http://2011.igem.org/wiki/index.php?title=Special:Contributions/98712149&feed=atom&limit=50&target=98712149&year=&month=2011.igem.org - User contributions [en]2024-03-29T11:13:46ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2011-10-06T04:04:05Z<p>98712149: </p>
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Gluconacetobacter hansenii which carries the acsABCD protein has the ability to produce cellulose is our main character in our project. Our project is to transform acsABCD genes into E. coli to monitor the production of cellulose production. We collect the protein produced by the transformed E.coli and Gluconacetobacter hansenii in different time point to monitor the expression of acsABCD gene products and the production of cellulose.<br />
<br><br />
<br><br />
<br><br />
<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
The protein purification from <i>E.Coli</i> with acsAB and with acsCD is use to run the SDS-PAGE in order to compare the expression of every protein sample. The expression is then use to predict and calculate the amount of protein produced by the <i>Gluconacetobacter hansenii</i>.<br />
<br><br />
<br><br />
<br />
<br />
Gluconacetobacter hansenii has been cultured in the HS medium for 3 days, the growth curve is recorded. On the 3rd day, the cellulose is collected and dried to weigh the nett weight. The highest record of the cellulose collected is 1.0032g by 50cc of Gluconacetobacter hansenii. Finally, 1c.c. of the bacterial medium is extract as positive control. While the E.coli without any extra genes will be the negative control.<br />
<br />
The growth curve from both pathways is recorded.<br />
<br />
<br> <br />
<br />
Below is the gene we improved. Their productivity is compare with our positive control gene.<br />
<br />
<br>1.BBa_K571004 + acsAB<br />
<br>2.BBa_K571004 + acsCD<br />
<br>3.acsAB <br />
<br>4.acsCD<br />
<br><br />
https://static.igem.org/mediawiki/2011/f/f9/TCU_MD2.jpg<br />
https://static.igem.org/mediawiki/2011/9/94/TCU_MD1.jpg<br />
<br><br />
<br><br />
The productivity of Gluconacetobacter hansenii on the 3rd day is depended on the gene acsAB and acsCD. <br />
<br><br />
E.coli : Gluconacetobacter hansenii = 163.502Unit : 154.965Unit <br />
<br><br />
The ratio of acsAB acsCD to E.coli is 1.055<br />
<br><br />
While the productivity of Gluconacetobacter hansenii is 1.0032g/3 days. Therefore, we can know that E.Coli has the ability to produce cellulose. In other words, the productivity is 1.0082g/ml.<br />
<br><br />
<br><br />
'''The formula !!!'''<br />
<br><br />
<br><br />
163.502/154.96=1.055(constant)<br />
<br><br />
1.055×OD(E.coli)×1×10^6=cellouse g /ml E.coli<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br><br />
<br> Run 2 SDS-PAGE. One with promoter R0011, one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the <i>E. coli</i> which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
<br><br />
<font size=3><b>Method 2 : Benedict’s test</b></font><br />
<br><br />
<br><br />
The bacterial culture which have been induced were collected and the bacteria is to remove, the end product is added with cellulase until the cellulose is fully decomposed. Benedict solution caused the formation of brick red precipitate, the concentration of cellulose is then tested with OD645.<br />
<br><br />
<br><br />
Cellulase is used to break down the cellulose into monosaccharide. Cellulose is a glucose polymer connected through a beta (1-4) glycosidic linkages. Benedict’s test is carried out to test the presence of reducing sugar, such as. The reducing sugar reduces copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I) oxide precipitate. The color would range from green to brick red respectively depends on the amount of reducing sugar present in the solution. It can detect the concentration of the reducing sugar under the absorbance condition <sub>OD645</sub>.<br />
<br />
Cellulose (beta-1,4 glucan) is the most plentiful biopolymer in nature and is an crucial raw material for many industries. It is synthesized as extracellular fibrils by cellulose synthase not only in plants but also in some bacteria. <br />
<br />
Bacteria with cellulose synthase gene use Isopropyl-β- D -1-thiogalactopyranoside (IPTG) as the inducer stimulating the production of protein. In our system, IPTG act as the inducer of the R0011 promoter, which then activate the operon. The activated acs operon encodes the cellulose synthase to synthesize cellulose. The reaction lasts two hours and every two hour we would need to collect the purified cellulose. The nett weight is recorded and the mechanism of cellulose activity on pure cellulose substrates is identified. Lastly, the Benedict’s solution is added to find out the absorbance (optical density [O.D.]) value, in order to calculate the amount of monosaccharide that cellulose can produce.<br />
<br><br />
<br><br />
<html><object type="application/x-shockwave-flash" height="450" width=650" data="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf"><br />
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=='''Bacterial cellulose'''==<br />
[[File:Big1.JPG|center|300px]]<br />
[[File:Big2.JPG|center|300px]]<br />
<br><html><div align="center">the sample of bacterial cellulose that we collected in 3 days</div></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
[[File:111456.jpg|center|500px]]<br />
<br><html><div align="center">bacterial cellulose under EM<br><br><br><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2011-10-06T03:59:24Z<p>98712149: </p>
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<br><br />
<br><br />
Gluconacetobacter hansenii which carries the acsABCD protein has the ability to produce cellulose is our main character in our project. Our project is to transform acsABCD genes into E. coli to monitor the production of cellulose production. We collect the protein produced by the transformed E.coli and Gluconacetobacter hansenii in different time point to monitor the expression of acsABCD gene products and the production of cellulose.<br />
<br><br />
<br><br />
<br><br />
<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
The protein purification from <i>E.Coli</i> with acsAB and with acsCD is use to run the SDS-PAGE in order to compare the expression of every protein sample. The expression is then use to predict and calculate the amount of protein produced by the <i>Gluconacetobacter hansenii</i>.<br />
<br />
The protein purification from E.Coli with acsAB and with acsCD is use to run the SDS-PAGE in order to compare the expression of every protein sample. The expression is then use to predict and calculate the amount of protein produced by the Gluconacetobacter hansenii.<br />
<br />
<br />
<br />
Gluconacetobacter hansenii has been cultured in the HS medium for 3 days, the growth curve is recorded. On the 3rd day, the cellulose is collected and dried to weigh the nett weight. The highest record of the cellulose collected is 1.0032g by 50cc of Gluconacetobacter hansenii. Finally, 1c.c. of the bacterial medium is extract as positive control. While the E.coli without any extra genes will be the negative control.<br />
<br />
The growth curve from both pathways is recorded.<br />
<br />
<br> <br />
<br />
Below is the gene we improved. Their productivity is compare with our positive control gene.<br />
<br />
<br>1.BBa_K571004 + acsAB<br />
<br>2.BBa_K571004 + acsCD<br />
<br>3.acsAB <br />
<br>4.acsCD<br />
<br><br />
https://static.igem.org/mediawiki/2011/f/f9/TCU_MD2.jpg<br />
https://static.igem.org/mediawiki/2011/9/94/TCU_MD1.jpg<br />
<br><br />
<br><br />
The productivity of Gluconacetobacter hansenii on the 3rd day is depended on the gene acsAB and acsCD. <br />
<br><br />
E.coli : Gluconacetobacter hansenii = 163.502Unit : 154.965Unit <br />
<br><br />
The ratio of acsAB acsCD to E.coli is 1.055<br />
<br><br />
While the productivity of Gluconacetobacter hansenii is 1.0032g/3 days. Therefore, we can know that E.Coli has the ability to produce cellulose. In other words, the productivity is 1.0082g/ml.<br />
<br><br />
<br><br />
'''The formula !!!'''<br />
<br><br />
<br><br />
163.502/154.96=1.055(constant)<br />
<br><br />
1.055×OD(E.coli)×1×10^6=cellouse g /ml E.coli<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
<br> Run 2 SDS-PAGE. One with promoter R0011, one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the <i>E. coli</i> which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
<br><br />
<font size=3><b>Method 2 : Benedict’s test</b></font><br />
<br><br />
<br><br />
The bacterial culture which have been induced were collected and the bacteria is to remove, the end product is added with cellulase until the cellulose is fully decomposed. Benedict solution caused the formation of brick red precipitate, the concentration of cellulose is then tested with OD645.<br />
<br><br />
<br><br />
Cellulase is used to break down the cellulose into monosaccharide. Cellulose is a glucose polymer connected through a beta (1-4) glycosidic linkages. Benedict’s test is carried out to test the presence of reducing sugar, such as. The reducing sugar reduces copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I) oxide precipitate. The color would range from green to brick red respectively depends on the amount of reducing sugar present in the solution. It can detect the concentration of the reducing sugar under the absorbance condition <sub>OD645</sub>.<br />
<br />
Cellulose (beta-1,4 glucan) is the most plentiful biopolymer in nature and is an crucial raw material for many industries. It is synthesized as extracellular fibrils by cellulose synthase not only in plants but also in some bacteria. <br />
<br />
Bacteria with cellulose synthase gene use Isopropyl-β- D -1-thiogalactopyranoside (IPTG) as the inducer stimulating the production of protein. In our system, IPTG act as the inducer of the R0011 promoter, which then activate the operon. The activated acs operon encodes the cellulose synthase to synthesize cellulose. The reaction lasts two hours and every two hour we would need to collect the purified cellulose. The nett weight is recorded and the mechanism of cellulose activity on pure cellulose substrates is identified. Lastly, the Benedict’s solution is added to find out the absorbance (optical density [O.D.]) value, in order to calculate the amount of monosaccharide that cellulose can produce.<br />
<br><br />
<br><br />
<br><br />
<html><object type="application/x-shockwave-flash" height="450" width=650" data="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf" /><br />
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=='''Bacterial cellulose'''==<br />
[[File:Big1.JPG|center|300px]]<br />
[[File:Big2.JPG|center|300px]]<br />
