <I>E. coli</I> when the samples contained different concentration of BPA and NP from 5mM to 5nM (Figure 17 and 18). This result indicates that when the concentration of EDCs decreases, the density of <I>E. coli</I> will decrease as well.
-</p>
-</font>
-<p>
+	<p>
-<img width="75%" src="https://static.igem.org/mediawiki/2017/3/32/T--NTHU_Taiwan--Results--bpa_500.png">
+	<img width="30%" src="https://static.igem.org/mediawiki/2017/d/d7/T--NTHU_Taiwan--Results--inp.png">
-</p>
+	</p>
-<p>
+	<p><center><font size=2>
-<center>
+	Figure 13. 1 : Fragments of <I>E. coli</I> with gene of RFP-INP ; 2:  Fragments of <I>E. coli</I> with gene of RFP
-<font size=2>
+	</font></center></p>
-Figure 17. The samples with different concentration of BPA under 500X microscope
-</font>
-</center>
-</p>
-<p>
-<img width="75%" src="https://static.igem.org/mediawiki/2017/8/81/T--NTHU_Taiwan--Results--np_500.png">
-</p>
-<p>
-<center>
-<font size=2>
-Figure 18. The samples with different concentration of NP under 500X microscope
-</font>
-</center>
-</p>
-<br>
-<font size="4">
+	<p>
-<p>
+	Characterization of detection system
-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 <I>E. coli</I> with samples containing the different concentration of BPA and NP from 5mM to 5nM.(Figure 19 and 20) From the result of observation, we can find the similar tendency as the sample without trehalose.
+	</p>
-</p>
-</font>
-<p>
-<img width="75%" src="https://static.igem.org/mediawiki/2017/7/70/T--NTHU_Taiwan--Results--tbpa_500.png">
-</p>
-<p>
-<center>
-<font size=2>
-Figure 19. The samples with different concentration of BPA in 0.1M trehalose under 500X microscope
-</font>
-</center>
-</p>
+	<p>
+	To prove that our detection system can distinguish the sample has EDCs or not, we used IR spectrum to measure specific bonds on <I>E. coli</I> and verify the detection function.(Table 1)
+	</p>
-<p>
-<img width="75%" src="https://static.igem.org/mediawiki/2017/c/c9/T--NTHU_Taiwan--Results--tnp_500.png">
-</p>
-<p>
+	<p>
-<center>
+	<img width="50%" src="https://static.igem.org/mediawiki/2017/7/71/T--NTHU_Taiwan--Results--table_1.png">
-<font size=2>
+	</p>
-Figure 20.  the samples with different concentration of NP in 0.1M trehalose under 500X microscope
-</font>
-</center>
-</p>
-<br>
-<font size="4">
+	<p><center><font size=2>
-<p>
+	Table 1. The IR signals for <I>E. coli</I>
-To clarify the relationship between the concentration of EDCs and the density of <I>E. coli</I>, we use image J to count the amount of <I>E. coli</I> on the surface. (Figure 21-24) From the results of imageJ, we can get the similar tendency as the result from the pictures of the microscope. However, the density of <I>E. coli</I> decreases in the presence of 0.1 M trehalose, and this result suggested that although trehalose can improve the time to store lyophilized <I>E. coli</I>, it can decrease the precision of our detection system.
+	</font></center></p>
-</p>
-</font>
-<p>
-<img width="55%" src="https://static.igem.org/mediawiki/2017/7/71/21.png">
-</p>
-<p>
-<center>
-<font size=2>
-Figure 21. Samples of BPA
-</font>
-</center>
-</p>
-<p>
+	<p>
-<img width="55%" src="https://static.igem.org/mediawiki/2017/2/2f/T--NTHU_Taiwan--Results--np-22.png">
+	In the beginning, we try to use live cells for the detection system. However, we found that all the samples have the information of <I>E. coli</I>. Showing that there is no difference between the sample with 5mM EDCs and the sample with no EDCs. (Figure 14)
-</p>
+	</p>
-<p>
-<center>
-<font size=2>
-Figure 22. Samples of NP
-</font>
-</center>
-</p>
-<p>
+	<p>
-<img width="55%" src="https://static.igem.org/mediawiki/2017/5/5e/T--NTHU_Taiwan--Results--tbpa-23.png">
+	<img width="75%"src="https://static.igem.org/mediawiki/2017/1/1f/T--NTHU_Taiwan--Results--ir.png">
-</p>
+	</p>
-<p>
+	<p><center><font size=2>
-<center>
+	Figure 14. The IR spectrum of samples in different conditions (live <I>E. coli</I>)
-<font size=2>
+	</font></center></p>
-Figure 23. Samples of BPA in 0.1M trehalose
-</font>
-</center>
-</p>
-<p>
-<img width="55%" src="https://static.igem.org/mediawiki/2017/5/55/T--NTHU_Taiwan--Results--tnp-24.png">
-</p>
-<p>
-<center>
-<font size=2>
-Figure 24. Samples of NP in 0.1M trehalose
-</font>
-</center>
-</p>
-<br>
-<p>
-<b>
-Functional test of ER-alpha
-</b>
-</p>
-<br>
-<p>
+	<p>
-To ensure the function of ER-alpha on the surface of <I>E. coli</I>, we compared the difference between the BL-21 <I>E. coli</I> and ER-alpha expressed <I>E. coli</I> in the different concentration of BPA and NP. (figure 25 and 26)From the results, we found that BL-21 can’t affect the outcomes of the detection system.
