Line 656: | Line 656: | ||
<p class="content">There are many possible processes of Pantide degradation we discussed below. (Figure 1) Pantide may have a chance to be reduced to a linear form by reductants or reductases. For both native form and linear form proteins, it may suffer hydrolysis and proteolysis, resulting in denaturing or amino acid cleavage. Also, UV light of sun also leads to Pantide degradation. Though the energy of UV light may not be not enough to break the covalent bonds efficiently, proteins still could undergo radiolytic oxidation.</p> | <p class="content">There are many possible processes of Pantide degradation we discussed below. (Figure 1) Pantide may have a chance to be reduced to a linear form by reductants or reductases. For both native form and linear form proteins, it may suffer hydrolysis and proteolysis, resulting in denaturing or amino acid cleavage. Also, UV light of sun also leads to Pantide degradation. Though the energy of UV light may not be not enough to break the covalent bonds efficiently, proteins still could undergo radiolytic oxidation.</p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/5/5e/NCTU_F1.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/5/5e/NCTU_F1.png" class="picture" style="width:50%;"> |
<p class="content-image" style="text-align:center !important;">Figure 1. Pantide degradation process</p> | <p class="content-image" style="text-align:center !important;">Figure 1. Pantide degradation process</p> | ||
</div> | </div> | ||
Line 674: | Line 674: | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/4/4b/NCTU_111111.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/4/4b/NCTU_111111.png" class="picture"style="width:50%;"> |
</div> | </div> | ||
Line 682: | Line 682: | ||
<p class="content">We tested the chemical stability of both native and linear types of Hv1a and Hv1a-lectin in neutral PBS solvent (phosphate buffered saline, pH=7.4) in 4 ℃; for one day and seven days. SDS-PAGE showed the remained protein concentration and calculated by software ImageJ. (Figure 2, Figure 3)</p> | <p class="content">We tested the chemical stability of both native and linear types of Hv1a and Hv1a-lectin in neutral PBS solvent (phosphate buffered saline, pH=7.4) in 4 ℃; for one day and seven days. SDS-PAGE showed the remained protein concentration and calculated by software ImageJ. (Figure 2, Figure 3)</p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/b/bd/NCTU_F2.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/b/bd/NCTU_F2.png" class="picture"style="width:60%;"> |
<p class="content-image" style="text-align:center !important;">Figure 2. SDS-PAGE gel and the concentrations of hydrolysis test to Hv1a (5.3 kDa). The samples were marked on the top of the gel.</p> | <p class="content-image" style="text-align:center !important;">Figure 2. SDS-PAGE gel and the concentrations of hydrolysis test to Hv1a (5.3 kDa). The samples were marked on the top of the gel.</p> | ||
</div> | </div> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/4/45/NCTU_F3.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/4/45/NCTU_F3.png" class="picture" style="width:60%;"> |
<p class="content-image" style="text-align:center !important;">Figure 3. SDS-PAGE gel and the concentrations of hydrolysis test to Hv1a-lectin (HL, 17.1 kDa). The samples were marked on the top of the gel.</p> | <p class="content-image" style="text-align:center !important;">Figure 3. SDS-PAGE gel and the concentrations of hydrolysis test to Hv1a-lectin (HL, 17.1 kDa). The samples were marked on the top of the gel.</p> | ||
</div> | </div> | ||
Line 704: | Line 704: | ||
<p class="content">The proteolysis process is irreversible in vivo because the product, linear form protein, is degraded quickly. We used differential equation to describe the major proteolysis process:</p> | <p class="content">The proteolysis process is irreversible in vivo because the product, linear form protein, is degraded quickly. We used differential equation to describe the major proteolysis process:</p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/a/a9/NCTU_222222.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/a/a9/NCTU_222222.png" class="picture" style="width:50%;"> |
</div> | </div> | ||
<p class="content">Where [<i>P </i>]: the concentation of Pantide, <i>V<sub>m,p </sub></i> : the maximum reaction rate of proteolysis, which is equal to the product of the concentration of total enzyme and turnover number <i>k<sub>cat </sub></i> of specific protease and substrate, <i>K<sub>M,p </sub></i>: Michaelis constant, which is the substrate concentration at which the reaction rate is half of <i>V<sub>m,p </sub></i></p> | <p class="content">Where [<i>P </i>]: the concentation of Pantide, <i>V<sub>m,p </sub></i> : the maximum reaction rate of proteolysis, which is equal to the product of the concentration of total enzyme and turnover number <i>k<sub>cat </sub></i> of specific protease and substrate, <i>K<sub>M,p </sub></i>: Michaelis constant, which is the substrate concentration at which the reaction rate is half of <i>V<sub>m,p </sub></i></p> | ||