<br><html><div align="center">the sample of bacterial cellulose that we collected in 3 days</div></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
[[File:111456.jpg|center|500px]]<br />
<br><html><div align="center">bacterial cellulose under EM<br><br><br><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2011-10-06T03:54:08Z<p>98712149: </p>
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<br><br />
<br><br />
Gluconacetobacter hansenii which carries the acsABCD protein has the ability to produce cellulose is our main character in our project. Our project is to transform acsABCD genes into E. coli to monitor the production of cellulose production. We collect the protein produced by the transformed E.coli and Gluconacetobacter hansenii in different time point to monitor the expression of acsABCD gene products and the production of cellulose.<br />
<br><br />
<br>E.coli : Gluconacetobacter hansenii = 163.502Unit : 154.965Unit <br />
The ratio is 1.055<br />
While the productivity of Gluconacetobacter hansenii is 1.0032g/3 days. Therefore, we can know that E.Coli has the ability to produce cellulose. In other words, the productivity is 1.0082g/ml.<br />
<br><br />
<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
The protein purification from <i>E.Coli</i> with acsAB and with acsCD is use to run the SDS-PAGE in order to compare the expression of every protein sample. The expression is then use to predict and calculate the amount of protein produced by the <i>Gluconacetobacter hansenii</i>.<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
<br> Run 2 SDS-PAGE. One with promoter R0011, one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the <i>E. coli</i> which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
<br><br />
<font size=3><b>Method 2 : Benedict’s test</b></font><br />
<br><br />
<br><br />
The bacterial culture which have been induced were collected and the bacteria is to remove, the end product is added with cellulase until the cellulose is fully decomposed. Benedict solution caused the formation of brick red precipitate, the concentration of cellulose is then tested with OD645.<br />
<br><br />
<br><br />
Cellulase is used to break down the cellulose into monosaccharide. Cellulose is a glucose polymer connected through a beta (1-4) glycosidic linkages. Benedict’s test is carried out to test the presence of reducing sugar, such as. The reducing sugar reduces copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I) oxide precipitate. The color would range from green to brick red respectively depends on the amount of reducing sugar present in the solution. It can detect the concentration of the reducing sugar under the absorbance condition <sub>OD645</sub>.<br />
<br />
Cellulose (beta-1,4 glucan) is the most plentiful biopolymer in nature and is an crucial raw material for many industries. It is synthesized as extracellular fibrils by cellulose synthase not only in plants but also in some bacteria. <br />
<br />
Bacteria with cellulose synthase gene use Isopropyl-β- D -1-thiogalactopyranoside (IPTG) as the inducer stimulating the production of protein. In our system, IPTG act as the inducer of the R0011 promoter, which then activate the operon. The activated acs operon encodes the cellulose synthase to synthesize cellulose. The reaction lasts two hours and every two hour we would need to collect the purified cellulose. The nett weight is recorded and the mechanism of cellulose activity on pure cellulose substrates is identified. Lastly, the Benedict’s solution is added to find out the absorbance (optical density [O.D.]) value, in order to calculate the amount of monosaccharide that cellulose can produce.<br />
<br><br />
<br><br />
<br><br />
<html><object type="application/x-shockwave-flash" height="450" width=650" data="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf"><br />
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=='''Bacterial cellulose'''==<br />
[[File:Big1.JPG|center|300px]]<br />
[[File:Big2.JPG|center|300px]]<br />
<br><html><div align="center">the sample of bacterial cellulose that we collected in 3 days</div></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
[[File:111456.jpg|center|500px]]<br />
<br><html><div align="center">bacterial cellulose under EM<br><br><br><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Project/PhotoPaperTeam:TzuChiU Formosa/Project/PhotoPaper2011-10-06T03:48:29Z<p>98712149: </p>
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<font color="#228B22" size=6>Photopaper</font><br />
<br><br><font color="#000080" size=5>Abstract</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
For most countries, paper-making has been a traditional but indispensable industry. Wood pulp is the major raw material for paper-making ,moreover,the complicated processes toward paper-making may contribute to environmental pollution. Acetobacter xylinum is a bacterium which produces bacterial cellulose. It has an acs operon, consisting of genes that called acsAB, acsC, and acsD. These genes interact with each other and synthesize cellulose synthase, an enzyme that transforms UDP-glucose into cellulose. What we want to do is to choose cyanobacteria which provides glucose through photosynthesis. Therefore, we want to use cyanobacteria as the host, then introduce the acs operon genes in it and produce bacterial cellulose by expressing this series of genes. With manufacturing processes, we believe this project can develop into a new and eco-friendly technology of papermaking.<br />
<br />
<br />
<br />
<br><br><font color="#000080" size=5>Background</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><font size=4><b>a. about cellulose biosynthesis</b></font><br />
<br>Bacterial cellulose is a form of cellulose which is produced by bacteria. It has the same molecular formula as green plants, which is a polysaccharide with thousands of β(1→4) linked D-glucose units. Cellulose biosynthesis is a carbohydrate metabolism with several enzyme regulations, and the key factor of this pathway is cellulose synthase, which participates in the transition from UDP-glucose to cellulose. The ligand of cellulose synthase is cyclic di-GMP, an effector forming from 2 molecules of GTP. The enzyme is activated by the binding of c-di-GMP with cellulose synthase, turning UDP-glucose into UDP, meanwhile form β-1,4-glucan chains, which are then used to synthesize cellulose.<br />
<br><br />
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<br><br />
<br><font size=4><b>b.about acs operon</b></font><br />
<br><br />
[[File:Screen shot 2011-10-06 at 上午5.45.47.png|right|350px]]<br />
<i>Gluconacetobacter hansenii</i> ATCC23769 has been characterized as a model organism for cellulose biosynthesis. The cluster of genes which play the role of producing cellulose is acs operon. It contains three major genes, acsAB, acsC and acsD, and the final product is cellulose synthase. The acsAB catalyzes the formation of bacterial cellulose; acsA is the catalytic subunit which utilizes UDP-glucose to form the basic unit of cellulose; while acsB provide the regulatory subunit which has a cyclic di-GMP binding domain. The acsC gene is the main composite in the formation of the membrane complex of cellulose synthase and proposed to be involved in the export of the polymer across the bacterial cell wall.The acsD gene is involved in the crystallization of the mature cellulose by cleaves the intrastrand ß-1,4 linkages in the cellulose chain and is proposed to have a role in the release of the growing polymer from the cell.<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
<br><br><font color="#000080" size=5>System Design</font><br><br />
<br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
<font size = 4><b>1.Biobricks</b></font><br><br><br />
<br />
Because of the long sequence of complete acs operon, we designed several pairs of<br />
primers to amplify acsAB and acsCD, the products are then purified and digested with<br />
EcoRI and SpeI, next ligated with the pSB1C3 cut by EcoRI-SpeI, which pSB1C3 is the<br />
iGEM 2011 standard backbone.<br />
[[File:Screen shot 2011-10-06 at 上午10.32.16.png|center|600px]]<br />
<br><br />
<br><br />
<font size = 4><b>2. Expression</b></font><br><br><br />
<br />
The recombinant plasmids would be introduced into <i>E. coli</i> for testing and modelling<br />
before transforming into <i>R. rubrum.</i> The IPTG-induced promoter is cloned into biobricks so<br />
that the acs operon expression would be regulated by the addition of IPTG.<br />
As to <i>R. rubrum</i>, the recombinant plasmids would be introduced by electroporation. The<br />
glucose which produced by photosynthesis would be the raw material for cellulose<br />
synthesis.<br />
<br><br />
<br><br />
<font size = 4><b>3. Modelling<br />
</b></font><br><br><br />
Observation of the performance of acs ABCD protein during PAGE can predict the<br />
productivity of cellulose, run 2 SDS-PAGE, one according to the reaction time and another<br />
according to the presence of promoter.<br />
The bacterial culture which have been induced were collected and the bacteria is to<br />
remove, the end product is added with cellulase until the cellulose is fully decomposed.<br />
Benedict solution caused the formation of brick red precipitate, the concentration of<br />
cellulose is then tested with OD645.<br />
[[File:Cellulose.jpg|center|700px]]<br />
<html><br />
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<br />
<br><br><font color="#000080" size=5>Result</font><br />
<Hr Align="left" width="100%" size=2><br />
<br />
<br><br />
<br />
<br />
Every wanted gene is successfully cloned. Beside the anticipated parts, we also<br />
constructed other biobricks which carry more insert genes. Transformation of every<br />
biobricks in E. coli is accomplished too.<br />
<br />
[[File:Screen shot 2011-10-06 at 上午11.12.32.png|400px]]<br />
[[File:Screen shot 2011-10-06 at 上午11.16.31.png|400px]]<br />
<br />
<br><br />
The SDS-PAGE is run twice. One is with promoter and another one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the <i>E. coli</i> which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
<br><br />
<br><br />
<br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br><font color="#000080" size=5>References</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
1.Rune Standal, Tore-geir Iversen, Dag H. Coucheron, Espen Fjaervik, Janet M. Blantny, Svein Valla. A New Gene Required for Cellulose Production and a Gen Encoding Cellulolytic Activity in Acetobacter xylinum Are Colocalized with the bcs operon. Journal of Bacteriology. Feb, 1994.<br />
<br><br />