+	To decrease the activity of <I>E. coli</I>, we tried to freeze <I>E. coli</I> 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 <I>E. coli</I>.(Figure 15) This result can prove that our detection system can detect EDCs in the water.
-</p>
+	</p>
-<p>
-<img width="55%" src="https://static.igem.org/mediawiki/2017/f/f8/T--NTHU_Taiwan--Result--BPA.png">
-</p>
-<p>
+	<p>
-<center>
+	<img width="75%" src="https://static.igem.org/mediawiki/2017/6/6c/T--NTHU_Taiwan--Results--ir2.png">
-<font size=2>
+	</p>
-Figure 25. Samples of BPA
-</font>
-</center>
-</p>
-<p>
+	<p><center><font size=2>
-<img width="55%" src="https://static.igem.org/mediawiki/2017/c/c8/T--NTHU_Taiwan--Result--NP.png">
+	Figure 15. The IR spectrum of samples in different conditions (freezed <I>E. coli</I>)
-</p>
+	</font></center></p>
-<p>
-<center>
-<font size=2>
-Figure 26. Sample of NP
-</font>
-</center>
-</p>
-<br>
-<p>
+	<p>Proof of concept: quantifying a number of EDCs in the water</p>
-<b>
-The limitation of our detection system
-</b>
-</p>
-<br>
-<font size="4">
-<p>
-To further understand the limitation of the concentration that our system can achieve, we observed the density of <I>E. coli</I> in four different conditions of background. We find that when there are no EDCs in the sample, the density of <I>E. coli</I> is close to the concentration of EDCs below 5µM. Showing that our detection system can’t measure the concentration of EDCs less than 5µM.(Figure 25 and Table 2)
-</p>
-</font>
-<p>
-<img width="35%" src="https://static.igem.org/mediawiki/2017/b/be/T--NTHU_Taiwan--Results--4%2A25.png">
-</p>
-<p>
-<center>
-<font size=2>
-Figure 27. The background of 4 different conditions
-</font>
-</center>
-</p>
-<p>
+	<p>
-<img width="80%" src="https://static.igem.org/mediawiki/2017/6/6f/T--NTHU_Taiwan--Results---t-26.png">
+	To prove our detection system can measure the concentration of EDCs, we use crystal violet to stain <I>E. coli</I> on the surface of gold and then observe the density of stained <I>E. coli</I> under microscope. Due to the surface tension force of water, there are lots of <I>E. coli</I> remained around the drop edge of sample, and we only observed the area in the middle of the sample to reduce the error. (Figure 16)
-</p>
+	</p>
-<p>
-<center>
-<font size=2>
-Table 2. The amounts of <I>E. coli</I> in the 4 different backgrounds
-</font>
-</center>
-</p>
-<br>
-<h1>
+	<p>
-Part III  Future Work
+	<img src="https://static.igem.org/mediawiki/2017/2/29/T--NTHU_Taiwan--Results--demon.png">
-</h1>
+	</p>
+	<p><center><font size=2>
+	Figure 16. Tthe sample under 50X microscope
+	</font></center></p>
-<font size="4">
-<p>
-. 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)
-</p>
-</font>
-<p>
-<img src="https://static.igem.org/mediawiki/2017/b/be/T--NTHU_Taiwan--Results--f-1.png">
-</p>
-<p>
+	<p>
-<center>
+	We observed the density of <I>E. coli</I> when the samples contained different concentration of BPA and NP from 5mM to 5nM (Figure 17 and 18). This result indicates that when the concentration of EDCs decreases, the density of <I>E. coli</I> will decrease as well.
-<font size=2>
+	</p>
-Figure 28. Expression of GFP in the <I>E. coli</I> for detection.