Line 712: | Line 712: | ||
<p class="content">For the first experiment, we dissolved the protein solutions in neutral PBS (pH=7.5) solvent, applied with 0.25% trypsin-EDTA(1:250), a serine protease, and then incubated the samples in work temperature 37℃ for one day. (Figure 4, Figure 5)</p> | <p class="content">For the first experiment, we dissolved the protein solutions in neutral PBS (pH=7.5) solvent, applied with 0.25% trypsin-EDTA(1:250), a serine protease, and then incubated the samples in work temperature 37℃ for one day. (Figure 4, Figure 5)</p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/f/fb/NCTU_F4.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/f/fb/NCTU_F4.png" class="picture" style="width:60%;"> |
<p class="content-image" style="text-align:center !important;">Figure 4. SDS-PAGE gel and the concentrations of trypsin resistance test to Hv1a (5.3 kDa). The samples were marked on the top of the gel.</p> | <p class="content-image" style="text-align:center !important;">Figure 4. SDS-PAGE gel and the concentrations of trypsin resistance test to Hv1a (5.3 kDa). The samples were marked on the top of the gel.</p> | ||
</div> | </div> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/4/42/NCTU_F5.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/4/42/NCTU_F5.png" class="picture" style="width:60%;"> |
<p class="content-image" style="text-align:center !important;">Figure 5. SDS-PAGE gel and the concentrations of trypsin resistance test to Hv1a-lectin (HL, 17.1 kDa). The samples were marked on the top of the gel.</p> | <p class="content-image" style="text-align:center !important;">Figure 5. SDS-PAGE gel and the concentrations of trypsin resistance test to Hv1a-lectin (HL, 17.1 kDa). The samples were marked on the top of the gel.</p> | ||
</div> | </div> | ||
+ | |||
<p class="content">Compared with the sample incubated for one day without trypsin treatment, native Hv1a and Hv1a-lectin showed the resistance to trypsin protease (111% and 100%), but linear proteins were degraded by proteolysis (67% and 18%).</p> | <p class="content">Compared with the sample incubated for one day without trypsin treatment, native Hv1a and Hv1a-lectin showed the resistance to trypsin protease (111% and 100%), but linear proteins were degraded by proteolysis (67% and 18%).</p> | ||
+ | |||
<p class="content">We next tested the proteolysis rate of only linear proteins in the period of four hours, and we drew the Lineweaver–Burk plot (also called “double reciprocal plot”) (Figure 6) to obtain <i>V<sub>m,p </sub></i> and <i>K<sub>M,p </sub></i> for linear form Hv1a and Hv1a-lectin. (Table 1)</p> | <p class="content">We next tested the proteolysis rate of only linear proteins in the period of four hours, and we drew the Lineweaver–Burk plot (also called “double reciprocal plot”) (Figure 6) to obtain <i>V<sub>m,p </sub></i> and <i>K<sub>M,p </sub></i> for linear form Hv1a and Hv1a-lectin. (Table 1)</p> | ||
<div> | <div> | ||
− | + | <img src="https://static.igem.org/mediawiki/2016/f/fb/F6.png" class="picture" style="width:60%;"> | |
<p class="content-image" style="text-align:center !important;">Figure 6. Lineweaver–Burk plot of proteolysis test of linear Hv1a (blue line) and Hv1a-lectin (orange line). The horizontal axis represents the reciprocal of substrate concentration, and the longitudinal axis represents the reciprocal of rate. The straight line’s x-intercept means the reciprocal of <i>-K<sub>M,p </sub></i>, and y-intercept means the reciprocal of <i>V<sub>m,p</sub> </i>.</p> | <p class="content-image" style="text-align:center !important;">Figure 6. Lineweaver–Burk plot of proteolysis test of linear Hv1a (blue line) and Hv1a-lectin (orange line). The horizontal axis represents the reciprocal of substrate concentration, and the longitudinal axis represents the reciprocal of rate. The straight line’s x-intercept means the reciprocal of <i>-K<sub>M,p </sub></i>, and y-intercept means the reciprocal of <i>V<sub>m,p</sub> </i>.</p> | ||
</div> | </div> | ||
+ | |||
+ | |||
+ | |||
<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/c/c9/NCTU_T1.png" class="picture"> | <img src="https://static.igem.org/mediawiki/2016/c/c9/NCTU_T1.png" class="picture"> | ||
Line 734: | Line 739: | ||
− | <!-5--> | + | <!--5--> |
<div class="modelingPart"> | <div class="modelingPart"> | ||
<h2 class="content-1" id="titleP" style="color:#FFFF82">V. UV radiolytic oxidation test</h2> | <h2 class="content-1" id="titleP" style="color:#FFFF82">V. UV radiolytic oxidation test</h2> | ||
Line 742: | Line 747: | ||