2.Inder M. Saxena, Krystyna Kudlicka, Kazuo Okuda, R. Malcolm Brown, JR. Characterization of Genes in the Cellulose-Synthesizing Operon(acs operon) of Acetobacter xylinum: Implications for Cellulose Crystallization. Journal of Bacteriology. Sep,1994.<br />
<br><br />
3.Shin Kawano, Kenji Tajima, Yukako Uemori, Hitomi Yamashita, Tomoki Erata, Masanobu Munekata, Mitsuo Takai. DNA Research. October, 2002. <br />
<br><br />
4.Joel Weadge, Wilfrid Laurier University.</div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Project/PhotoPaperTeam:TzuChiU Formosa/Project/PhotoPaper2011-10-06T03:40:05Z<p>98712149: </p>
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<font color="#228B22" size=6>Photopaper</font><br />
<br><br><font color="#000080" size=5>Abstract</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
For most countries, paper-making has been a traditional but indispensable industry. Wood pulp is the major raw material for paper-making ,moreover,the complicated processes toward paper-making may contribute to environmental pollution. Acetobacter xylinum is a bacterium which produces bacterial cellulose. It has an acs operon, consisting of genes that called acsAB, acsC, and acsD. These genes interact with each other and synthesize cellulose synthase, an enzyme that transforms UDP-glucose into cellulose. What we want to do is to choose cyanobacteria which provides glucose through photosynthesis. Therefore, we want to use cyanobacteria as the host, then introduce the acs operon genes in it and produce bacterial cellulose by expressing this series of genes. With manufacturing processes, we believe this project can develop into a new and eco-friendly technology of papermaking.<br />
<br />
<br />
<br />
<br><br><font color="#000080" size=5>Background</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><font size=4><b>a. about cellulose biosynthesis</b></font><br />
<br>Bacterial cellulose is a form of cellulose which is produced by bacteria. It has the same molecular formula as green plants, which is a polysaccharide with thousands of β(1→4) linked D-glucose units. Cellulose biosynthesis is a carbohydrate metabolism with several enzyme regulations, and the key factor of this pathway is cellulose synthase, which participates in the transition from UDP-glucose to cellulose. The ligand of cellulose synthase is cyclic di-GMP, an effector forming from 2 molecules of GTP. The enzyme is activated by the binding of c-di-GMP with cellulose synthase, turning UDP-glucose into UDP, meanwhile form β-1,4-glucan chains, which are then used to synthesize cellulose.<br />
<br><br />
<html><br />
<object type="application/x-shockwave-flash" height="800" width="600" data="https://static.igem.org/mediawiki/2011/6/61/Cellulose_biosynthesis.swf"><br />
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<br />
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<br />
<br><br />
<br><font size=4><b>b.about acs operon</b></font><br />
<br><br />
[[File:Screen shot 2011-10-06 at 上午5.45.47.png|right|350px]]<br />
<i>Gluconacetobacter hansenii</i> ATCC23769 has been characterized as a model organism for cellulose biosynthesis. The cluster of genes which play the role of producing cellulose is acs operon. It contains three major genes, acsAB, acsC and acsD, and the final product is cellulose synthase. The acsAB catalyzes the formation of bacterial cellulose; acsA is the catalytic subunit which utilizes UDP-glucose to form the basic unit of cellulose; while acsB provide the regulatory subunit which has a cyclic di-GMP binding domain. The acsC gene is the main composite in the formation of the membrane complex of cellulose synthase and proposed to be involved in the export of the polymer across the bacterial cell wall.The acsD gene is involved in the crystallization of the mature cellulose by cleaves the intrastrand ß-1,4 linkages in the cellulose chain and is proposed to have a role in the release of the growing polymer from the cell.<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
<br><br><font color="#000080" size=5>System Design</font><br><br />
<br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
<font size = 4><b>1.Biobricks</b></font><br><br><br />
<br />
Because of the long sequence of complete acs operon, we designed several pairs of<br />
primers to amplify acsAB and acsCD, the products are then purified and digested with<br />
EcoRI and SpeI, next ligated with the pSB1C3 cut by EcoRI-SpeI, which pSB1C3 is the<br />
iGEM 2011 standard backbone.<br />
[[File:Screen shot 2011-10-06 at 上午10.32.16.png|center|600px]]<br />
<br><br />
<br><br />
<font size = 4><b>2. Expression</b></font><br><br><br />
<br />
The recombinant plasmids would be introduced into E. coli for testing and modelling<br />
before transforming into R. rubrum. The IPTG-induced promoter is cloned into biobricks so<br />
that the acs operon expression would be regulated by the addition of IPTG.<br />
As to R. rubrum, the recombinant plasmids would be introduced by electroporation. The<br />
glucose which produced by photosynthesis would be the raw material for cellulose<br />
synthesis.<br />
<br><br />
<br><br />
<font size = 4><b>3. Modelling<br />
</b></font><br><br><br />
Observation of the performance of acs ABCD protein during PAGE can predict the<br />
productivity of cellulose, run 2 SDS-PAGE, one according to the reaction time and another<br />
according to the presence of promoter.<br />
The bacterial culture which have been induced were collected and the bacteria is to<br />
remove, the end product is added with cellulase until the cellulose is fully decomposed.<br />
Benedict solution caused the formation of brick red precipitate, the concentration of<br />
cellulose is then tested with OD645.<br />
[[File:Cellulose.jpg|center|700px]]<br />
<html><br />
<object type="application/x-shockwave-flash" height="400" width="800" data="https://static.igem.org/mediawiki/2011/3/39/Overview.swf"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2011/3/39/Overview.swf" /><br />
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</object><br />
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<br />
<br><br><font color="#000080" size=5>Result</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
The protein purification from <i>E.Coli</i> with acsAB and with acsCD is use to run the SDS-PAGE in order to compare the expression of every protein sample. The expression is then use to predict and calculate the amount of protein produced by the <i>Gluconacetobacter hansenii</i>.<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
The SDS-PAGE is run twice. One is with promoter and another one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the <i>E. coli</i> which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
Every wanted gene is successfully cloned. Beside the anticipated parts, we also<br />
constructed other biobricks which carry more insert genes. Transformation of every<br />
biobricks in E. coli is accomplished too.<br />
<br />
[[File:Screen shot 2011-10-06 at 上午11.12.32.png|400px]]<br />
[[File:Screen shot 2011-10-06 at 上午11.16.31.png|400px]]<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br><font color="#000080" size=5>References</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
1.Rune Standal, Tore-geir Iversen, Dag H. Coucheron, Espen Fjaervik, Janet M. Blantny, Svein Valla. A New Gene Required for Cellulose Production and a Gen Encoding Cellulolytic Activity in Acetobacter xylinum Are Colocalized with the bcs operon. Journal of Bacteriology. Feb, 1994.<br />
<br><br />
2.Inder M. Saxena, Krystyna Kudlicka, Kazuo Okuda, R. Malcolm Brown, JR. Characterization of Genes in the Cellulose-Synthesizing Operon(acs operon) of Acetobacter xylinum: Implications for Cellulose Crystallization. Journal of Bacteriology. Sep,1994.<br />
<br><br />
3.Shin Kawano, Kenji Tajima, Yukako Uemori, Hitomi Yamashita, Tomoki Erata, Masanobu Munekata, Mitsuo Takai. DNA Research. October, 2002. <br />
<br><br />
4.Joel Weadge, Wilfrid Laurier University.</div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2011-10-06T03:34:41Z<p>98712149: </p>
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<br><br />
Gluconacetobacter hansenii which carries the acsABCD protein has the ability to produce cellulose is our main character in our project. Our project is to transform acsABCD genes into E. coli to monitor the production of cellulose production. We collect the protein produced by the transformed E.coli and Gluconacetobacter hansenii in different time point to monitor the expression of acsABCD gene products and the production of cellulose.<br />
<br><br />
<br><br />
<br><br />
<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
The protein purification from <i>E.Coli</i> with acsAB and with acsCD is use to run the SDS-PAGE in order to compare the expression of every protein sample. The expression is then use to predict and calculate the amount of protein produced by the <i>Gluconacetobacter hansenii</i>.<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
The SDS-PAGE is run twice. One is with promoter and another one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the <i>E. coli</i> which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
<br><br />
<font size=3><b>Method 2 : Benedict’s test</b></font><br />
<br><br />
<br><br />
The bacterial culture which have been induced were collected and the bacteria is to remove, the end product is added with cellulase until the cellulose is fully decomposed. Benedict solution caused the formation of brick red precipitate, the concentration of cellulose is then tested with OD645.<br />
<br><br />
<br><br />
Cellulase is used to break down the cellulose into monosaccharide. Cellulose is a glucose polymer connected through a beta (1-4) glycosidic linkages. Benedict’s test is carried out to test the presence of reducing sugar, such as. The reducing sugar reduces copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I) oxide precipitate. The color would range from green to brick red respectively depends on the amount of reducing sugar present in the solution. It can detect the concentration of the reducing sugar under the absorbance condition <sub>OD645</sub>.<br />
<br />
Cellulose (beta-1,4 glucan) is the most plentiful biopolymer in nature and is an crucial raw material for many industries. It is synthesized as extracellular fibrils by cellulose synthase not only in plants but also in some bacteria. <br />
<br />