-</font>
-</center>
-</p>
-<br>
-<font size="4">
-<p>
-. Since we don’t have time to construct the detection system to measure the change of fluorescence or surface plasmon resonance, we will construct the detection system to estimate the concentration of EDCs in the water precisely. (Figure 27)
-</p>
-</font>
-<p>
+	<p>
-<img src="https://static.igem.org/mediawiki/2017/f/f7/T--NTHU_Taiwan--Results--f-2.png">
+	<img width="75%" src="https://static.igem.org/mediawiki/2017/3/32/T--NTHU_Taiwan--Results--bpa_500.png">
-</p>
+	</p>
-<p>
+	<p><center><font size=2>
-<center>
+	Figure 17. The samples with different concentration of BPA under 500X microscope
-<font size=2>
+	</font></center></p>
-Figure 29. Using the change of  fluorescence to estimate the precise concentration of EDCs in the water
-</font>
-</center>
-</p>
-</center>
+	<p>
-</body>
+	<img width="75%" src="https://static.igem.org/mediawiki/2017/8/81/T--NTHU_Taiwan--Results--np_500.png">
+	</p>
+	<p><center><font size=2>
-</div>
+	Figure 18. The samples with different concentration of NP under 500X microscope
+	</font></center></p>
-<script type="Text/JavaScript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.6/jquery.min.js"></script>
-<script>
-	// This is the jquery part of your template.
+	<p>
-	// Try not modify any of this code too much since it makes your menu work.
+	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 <I>E. coli</I> with samples containing the different concentration of BPA and NP from 5mM to 5nM.(Figure 19 and 20) From the result of observation, we can find the similar tendency as the sample without trehalose.
+	</p>
-	$(document).ready(function() {
-		$("#HQ_page").attr('id','');
+	<p>
+	<img width="75%" src="https://static.igem.org/mediawiki/2017/7/70/T--NTHU_Taiwan--Results--tbpa_500.png">
+	</p>
-		// call the functions that control the menu
+	<p><center><font size=2>
-		menu_functionality();
+	Figure 19. The samples with different concentration of BPA in 0.1M trehalose under 500X microscope
-		hide_show_menu();
+	</font></center></p>
+	<p>
+	<img width="75%" src="https://static.igem.org/mediawiki/2017/c/c9/T--NTHU_Taiwan--Results--tnp_500.png">
+	</p>
-		//this function controls the expand and collapse buttons of the menu and changes the +/- symbols
+	<p><center><font size=2>
-		function menu_functionality() {
+	Figure 20.  the samples with different concentration of NP in 0.1M trehalose under 500X microscope
+	</font></center></p>
-			//when clicking on a "menu_button", it will change the "+/-" accordingly and it will show/hide the corresponding submenu
-			$(".menu_button").click(function(){
-				// add or remove the class "open" , this class holds the "-"
-				$(this).children().toggleClass("open");
-				// show or hide the submenu
-				$(this).next('.submenu_wrapper').fadeToggle(400);
-			});
-			// when the screen size is smaller than 800px, the display_menu_control button appears and will show/hide the whole menu
+	<p>
-			$("#display_menu_control").click(function(){
+	To clarify the relationship between the concentration of EDCs and the density of <I>E. coli</I>, we use image J to count the amount of <I>E. coli</I> on the surface. (Figure 21-24) From the results of imageJ, we can get the similar tendency as the result from the pictures of the microscope. However, the density of <I>E. coli</I> decreases in the presence of 0.1 M trehalose, and this result suggested that although trehalose can improve the time to store lyophilized <I>E. coli</I>, it can decrease the precision of our detection system.