<p class="content">Ionizing radiation causes the radiolysis of water is, the major process is shown below. (Figure 7)</p> | <p class="content">Ionizing radiation causes the radiolysis of water is, the major process is shown below. (Figure 7)</p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/a/ac/NCTU_F7.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/a/ac/NCTU_F7.png" class="picture" style="width:50%;"> |
<p class="content-image" style="text-align:center !important;">Figure 7. The radiolysis process of water.</p> | <p class="content-image" style="text-align:center !important;">Figure 7. The radiolysis process of water.</p> | ||
</div> | </div> | ||
<p class="content">Though the real mechanism that radicals attack to protein is quite complex, we can simply indicate the rate of protein been attacked by an unknown power, n, of the total concentration of radicals. Then we derived the rate formula as differential equations.</p> | <p class="content">Though the real mechanism that radicals attack to protein is quite complex, we can simply indicate the rate of protein been attacked by an unknown power, n, of the total concentration of radicals. Then we derived the rate formula as differential equations.</p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/6/64/NCTU_333333.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/6/64/NCTU_333333.png" class="picture" style="width:50%;"> |
</div> | </div> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/c/c9/NCTU_444444.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/c/c9/NCTU_444444.png" class="picture" style="width:50%;"> |
</div> | </div> | ||
<p class="content"> Where [<i>radical </i>]: the total concentation of radicals, [<i>P </i>]: the concentation of Pantide, <i>G<sub>γ </sub></i>: the dose rate of absorbing γ-ray (radiation energy absorption rate per mass, for water, 1.42 Gy/s) [5], <i>A<sub>γ </sub></i>: the number of radicals created per energy (for water, 0.045 μmol/J) [5], <i>I </i>: the intensity of UVB from sunlight measured by UV Sensor (UVM30A), <i>R<sub>T </sub></i>: the rate constant of radicals termination, which is equal to 2.365×10<sup>-7 </sup>mol<sup>-1 </sup>s<sup>-1 </sup> [6], <i>K<sub>UV </sub></i>: the rate const ant of UV radiolytic oxidation to protiens, which is set to 44 </sup>mol<sup>-1 </sup>s<sup>-1 </sup>at the beginning [6]</p> | <p class="content"> Where [<i>radical </i>]: the total concentation of radicals, [<i>P </i>]: the concentation of Pantide, <i>G<sub>γ </sub></i>: the dose rate of absorbing γ-ray (radiation energy absorption rate per mass, for water, 1.42 Gy/s) [5], <i>A<sub>γ </sub></i>: the number of radicals created per energy (for water, 0.045 μmol/J) [5], <i>I </i>: the intensity of UVB from sunlight measured by UV Sensor (UVM30A), <i>R<sub>T </sub></i>: the rate constant of radicals termination, which is equal to 2.365×10<sup>-7 </sup>mol<sup>-1 </sup>s<sup>-1 </sup> [6], <i>K<sub>UV </sub></i>: the rate const ant of UV radiolytic oxidation to protiens, which is set to 44 </sup>mol<sup>-1 </sup>s<sup>-1 </sup>at the beginning [6]</p> | ||
<p class="content">We then used software MATLAB to simulate the degradation rate by UV radiolytic oxidation on the intensity of UVB from sunlight. The results showed that the degradation rate increases as rising intensity whatever n is, and eventually it tends to be fully degraded. (Figure 8)</p> | <p class="content">We then used software MATLAB to simulate the degradation rate by UV radiolytic oxidation on the intensity of UVB from sunlight. The results showed that the degradation rate increases as rising intensity whatever n is, and eventually it tends to be fully degraded. (Figure 8)</p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/8/87/F8.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/8/87/F8.png" class="picture" style="width:60%;"> |
<p class="content-image" style="text-align:center !important;">Figure 8. The simulation of degradation rate by UV radiolytic oxidation on the intensity of UVB from sunlight.</p> | <p class="content-image" style="text-align:center !important;">Figure 8. The simulation of degradation rate by UV radiolytic oxidation on the intensity of UVB from sunlight.</p> | ||
</div> | </div> | ||
Line 761: | Line 766: | ||
<p class="content">We applied the four kinds of native protein solutions to the UVB light from UV transilluminator (302 nm, 50 mW/m<sup>2 </sup>) in the period of 2 hours, where the environment temperature was 36.8℃ in average. The results showed the six proteins degraded under UVB light treatment and the tendency of the reduction of proteins corresponded to our model. (Figure 9)</p> | <p class="content">We applied the four kinds of native protein solutions to the UVB light from UV transilluminator (302 nm, 50 mW/m<sup>2 </sup>) in the period of 2 hours, where the environment temperature was 36.8℃ in average. The results showed the six proteins degraded under UVB light treatment and the tendency of the reduction of proteins corresponded to our model. (Figure 9)</p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/f/fe/F9.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/f/fe/F9.png" class="picture" style="width:60%;"> |