Bacteria with cellulose synthase gene use Isopropyl-β- D -1-thiogalactopyranoside (IPTG) as the inducer stimulating the production of protein. In our system, IPTG act as the inducer of the R0011 promoter, which then activate the operon. The activated acs operon encodes the cellulose synthase to synthesize cellulose. The reaction lasts two hours and every two hour we would need to collect the purified cellulose. The nett weight is recorded and the mechanism of cellulose activity on pure cellulose substrates is identified. Lastly, the Benedict’s solution is added to find out the absorbance (optical density [O.D.]) value, in order to calculate the amount of monosaccharide that cellulose can produce.<br />
<br><br />
<br><br />
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=='''Bacterial cellulose'''==<br />
[[File:Big1.JPG|center|300px]]<br />
[[File:Big2.JPG|center|300px]]<br />
<br><html><div align="center">the sample of bacterial cellulose that we collected in 3 days</div></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
[[File:111456.jpg|center|500px]]<br />
<br><html><div align="center">bacterial cellulose under EM<br><br><br><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Project/PhotoPaperTeam:TzuChiU Formosa/Project/PhotoPaper2011-10-06T03:33:39Z<p>98712149: </p>
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<font color="#228B22" size=6>Photopaper</font><br />
<br><br><font color="#000080" size=5>Abstract</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
For most countries, paper-making has been a traditional but indispensable industry. Wood pulp is the major raw material for paper-making ,moreover,the complicated processes toward paper-making may contribute to environmental pollution. Acetobacter xylinum is a bacterium which produces bacterial cellulose. It has an acs operon, consisting of genes that called acsAB, acsC, and acsD. These genes interact with each other and synthesize cellulose synthase, an enzyme that transforms UDP-glucose into cellulose. What we want to do is to choose cyanobacteria which provides glucose through photosynthesis. Therefore, we want to use cyanobacteria as the host, then introduce the acs operon genes in it and produce bacterial cellulose by expressing this series of genes. With manufacturing processes, we believe this project can develop into a new and eco-friendly technology of papermaking.<br />
<br />
<br />
<br />
<br><br><font color="#000080" size=5>Background</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><font size=4><b>a. about cellulose biosynthesis</b></font><br />
<br>Bacterial cellulose is a form of cellulose which is produced by bacteria. It has the same molecular formula as green plants, which is a polysaccharide with thousands of β(1→4) linked D-glucose units. Cellulose biosynthesis is a carbohydrate metabolism with several enzyme regulations, and the key factor of this pathway is cellulose synthase, which participates in the transition from UDP-glucose to cellulose. The ligand of cellulose synthase is cyclic di-GMP, an effector forming from 2 molecules of GTP. The enzyme is activated by the binding of c-di-GMP with cellulose synthase, turning UDP-glucose into UDP, meanwhile form β-1,4-glucan chains, which are then used to synthesize cellulose.<br />
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<br><font size=4><b>b.about acs operon</b></font><br />
<br><br />
[[File:Screen shot 2011-10-06 at 上午5.45.47.png|right|350px]]<br />
<i>Gluconacetobacter hansenii</i> ATCC23769 has been characterized as a model organism for cellulose biosynthesis. The cluster of genes which play the role of producing cellulose is acs operon. It contains three major genes, acsAB, acsC and acsD, and the final product is cellulose synthase. The acsAB catalyzes the formation of bacterial cellulose; acsA is the catalytic subunit which utilizes UDP-glucose to form the basic unit of cellulose; while acsB provide the regulatory subunit which has a cyclic di-GMP binding domain. The acsC gene is the main composite in the formation of the membrane complex of cellulose synthase and proposed to be involved in the export of the polymer across the bacterial cell wall.The acsD gene is involved in the crystallization of the mature cellulose by cleaves the intrastrand ß-1,4 linkages in the cellulose chain and is proposed to have a role in the release of the growing polymer from the cell.<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
<br><br><font color="#000080" size=5>System Design</font><br><br />
<br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
<font size = 4><b>1.Biobricks</b></font><br><br><br />
<br />
Because of the long sequence of complete acs operon, we designed several pairs of<br />
primers to amplify acsAB and acsCD, the products are then purified and digested with<br />
EcoRI and SpeI, next ligated with the pSB1C3 cut by EcoRI-SpeI, which pSB1C3 is the<br />
iGEM 2011 standard backbone.<br />
[[File:Screen shot 2011-10-06 at 上午10.32.16.png|center|600px]]<br />
<br><br />
<br><br />
<font size = 4><b>2. Expression</b></font><br><br><br />
<br />
The recombinant plasmids would be introduced into E. coli for testing and modelling<br />
before transforming into R. rubrum. The IPTG-induced promoter is cloned into biobricks so<br />
that the acs operon expression would be regulated by the addition of IPTG.<br />
As to R. rubrum, the recombinant plasmids would be introduced by electroporation. The<br />
glucose which produced by photosynthesis would be the raw material for cellulose<br />
synthesis.<br />
<br><br />
<br><br />
<font size = 4><b>3. Modelling<br />
</b></font><br><br><br />
Observation of the performance of acs ABCD protein during PAGE can predict the<br />
productivity of cellulose, run 2 SDS-PAGE, one according to the reaction time and another<br />
according to the presence of promoter.<br />
The bacterial culture which have been induced were collected and the bacteria is to<br />
remove, the end product is added with cellulase until the cellulose is fully decomposed.<br />
Benedict solution caused the formation of brick red precipitate, the concentration of<br />
cellulose is then tested with OD645.<br />
[[File:Cellulose.jpg|center|700px]]<br />
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<br><br><font color="#000080" size=5>Result</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
The protein purification from <i>E.Coli</i> with acsAB and with acsCD is use to run the SDS-PAGE in order to compare the expression of every protein sample. The expression is then use to predict and calculate the amount of protein produced by the <i>Gluconacetobacter hansenii</i>.<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
The SDS-PAGE is run twice. One is with promoter and another one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the <i>E. coli</i> which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
Every wanted gene is successfully cloned. Beside the anticipated parts, we also<br />
constructed other biobricks which carry more insert genes. Transformation of every<br />
biobricks in E. coli is accomplished too.<br />
<br />
<br />
<br><br><font color="#000080" size=5>References</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
1.Rune Standal, Tore-geir Iversen, Dag H. Coucheron, Espen Fjaervik, Janet M. Blantny, Svein Valla. A New Gene Required for Cellulose Production and a Gen Encoding Cellulolytic Activity in Acetobacter xylinum Are Colocalized with the bcs operon. Journal of Bacteriology. Feb, 1994.<br />
<br><br />
2.Inder M. Saxena, Krystyna Kudlicka, Kazuo Okuda, R. Malcolm Brown, JR. Characterization of Genes in the Cellulose-Synthesizing Operon(acs operon) of Acetobacter xylinum: Implications for Cellulose Crystallization. Journal of Bacteriology. Sep,1994.<br />
<br><br />
3.Shin Kawano, Kenji Tajima, Yukako Uemori, Hitomi Yamashita, Tomoki Erata, Masanobu Munekata, Mitsuo Takai. DNA Research. October, 2002. <br />
<br><br />
4.Joel Weadge, Wilfrid Laurier University.<br />
<br />
<br />
<br />
<br />
</html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2011-10-06T03:32:47Z<p>98712149: </p>
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Gluconacetobacter hansenii which carries the acsABCD protein has the ability to produce cellulose is our main character in our project. Our project is to transform acsABCD genes into E. coli to monitor the production of cellulose production. We collect the protein produced by the transformed E.coli and Gluconacetobacter hansenii in different time point to monitor the expression of acsABCD gene products and the production of cellulose.<br />
<br><br />
<br><br />
<br><br />
<br />
<br><br />
<br><br />
<font size=3><b>Method 2 : Benedict’s test</b></font><br />
<br><br />
<br><br />
The bacterial culture which have been induced were collected and the bacteria is to remove, the end product is added with cellulase until the cellulose is fully decomposed. Benedict solution caused the formation of brick red precipitate, the concentration of cellulose is then tested with OD645.<br />
<br><br />
<br><br />
Cellulase is used to break down the cellulose into monosaccharide. Cellulose is a glucose polymer connected through a beta (1-4) glycosidic linkages. Benedict’s test is carried out to test the presence of reducing sugar, such as. The reducing sugar reduces copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I) oxide precipitate. The color would range from green to brick red respectively depends on the amount of reducing sugar present in the solution. It can detect the concentration of the reducing sugar under the absorbance condition <sub>OD645</sub>.<br />
<br />
Cellulose (beta-1,4 glucan) is the most plentiful biopolymer in nature and is an crucial raw material for many industries. It is synthesized as extracellular fibrils by cellulose synthase not only in plants but also in some bacteria. <br />
<br />
Bacteria with cellulose synthase gene use Isopropyl-β- D -1-thiogalactopyranoside (IPTG) as the inducer stimulating the production of protein. In our system, IPTG act as the inducer of the R0011 promoter, which then activate the operon. The activated acs operon encodes the cellulose synthase to synthesize cellulose. The reaction lasts two hours and every two hour we would need to collect the purified cellulose. The nett weight is recorded and the mechanism of cellulose activity on pure cellulose substrates is identified. Lastly, the Benedict’s solution is added to find out the absorbance (optical density [O.D.]) value, in order to calculate the amount of monosaccharide that cellulose can produce.<br />
<br><br />
<br><br />
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=='''Bacterial cellulose'''==<br />
[[File:Big1.JPG|center|300px]]<br />
[[File:Big2.JPG|center|300px]]<br />
<br><html><div align="center">the sample of bacterial cellulose that we collected in 3 days</div></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
[[File:111456.jpg|center|500px]]<br />