-				$('#menu_content').fadeToggle(400);
+	</p>
-			});
-			// call the current page highlight function
-			highlight_current_page();
-		}
+	<p>
+	<img width="55%" src="https://static.igem.org/mediawiki/2017/7/71/21.png">
+	</p>
-		// call the highlight current page function to show it on the menu with a different background color
+	<p><center><font size=2>
-		function highlight_current_page() {
+	Figure 21. Samples of BPA
+	</font></center></p>
-			// select a page from the menu based on the id assigned to it and the current page name and add the class "current page" to make it change background color
+	<p>
-			$("#"+  wgPageName.substring(wgPageName.lastIndexOf("/")+1, wgPageName.length ) + "_page").addClass("current_page");
+	<img width="55%" src="https://static.igem.org/mediawiki/2017/2/2f/T--NTHU_Taiwan--Results--np-22.png">
+	</p>
-			// now that the current_page class has been added to a menu item, make the submenu fade in
+	<p><center><font size=2>
-			$(".current_page").parents(".submenu_wrapper").fadeIn(400);
+	Figure 22. Samples of NP
-			// change the +/- symbol of the corresponding menu button
+	</font></center></p>
-			$(".current_page").parents(".submenu_wrapper").prev().children().toggleClass("open");
-		}
+	<p>
+	<img width="55%" src="https://static.igem.org/mediawiki/2017/5/5e/T--NTHU_Taiwan--Results--tbpa-23.png">
+	</p>
+	<p><center><font size=2>
+	Figure 23. Samples of BPA in 0.1M trehalose
+	</font></center></p>
-		// allow button on the black menu bar to show/hide the side menu
+	<p>
-		function hide_show_menu() {
+	<img width="55%" src="https://static.igem.org/mediawiki/2017/5/55/T--NTHU_Taiwan--Results--tnp-24.png">
+	</p>
-			// in case you preview mode is selected, the menu is hidden for better visibility
-			if (window.location.href.indexOf("submit") >= 0) {
-				$(".igem_2017_menu_wrapper").hide();
-			}
-			// if the black menu bar has been loaded
+	<p><center><font size=2>
-  			if (document.getElementById('bars_item')) {
+	Figure 24. Samples of NP in 0.1M trehalose
+	</font></center></p>
-				// when the "bars_item" has been clicked
-				$("#bars_item").click(function() {
-					$("#sideMenu").hide();
-					// show/hide the menu wrapper
+	<p>Functional test of ER-alpha</p>
-					$(".igem_2017_menu_wrapper").fadeToggle("100");
-				});
-  			}
-			// because the black menu bars loads at a different time than the rest of the page, this function is set on a time out so it can run again in case it has not been loaded yet
-			else {
-    				setTimeout(hide_show_menu, 15);
-			}
-		}
+	<p>
+	To ensure the function of ER-alpha on the surface of <I>E. coli</I>, we compared the difference between the BL-21 <I>E. coli</I> and ER-alpha expressed <I>E. coli</I> in the different concentration of BPA and NP. (figure 25 and 26)From the results, we found that BL-21 can’t affect the outcomes of the detection system.
+	</p>
-	});
+	<p>
+	<img width="55%" src="https://static.igem.org/mediawiki/2017/f/f8/T--NTHU_Taiwan--Result--BPA.png">
+	</p>
+	<p><center><font size=2>
+	Figure 25. Samples of BPA
+	</font></center></p>
-</script>
+	<p>
+	<img width="55%" src="https://static.igem.org/mediawiki/2017/c/c8/T--NTHU_Taiwan--Result--NP.png">
+	</p>
+	<p><center><font size=2>
+	Figure 26. Sample of NP
+	</font></center></p>
+	<p>The limitation of our detection system</p>
+	<p>
+	To further understand the limitation of the concentration that our system can achieve, we observed the density of <I>E. coli</I> in four different conditions of background. We find that when there are no EDCs in the sample, the density of <I>E. coli</I> is close to the concentration of EDCs below 5µM. Showing that our detection system can’t measure the concentration of EDCs less than 5µM.(Figure 25 and Table 2)
+	</p>
+	<p>
+	<img width="35%" src="https://static.igem.org/mediawiki/2017/b/be/T--NTHU_Taiwan--Results--4%2A25.png">
+	</p>
+	<p><center><font size=2>
+	Figure 27. The background of 4 different conditions
+	</font></center></p>
+	<p>
+	<img width="80%" src="https://static.igem.org/mediawiki/2017/6/6f/T--NTHU_Taiwan--Results---t-26.png">
+	</p>
+	<p><center><font size=2>
+	Table 2. The amounts of <I>E. coli</I> in the 4 different backgrounds
+	</font></center></p>
+	<center><h1>
+	Part III  Future Work
+	</center></h1>
+	<p>
+. 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)
+	</p>
+	<p>
+	<img src="https://static.igem.org/mediawiki/2017/b/be/T--NTHU_Taiwan--Results--f-1.png">
+	</p>
+	<p><center><font size=2>
+	Figure 28. Expression of GFP in the <I>E. coli</I> for detection.
+	</font></center></p>
+	<p>
+. Since we don’t have time to construct the detection system to measure the change of fluorescence or surface plasmon resonance, we will construct the detection system to estimate the concentration of EDCs in the water precisely. (Figure 27)
+	</p>
+	<p>
+	<img src="https://static.igem.org/mediawiki/2017/f/f7/T--NTHU_Taiwan--Results--f-2.png">
+	</p>
+	<p><center><font size=2>
+	Figure 29. Using the change of fluorescence to estimate the precise concentration of EDCs in the water
+	</font></center></p>
+	</div>
+</div>
 </html>

Difference between revisions of "Team:NTHU Taiwan/Results"

Revision as of 13:18, 31 October 2017

Results

PART I Degradation

PART II Detection

Part III Future Work