<p class="content-image" style="text-align:center !important;">Figure 9. The degradation rate of four proteins by UV radiolytic oxidation as time goes on.</p> | <p class="content-image" style="text-align:center !important;">Figure 9. The degradation rate of four proteins by UV radiolytic oxidation as time goes on.</p> | ||
</div> | </div> | ||
Line 782: | Line 787: | ||
<p class="content">The whole equation of degradation rate could be expressed by the summation of the rates of three possible degrade processes, that is hydrolysis, proteolysis, and UV radiolytic oxidation, and the rate that proteins transfer from native form to linear form indicated as <i>R<sub>SS </sub></i></p> | <p class="content">The whole equation of degradation rate could be expressed by the summation of the rates of three possible degrade processes, that is hydrolysis, proteolysis, and UV radiolytic oxidation, and the rate that proteins transfer from native form to linear form indicated as <i>R<sub>SS </sub></i></p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/5/5a/NCTU_555555.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/5/5a/NCTU_555555.png" class="picture" style="width:50%;"> |
</div> | </div> | ||
<p class="content">However, according to the previous experiments, if we only considered about the protein in native form, because of the high chemical stability and protease resistance, <i>K<sub>h </sub></i> and <i>V<sub>m,p </sub></i> is much smaller than <i>K<sub>UV </sub></i>, and the first two terms on the right side of the equal sign is relative insignificant. As for the reduction of disulfide bonds, since proteins are most stable in their favorable dimensional structure, it does not tend to break this strong bond down, so we assumed that <i>R<sub>SS </sub></i> is not contributed a lot for degradation in the nature. Then the equation was simplified to only one term.</p> | <p class="content">However, according to the previous experiments, if we only considered about the protein in native form, because of the high chemical stability and protease resistance, <i>K<sub>h </sub></i> and <i>V<sub>m,p </sub></i> is much smaller than <i>K<sub>UV </sub></i>, and the first two terms on the right side of the equal sign is relative insignificant. As for the reduction of disulfide bonds, since proteins are most stable in their favorable dimensional structure, it does not tend to break this strong bond down, so we assumed that <i>R<sub>SS </sub></i> is not contributed a lot for degradation in the nature. Then the equation was simplified to only one term.</p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/c/c4/NCTU_666666.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/c/c4/NCTU_666666.png" class="picture" style="width:50%;"> |
</div> | </div> | ||
<p class="content">and</p> | <p class="content">and</p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/2/2a/NCTU_777777.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/2/2a/NCTU_777777.png" class="picture" style="width:50%;"> |
</div> | </div> | ||
Line 796: | Line 801: | ||
<p class="content">We put the samples on a wide square in a transparent and closed acrylic box outdoor for 4 hours at a different time in a day. Combined with UV intensity sensor, we got the remained protein concentration with the average UVB light intensity in each period. (Figure 11)</p> | <p class="content">We put the samples on a wide square in a transparent and closed acrylic box outdoor for 4 hours at a different time in a day. Combined with UV intensity sensor, we got the remained protein concentration with the average UVB light intensity in each period. (Figure 11)</p> | ||
<div> | <div> | ||
− | <img src="https://static.igem.org/mediawiki/2016/f/f0/F11.png" class="picture"> | + | <img src="https://static.igem.org/mediawiki/2016/f/f0/F11.png" class="picture" style="width:60%;"> |
<p class="content-image" style="text-align:center !important;">Figure 11. The remained protein concentration and the average UVB light intensity in each 4 hours at a different time in a day.</p> | <p class="content-image" style="text-align:center !important;">Figure 11. The remained protein concentration and the average UVB light intensity in each 4 hours at a different time in a day.</p> | ||
</div> | </div> |
Revision as of 01:44, 20 October 2016
![](https://static.igem.org/mediawiki/2016/0/01/NCTU_MODELING_BIG_PICTURE.png)
![](https://static.igem.org/mediawiki/2016/f/f8/Web.png)
![](https://static.igem.org/mediawiki/2016/6/65/Right_%281%29.png)