<br><html><div align="center">bacterial cellulose under EM<br><br><br><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2011-10-06T03:29:18Z<p>98712149: /* Bacterial cellulose */</p>
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<br><br />
Gluconacetobacter hansenii which carries the acsABCD protein has the ability to produce cellulose is our main character in our project. Our project is to transform acsABCD genes into E. coli to monitor the production of cellulose production. We collect the protein produced by the transformed E.coli and Gluconacetobacter hansenii in different time point to monitor the expression of acsABCD gene products and the production of cellulose.<br />
<br><br />
<br><br />
<br><br />
<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
The protein purification from <i>E.Coli</i> with acsAB and with acsCD is use to run the SDS-PAGE in order to compare the expression of every protein sample. The expression is then use to predict and calculate the amount of protein produced by the <i>Gluconacetobacter hansenii</i>.<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
The SDS-PAGE is run twice. One is with promoter and another one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the <i>E. coli</i> which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
<br><br />
<font size=3><b>Method 2 : Benedict’s test</b></font><br />
<br><br />
<br><br />
The bacterial culture which have been induced were collected and the bacteria is to remove, the end product is added with cellulase until the cellulose is fully decomposed. Benedict solution caused the formation of brick red precipitate, the concentration of cellulose is then tested with OD645.<br />
<br><br />
<br><br />
Cellulase is used to break down the cellulose into monosaccharide. Cellulose is a glucose polymer connected through a beta (1-4) glycosidic linkages. Benedict’s test is carried out to test the presence of reducing sugar, such as. The reducing sugar reduces copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I) oxide precipitate. The color would range from green to brick red respectively depends on the amount of reducing sugar present in the solution. It can detect the concentration of the reducing sugar under the absorbance condition <sub>OD645</sub>.<br />
<br />
Cellulose (beta-1,4 glucan) is the most plentiful biopolymer in nature and is an crucial raw material for many industries. It is synthesized as extracellular fibrils by cellulose synthase not only in plants but also in some bacteria. <br />
<br />
Bacteria with cellulose synthase gene use Isopropyl-β- D -1-thiogalactopyranoside (IPTG) as the inducer stimulating the production of protein. In our system, IPTG act as the inducer of the R0011 promoter, which then activate the operon. The activated acs operon encodes the cellulose synthase to synthesize cellulose. The reaction lasts two hours and every two hour we would need to collect the purified cellulose. The nett weight is recorded and the mechanism of cellulose activity on pure cellulose substrates is identified. Lastly, the Benedict’s solution is added to find out the absorbance (optical density [O.D.]) value, in order to calculate the amount of monosaccharide that cellulose can produce.<br />
<br><br />
<br><br />
<br><br />
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=='''Bacterial cellulose'''==<br />
[[File:Big1.JPG|center|300px]]<br />
[[File:Big2.JPG|center|300px]]<br />
<br><html><div align="center">the sample of bacterial cellulose that we collected in 3 days</div></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
[[File:111456.jpg|center|500px]]<br />
<br><html><div align="center">bacterial cellulose under EM<br><br><br><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2011-10-06T03:28:54Z<p>98712149: </p>
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<br><br />
Gluconacetobacter hansenii which carries the acsABCD protein has the ability to produce cellulose is our main character in our project. Our project is to transform acsABCD genes into E. coli to monitor the production of cellulose production. We collect the protein produced by the transformed E.coli and Gluconacetobacter hansenii in different time point to monitor the expression of acsABCD gene products and the production of cellulose.<br />
<br><br />
<br><br />
<br><br />
<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
The protein purification from <i>E.Coli</i> with acsAB and with acsCD is use to run the SDS-PAGE in order to compare the expression of every protein sample. The expression is then use to predict and calculate the amount of protein produced by the <i>Gluconacetobacter hansenii</i>.<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
The SDS-PAGE is run twice. One is with promoter and another one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the <i>E. coli</i> which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
<br><br />
<font size=3><b>Method 2 : Benedict’s test</b></font><br />
<br><br />
<br><br />
The bacterial culture which have been induced were collected and the bacteria is to remove, the end product is added with cellulase until the cellulose is fully decomposed. Benedict solution caused the formation of brick red precipitate, the concentration of cellulose is then tested with OD645.<br />
<br><br />
<br><br />
Cellulase is used to break down the cellulose into monosaccharide. Cellulose is a glucose polymer connected through a beta (1-4) glycosidic linkages. Benedict’s test is carried out to test the presence of reducing sugar, such as. The reducing sugar reduces copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I) oxide precipitate. The color would range from green to brick red respectively depends on the amount of reducing sugar present in the solution. It can detect the concentration of the reducing sugar under the absorbance condition <sub>OD645</sub>.<br />
<br />
Cellulose (beta-1,4 glucan) is the most plentiful biopolymer in nature and is an crucial raw material for many industries. It is synthesized as extracellular fibrils by cellulose synthase not only in plants but also in some bacteria. <br />
<br />
Bacteria with cellulose synthase gene use Isopropyl-β- D -1-thiogalactopyranoside (IPTG) as the inducer stimulating the production of protein. In our system, IPTG act as the inducer of the R0011 promoter, which then activate the operon. The activated acs operon encodes the cellulose synthase to synthesize cellulose. The reaction lasts two hours and every two hour we would need to collect the purified cellulose. The nett weight is recorded and the mechanism of cellulose activity on pure cellulose substrates is identified. Lastly, the Benedict’s solution is added to find out the absorbance (optical density [O.D.]) value, in order to calculate the amount of monosaccharide that cellulose can produce.<br />
<br><br />
<br><br />
<br><br />
<html><object type="application/x-shockwave-flash" height="450" width=650" data="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf" /><br />
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</object><br />
</html><br />
=='''Bacterial cellulose'''==<br />
[[File:Big1.JPG|center|300px]]<br />
[[File:Big2.JPG|center|300px]]<br />
<br><html><div align="center">the sample of bacterial cellulose that we collected in 10 days</div></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
[[File:111456.jpg|center|500px]]<br />
<br><html><div align="center">bacterial cellulose under EM<br><br><br><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/PartsTeam:TzuChiU Formosa/Parts2011-10-06T03:23:44Z<p>98712149: </p>
<hr />
<div><html><br />
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<img src="https://static.igem.org/mediawiki/2011/a/a3/BACK.jpg" class="bg" height="100%" width="100%"><br />
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<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
<param name="wmode" value="transparent"><br />
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<style type="text/css"><br />
#headerimage{width:975px;margin-top:-18px;}<br />
#headerimage img{max-width:975px;}<br />
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.firstHeading{width:0;height:0;display:none;position:relative;top:0;left:0;margin:0;}<br />
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<br />
<br><br><font color="#000080" size=5>Description</font><br />
<Hr Align="left" width="100%" size=2><br />
<br><br />
We had design 8 biobricks for our project. Due to the long sequence of complete acs operon, we amplified the acsAB and acsCD gene respectively. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K571000 BBa_K571000] is the first biobrick with acsAB gene cloned into pSB1C3, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K571003 BBa_K571003] is the second one with acsCD as insert. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_K571004] is an improved part from the existing biobrick [http://partsregistry.org/Part:BBa_R0011 BBa_R0011]. We added a RBS at the end of the original sequence in order to get a better gene expression performance.<br />
<br />
As to the composite biobricks, we tried a few times for different arrangement of the parts to get the most ideal one. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K571005 BBa_K571005] was designed to work together with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K571006 BBa_K571006], as the former carries acsAB gene and the latter carries acsCD gene. Both of them were added the promoter([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_K571004]) for gene expression after being induced by IPTG. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K571007 BBa_K571007] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K571008 BBa_K571008] are biobricks with all elements cloned in them.<br />
<br />
All of the biobricks have been tested to make sure that the clonings are successful. Modelling and system construction are still proceeding and improving. <br />
<br />
<br><br />
<br><br><font color="#000080" size=5>Parts</font><br />
<Hr Align="left" width="100%" size=2><br />
<br />
New for iGEM 2010 is the ''groupparts'' tag. This tag will generate a table with all of the parts that your team adds to your team sandbox. Note that if you want to document a part you need to document it on the [http://partsregistry.org Registry], not on your team wiki.<br />
<br />
<groupparts>iGEM011 TzuChiU_Formosa</groupparts><br />
<br><br />
<br></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2011-10-06T03:22:30Z<p>98712149: </p>
<hr />
<div><html><br />
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<img src="https://static.igem.org/mediawiki/2011/a/a3/BACK.jpg" class="bg" height="100%" width="100%"><br />
<div id="wrap"><br />
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<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
</div><br />
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<style type="text/css"><br />
#headerimage{width:975px;margin-top:-18px;}<br />
#headerimage img{max-width:975px;}<br />
#top-section{height:0px;margin:0 auto !important;}<br />
.firstHeading{width:0;height:0;display:none;position:relative;top:0;left:0;margin:0;}<br />
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</style><br />
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<body><br />
<p><br />
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</object><br />
</p><br />
<br />
</html><br />
<br><br />
<br><br />
<br />
<br><br />
<br><br />
<br><br />
<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
The protein purification from <i>E.Coli</i> with acsAB and with acsCD is use to run the SDS-PAGE in order to compare the expression of every protein sample. The expression is then use to predict and calculate the amount of protein produced by the <i>Gluconacetobacter hansenii</i>.<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
The SDS-PAGE is run twice. One is with promoter and another one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the <i>E. coli</i> which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
<br><br />
<font size=3><b>Method 2 : Benedict’s test</b></font><br />
<br><br />
<br><br />
The bacterial culture which have been induced were collected and the bacteria is to remove, the end product is added with cellulase until the cellulose is fully decomposed. Benedict solution caused the formation of brick red precipitate, the concentration of cellulose is then tested with OD645.<br />
<br><br />
<br><br />
Cellulase is used to break down the cellulose into monosaccharide. Cellulose is a glucose polymer connected through a beta (1-4) glycosidic linkages. Benedict’s test is carried out to test the presence of reducing sugar, such as. The reducing sugar reduces copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I) oxide precipitate. The color would range from green to brick red respectively depends on the amount of reducing sugar present in the solution. It can detect the concentration of the reducing sugar under the absorbance condition <sub>OD645</sub>.<br />
<br />
Cellulose (beta-1,4 glucan) is the most plentiful biopolymer in nature and is an crucial raw material for many industries. It is synthesized as extracellular fibrils by cellulose synthase not only in plants but also in some bacteria. <br />
<br />
Bacteria with cellulose synthase gene use Isopropyl-β- D -1-thiogalactopyranoside (IPTG) as the inducer stimulating the production of protein. In our system, IPTG act as the inducer of the R0011 promoter, which then activate the operon. The activated acs operon encodes the cellulose synthase to synthesize cellulose. The reaction lasts two hours and every two hour we would need to collect the purified cellulose. The nett weight is recorded and the mechanism of cellulose activity on pure cellulose substrates is identified. Lastly, the Benedict’s solution is added to find out the absorbance (optical density [O.D.]) value, in order to calculate the amount of monosaccharide that cellulose can produce.<br />
<br><br />
<br><br />
<br><br />
<html><object type="application/x-shockwave-flash" height="450" width=650" data="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf" /><br />
<param name="quality" value="high" /><br />
<param name="wmode" value="transparent"><br />
</object><br />
</html><br />
=='''Bacterial cellulose'''==<br />
[[File:Big1.JPG|center|300px]]<br />
[[File:Big2.JPG|center|300px]]<br />
<br><html><div align="center">the sample of bacterial cellulose that we collected</div></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
[[File:111456.jpg|center|500px]]<br />
<br><html><div align="center">bacterial cellulose under EM<br><br><br><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2011-10-06T03:19:12Z<p>98712149: </p>
<hr />
<div><html><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/a/a3/BACK.jpg" class="bg" height="100%" width="100%"><br />
<div id="wrap"><br />
<div id="headerimage"><br />
<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
</div><br />
<br />
<style type="text/css"><br />
#headerimage{width:975px;margin-top:-18px;}<br />
#headerimage img{max-width:975px;}<br />
#top-section{height:0px;margin:0 auto !important;}<br />
.firstHeading{width:0;height:0;display:none;position:relative;top:0;left:0;margin:0;}<br />
img.bg{min-height: 100%;min-width: 1024px;width: 100%;height: auto;position: fixed;top: 0;left: 0;z-index:-1;}<br />
</style><br />
<br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="350" width=1024" data="https://static.igem.org/mediawiki/2011/f/fd/Menubar2.swf"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2011/f/fd/Menubar2.swf" /><br />
<param name="quality" value="high" /><br />
<param name="wmode" value="transparent"><br />
</object><br />
</p><br />
<br />
</html><br />
<br><br />
<br><br />
<br />
<br><br />
<br><br />
<br><br />
<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
The protein purification from <i>E.Coli</i> with acsAB and with acsCD is use to run the SDS-PAGE in order to compare the expression of every protein sample. The expression is then use to predict and calculate the amount of protein produced by the <i>Gluconacetobacter hansenii</i>.<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
The SDS-PAGE is run twice. One is with promoter and another one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the E. coli which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
<br><br />
<font size=3><b>Method 2 : Benedict’s test</b></font><br />
<br><br />
<br><br />
The bacterial culture which have been induced were collected and the bacteria is to remove, the end product is added with cellulase until the cellulose is fully decomposed. Benedict solution caused the formation of brick red precipitate, the concentration of cellulose is then tested with OD645.<br />
<br><br />
<br><br />
Cellulase is used to break down the cellulose into monosaccharide. Cellulose is a glucose polymer connected through a beta (1-4) glycosidic linkages. Benedict’s test is carried out to test the presence of reducing sugar, such as. The reducing sugar reduces copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I) oxide precipitate. The color would range from green to brick red respectively depends on the amount of reducing sugar present in the solution. It can detect the concentration of the reducing sugar under the absorbance condition <sub>OD645</sub>.<br />
<br />
Cellulose (beta-1,4 glucan) is the most plentiful biopolymer in nature and is an crucial raw material for many industries. It is synthesized as extracellular fibrils by cellulose synthase not only in plants but also in some bacteria. <br />
<br />
Bacteria with cellulose synthase gene use Isopropyl-β- D -1-thiogalactopyranoside (IPTG) as the inducer stimulating the production of protein. In our system, IPTG act as the inducer of the R0011 promoter, which then activate the operon. The activated acs operon encodes the cellulose synthase to synthesize cellulose. The reaction lasts two hours and every two hour we would need to collect the purified cellulose. The nett weight is recorded and the mechanism of cellulose activity on pure cellulose substrates is identified. Lastly, the Benedict’s solution is added to find out the absorbance (optical density [O.D.]) value, in order to calculate the amount of monosaccharide that cellulose can produce.<br />
<br><br />
<br><br />
<br><br />
<html><object type="application/x-shockwave-flash" height="450" width=650" data="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf" /><br />
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</object><br />
</html><br />
=='''Bacterial cellulose'''==<br />
[[File:Big1.JPG|center|300px]]<br />
[[File:Big2.JPG|center|300px]]<br />
<br><html><div align="center">the sample of bacterial cellulose that we collected</div></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
[[File:111456.jpg|center|500px]]<br />
<br><html><div align="center">bacterial cellulose under EM<br><br><br><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2011-10-06T03:15:56Z<p>98712149: /* Bacterial cellulose */</p>
<hr />
<div><html><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/a/a3/BACK.jpg" class="bg" height="100%" width="100%"><br />
<div id="wrap"><br />
<div id="headerimage"><br />
<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
</div><br />
<br />
<style type="text/css"><br />
#headerimage{width:975px;margin-top:-18px;}<br />
#headerimage img{max-width:975px;}<br />
#top-section{height:0px;margin:0 auto !important;}<br />
.firstHeading{width:0;height:0;display:none;position:relative;top:0;left:0;margin:0;}<br />
img.bg{min-height: 100%;min-width: 1024px;width: 100%;height: auto;position: fixed;top: 0;left: 0;z-index:-1;}<br />
</style><br />
<br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="350" width=1024" data="https://static.igem.org/mediawiki/2011/f/fd/Menubar2.swf"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2011/f/fd/Menubar2.swf" /><br />
<param name="quality" value="high" /><br />
<param name="wmode" value="transparent"><br />
</object><br />
</p><br />
<br />
</html><br />
<br><br />
<br><br />
<br />
<br><br />
<br><br />
<br><br />
<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
The SDS-PAGE is run twice. One is with promoter and another one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the E. coli which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
<br><br />
<font size=3><b>Method 2 : Benedict’s test</b></font><br />
<br><br />
<br><br />
The bacterial culture which have been induced were collected and the bacteria is to remove, the end product is added with cellulase until the cellulose is fully decomposed. Benedict solution caused the formation of brick red precipitate, the concentration of cellulose is then tested with OD645.<br />
<br><br />
<br><br />
Cellulase is used to break down the cellulose into monosaccharide. Cellulose is a glucose polymer connected through a beta (1-4) glycosidic linkages. Benedict’s test is carried out to test the presence of reducing sugar, such as. The reducing sugar reduces copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I) oxide precipitate. The color would range from green to brick red respectively depends on the amount of reducing sugar present in the solution. It can detect the concentration of the reducing sugar under the absorbance condition <sub>OD645</sub>.<br />
<br />
Cellulose (beta-1,4 glucan) is the most plentiful biopolymer in nature and is an crucial raw material for many industries. It is synthesized as extracellular fibrils by cellulose synthase not only in plants but also in some bacteria. <br />
<br />
Bacteria with cellulose synthase gene use Isopropyl-β- D -1-thiogalactopyranoside (IPTG) as the inducer stimulating the production of protein. In our system, IPTG act as the inducer of the R0011 promoter, which then activate the operon. The activated acs operon encodes the cellulose synthase to synthesize cellulose. The reaction lasts two hours and every two hour we would need to collect the purified cellulose. The nett weight is recorded and the mechanism of cellulose activity on pure cellulose substrates is identified. Lastly, the Benedict’s solution is added to find out the absorbance (optical density [O.D.]) value, in order to calculate the amount of monosaccharide that cellulose can produce.<br />
<br><br />
<br><br />
<br><br />
<html><object type="application/x-shockwave-flash" height="450" width=650" data="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2011/3/3e/Growth_curve.swf" /><br />
<param name="quality" value="high" /><br />
<param name="wmode" value="transparent"><br />
</object><br />
</html><br />
=='''Bacterial cellulose'''==<br />
[[File:Big1.JPG|center|300px]]<br />
[[File:Big2.JPG|center|300px]]<br />
<br><html><div align="center">the sample of bacterial cellulose that we collected</div></html><br />
<br><br />
<br><br />
<br><br />
<br><br />
[[File:111456.jpg|center|500px]]<br />
<br><html><div align="center">bacterial cellulose under EM<br><br><br><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2011-10-06T03:15:39Z<p>98712149: /* Bacterial cellulose */</p>
<hr />
<div><html><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/a/a3/BACK.jpg" class="bg" height="100%" width="100%"><br />
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<font size=3><b>Method 1 : SDS-PAGE</b></font><br />
<br><br />
<br><br />
Observe the performance of acs ABCD protein during PAGE to predict the productivity of cellulose, run 2 SDS-PAGE respectively, one according to the reaction time and another according to the presence of promoter.<br />
<br><br />
<br><br />
'''-difference of reaction time'''<br />
<br><br />
0hr, 4hrs, 8hrs, 10hrs, and 12hrs of bacterial culture were collected. Compare the high-performance phase of the acs ABCD protein, calculate the productivity of cellulose.<br />
<br />
<br><br />
<br><br />
'''-difference of promoter'''<br />
<br>[[File:Pro.jpg|center|800px]]<br />
The SDS-PAGE is run twice. One is with promoter and another one without promoter. The difference of the protein produced is used to predict productivity of cellulose. At 0 hr, the E. coli which carries promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsAB or promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K571004 BBa_k571004]) + acsCD all performed well. At 4hrs, the gene expression is shown as well, especially the gene which carries promoter+acsCD. The gene which carries promoter+acsCD expressed constantly until 10hrs. <br />
<br> <br />
<br><br />
The relative molecular mass of acsAB is about 168KDa, while the relative molecular mass of acsCD is about 155KDa. Therefore, we predict that the protein expression should be somewhere between this 2 bands. Therefore, we can conclude that the primer we designed is functional.<br />
<br><br />
1. Compare the positive control with acsAB gene and acsCD gene<br />
<br><br />
2. Calculate the amount of protein produced <br />
<br><br />
3. Predict the productivity of cellulose<br />
<br />
<br><br />
<br><br />
<font size=3><b>Method 2 : Benedict’s test</b></font><br />
<br><br />
<br><br />
The bacterial culture which have been induced were collected and the bacteria is to remove, the end product is added with cellulase until the cellulose is fully decomposed. Benedict solution caused the formation of brick red precipitate, the concentration of cellulose is then tested with OD645.<br />
<br><br />
<br><br />
Cellulase is used to break down the cellulose into monosaccharide. Cellulose is a glucose polymer connected through a beta (1-4) glycosidic linkages. Benedict’s test is carried out to test the presence of reducing sugar, such as. The reducing sugar reduces copper(II) ions in these test solutions to copper(I), which then forms a brick red copper(I) oxide precipitate. The color would range from green to brick red respectively depends on the amount of reducing sugar present in the solution. It can detect the concentration of the reducing sugar under the absorbance condition <sub>OD645</sub>.<br />
<br />
Cellulose (beta-1,4 glucan) is the most plentiful biopolymer in nature and is an crucial raw material for many industries. It is synthesized as extracellular fibrils by cellulose synthase not only in plants but also in some bacteria. <br />
<br />
Bacteria with cellulose synthase gene use Isopropyl-β- D -1-thiogalactopyranoside (IPTG) as the inducer stimulating the production of protein. In our system, IPTG act as the inducer of the R0011 promoter, which then activate the operon. The activated acs operon encodes the cellulose synthase to synthesize cellulose. The reaction lasts two hours and every two hour we would need to collect the purified cellulose. The nett weight is recorded and the mechanism of cellulose activity on pure cellulose substrates is identified. Lastly, the Benedict’s solution is added to find out the absorbance (optical density [O.D.]) value, in order to calculate the amount of monosaccharide that cellulose can produce.<br />
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=='''Bacterial cellulose'''==<br />
[[File:Big1.JPG|center|300px]]<br />
[[File:Big2.JPG|center|300px]]<br />
<br><html><div align="center">the sample of bacterial cellulose that we collected</div></html><br />
<br><br />
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<br><br />
<br><br />
[[File:111456.jpg|center|500px]]<br />
<br><html><div align="center">bacterial cellulose under EM<br />
</div></html></div>98712149http://2011.igem.org/File:111456.jpgFile:111456.jpg2011-10-06T03:11:49Z<p>98712149: </p>
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<div></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Acknowledgment/attributionsTeam:TzuChiU Formosa/Acknowledgment/attributions2011-10-06T03:04:57Z<p>98712149: </p>
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<font size=5><b>W</b></font><font size=3>e are proud to say that all the work was accomplished by the undergraduate <br />
<br><br>members of our iGEM team with mentorship from our advisors and professors.<br />
<br><br><br />
<br><br>A big, hearty thanks to all our instructors and advisors, for supervising us throughout <br />
<br><br>the project and keeping our team motivated and helping us in any way possible.</font><br />
<br><br><br><img height="500" width=650" src="https://static.igem.org/mediawiki/2011/f/f4/Fddddddb.gif"><br />
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</html></div>98712149http://2011.igem.org/File:Fddddddb.gifFile:Fddddddb.gif2011-10-06T03:04:14Z<p>98712149: </p>
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<div></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Notebook/photopaperTeam:TzuChiU Formosa/Notebook/photopaper2011-10-06T02:56:39Z<p>98712149: </p>
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<font color="#228B22" size=6>Photopaper</font><br />
<br><br><font color="#000080" size=4>Meeting Minutes</font><br />
<Hr Align="left" width="100%" size=2><br />
<font color="#000000" size=3><b>2011.02.24</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Team organization[[File:001.jpg|right|500px|caption]]<br />
*Brain storming<br />
**paper made by bacteria with add-ons such as colors, fragrance, etc.<br />
**"light up" the plants for replacing lamp posts.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.04</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Team advisory <br />
*Brain storming<br />
**Add-ons: colorful cellulose which produced by bacteria such as beta-carotene<br />
**information exchange with iGEM 2009 Cambridge team<br />
<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.14</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Task Allocation<br />
*Brain storming<br />
**Avatar - "light up" the plants by transfecting symbiotic bacteria which cloned into fluorescence gene<br />
**Eco-friendly warmer - biotic thermal pad<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.23</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
**Project : paperia <br />
**Option 1 : Culture bacteria which has pigment gene<br />
**Option 2 : Cellulose-producing bacteria secrete pigment into the medium<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.24</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Exp. procedure: <br />
** cloning of cellulose gene’s CDS<br />
** the product should operate within E. coli.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.06.22</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Due to some unforseen reason, the team decided to change their project.<br />
* New project: Biojenny <br />
-economical and humane way to produce paper in large quantities.<br><br />
-yeast to be our host<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.01</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Freeze > grin > genome DNA isolation > Cloning = silk protein gene<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.09</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* the connections between 3 silk proteins : Fibl Fibh P25<br />
* major proteins : H-chain, L-chain, P25<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.15</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Due to limited resources and techniques required, the team decided to switch back to the previous project. Paper making !<br />
* However it would be modified to be more innovative and creative.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.18</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Latest project : Photo paper<br />
* cyanobacteria is designed as the host, cellulose made up of the glucose produced by cyanobacteria could be one of the main attraction of the project.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.23</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* system modification to overcome the problems arises during preliminary round<br />
* Biobricks from Tokyo 2010 team will be utilized<br />
**regulator promoter in order to regulate the secretion of cellulose to solve the aggregation of the bacteria<br />
<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.09.15</b></font><br />
<br />
'''Genome miniprep'''<br><br />
Gluconacetobacter hansenii<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.09.18</b></font><br />
<br />
'''Gel/PCR DNA extraction'''<br><br />
Gluconacetobacter hansenii<br />
<br />
<br />
<br />
<font size=4 color="#000080"><b>Protocols</b></font><br />
<Hr Align="left" width="100%" size=3><br />
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<br>[[File:2-20110908-13.jpg|lefe|650px|caption]]<br />
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<br>[[File:3-20110910-11.jpg|lefe|650px|caption]]<br />
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<br>[[File:4-20110912-20.jpg|lefe|650px|caption]]<br />
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<br>[[File:13-20110929.jpg|lefe|650px|caption]]<br />
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<br></div>98712149http://2011.igem.org/File:14-20111001.jpgFile:14-20111001.jpg2011-10-06T02:56:07Z<p>98712149: </p>
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<div></div>98712149http://2011.igem.org/File:5-20110921.jpgFile:5-20110921.jpg2011-10-06T02:55:11Z<p>98712149: uploaded a new version of &quot;File:5-20110921.jpg&quot;</p>
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<div></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Acknowledgment/attributionsTeam:TzuChiU Formosa/Acknowledgment/attributions2011-10-06T02:53:29Z<p>98712149: </p>
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<font size=5><b>W</b></font><font size=3>e are proud to say that all the work was accomplished by the undergraduate <br />
<br><br>members of our iGEM team with mentorship from our advisors and professors.<br />
<br><br><br />
<br><br>A big, hearty thanks to all our instructors and advisors, for supervising us throughout <br />
<br><br>the project and keeping our team motivated and helping us in any way possible.</font><br />
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<div></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Acknowledgment/attributionsTeam:TzuChiU Formosa/Acknowledgment/attributions2011-10-06T02:10:59Z<p>98712149: </p>
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<font size=5><b>W</b></font><font size=3>e are proud to say that all the work was accomplished by the undergraduate <br />
<br><br>members of our iGEM team with mentorship from our advisors and professors.<br />
<br><br><br />
<br><br>A big, hearty thanks to all our instructors and advisors, for supervising us throughout <br />
<br><br>the project and keeping our team motivated and helping us in any way possible.</font><br />
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<font size=3><b>We are proud to say that all the work was accomplished by the undergraduate <br />
<br><br>members of our iGEM team with mentorship from our advisors and professors.<br />
<br><br><br />
<br><br>A big, hearty thanks to all our instructors and advisors, for supervising us throughout <br />
<br><br>the project and keeping our team motivated and helping us in any way possible.</b></font><br />
</div><br />
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<font size=3>We are proud to say that all the work was accomplished by the undergraduate <br />
<br><br>members of our iGEM team with mentorship from our advisors and professors.<br />
<br><br><br />
<br><br>A big, hearty thanks to all our instructors and advisors, for supervising us throughout <br />
<br><br>the project and keeping our team motivated and helping us in any way possible.</font><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Acknowledgment/attributionsTeam:TzuChiU Formosa/Acknowledgment/attributions2011-10-06T02:01:38Z<p>98712149: </p>
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<font size=3>We are proud to say that all the work was accomplished by the undergraduate <br />
<br><br>members of our iGEM team with mentorship from our advisors and professors.<br />
<br><br><br />
<br><br>A big, hearty thanks to all our instructors and advisors, for supervising us throughout <br />
<br><br>the project and keeping our team motivated and helping us in any way possible.</font><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Acknowledgment/attributionsTeam:TzuChiU Formosa/Acknowledgment/attributions2011-10-06T02:00:05Z<p>98712149: </p>
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<font size=3>We are proud to say that all the work was accomplished by the undergraduate <br />
<br><br>members of our iGEM team with mentorship from our advisors and professors.<br />
<br><br><br />
<br><br>A big, hearty thanks to all our instructors and advisors, for supervising us throughout <br />
<br><br>the project and keeping our team motivated and helping us in any way possible.</font><br />
</div></html></div>98712149http://2011.igem.org/File:Dsasdadsa.gifFile:Dsasdadsa.gif2011-10-06T01:59:30Z<p>98712149: </p>
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<font size=3>We are proud to say that all the work was accomplished by the undergraduate <br />
<br><br>members of our iGEM team with mentorship from our advisors and professors.<br />
<br><br><br />
<br><br>A big, hearty thanks to all our instructors and advisors, for supervising us throughout <br />
<br><br>the project and keeping our team motivated and helping us in any way possible.</font><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Acknowledgment/attributionsTeam:TzuChiU Formosa/Acknowledgment/attributions2011-10-06T01:55:13Z<p>98712149: </p>
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<font size=3>We are proud to say that all the work was accomplished by the undergraduate <br />
<br><br>members of our iGEM team with mentorship from our advisors and professors.<br />
<br><br><br />
<br><br>A big, hearty thanks to all our instructors and advisors, for supervising us throughout <br />
<br><br>the project and keeping our team motivated and helping us in any way possible.</font><br />
</div></html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Acknowledgment/attributionsTeam:TzuChiU Formosa/Acknowledgment/attributions2011-10-06T01:53:49Z<p>98712149: </p>
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<font size=3>We are proud to say that all the work was accomplished by the undergraduate <br />
<br><br>members of our iGEM team with mentorship from our advisors and professors.<br />
<br><br><br />
<br><br>A big, hearty thanks to all our instructors and advisors, for supervising us throughout <br />
<br><br>the project and keeping our team motivated and helping us in any way possible.</font></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Acknowledgment/attributionsTeam:TzuChiU Formosa/Acknowledgment/attributions2011-10-06T01:52:05Z<p>98712149: Created page with "<html> <img src="https://static.igem.org/mediawiki/2011/a/a3/BACK.jpg" class="bg" height="100%" width="100%"> <div id="wrap"> <div id="headerimage"> <img src ="https://2011.igem.org/wi..."</p>
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<font size=3>We are proud to say that all the work was accomplished by the undergraduate members of our iGEM team with mentorship from our advisors and professors.<br />
<br />
A big, hearty thanks to all our instructors and advisors, for supervising us throughout the project and keeping our team motivated and helping us in any way possible.</font></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:39:57Z<p>98712149: </p>
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<font color="#228B22" size=6><b>O</b></font><font size=3>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio <br />
<br><br>for helping us. We have done education on synthetic biology via one of the most <br />
<br><br>populat radio channel in Taiwan, <a href="http://radio.newdaai.tv/aod/index_in.php" target=_blank>DaAi Radio</a>.<br />
<br><br />
<br> <br />
<br><br>One of the staff was at our synthetic biology fair. She was impressed by<br />
<br><br>our idea and project, and hence arranged an interview with us.In the interview ,<br />
<br><br>we mainly discussed about synthetic biology, our university achievements and <br />
<br><br>also our current project. Our advisor, Eric Yang also shared his personal iGEM <br />
<br><br>experience with all the audience.</font></div> <br />
<br> <br />
<br><br />
</html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:36:06Z<p>98712149: </p>
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<br />
<font color="#228B22" size=6><b>O</b></font><font size=3>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us.<br />
<br>We have done education on synthetic biology via one of the most populat radio channel <br />
<br>in Taiwan, <a href="http://radio.newdaai.tv/aod/index_in.php" target=_blank>DaAi Radio</a>.<br />
<br><br />
<br> <br />
<br>One of the staff was at our synthetic biology fair. She was impressed by<br />
<br>our idea and project, and hence arranged an interview with us.In the interview ,<br />
<br>we mainly discussed about synthetic biology, our university achievements and <br />
<br>also our current project. Our advisor, Eric Yang also shared his personal iGEM <br />
<br>experience with all the audience.</font> <br />
<br> <br />
<br><br />
</html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:33:11Z<p>98712149: </p>
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</p><br />
</body><br />
<div align="center"><br />
<font color="#228B22" size=6><b>O</b></font><font size=3>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us.<br />
<br>We have done education on synthetic biology via one of the most populat radio channel <br />
<br>in Taiwan, <a href="http://radio.newdaai.tv/aod/index_in.php" target=_blank>DaAi Radio</a>.<br />
<br><br />
<br> <br />
<br>One of the staff was at our synthetic biology fair. She was impressed by <br />
<br>our idea and project, and hence arranged an interview with us.In the interview ,<br />
<br>we mainly discussed about synthetic biology, our university achievements and <br />
<br>also our current project. Our advisor, Eric Yang also shared his personal iGEM <br />
<br>experience with all the audience.</font> <br />
</div>><br> <br />
<br><br />
</html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:30:04Z<p>98712149: </p>
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<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
</div><br />
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<style type="text/css"><br />
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</p><br />
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<div align="center"><br />
<font color="#228B22" size=6><b>O</b></font><font size=3>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us. We have done education on synthetic biology via one of the most populat radio channel in Taiwan, <a href="http://radio.newdaai.tv/aod/index_in.php" target=_blank>DaAi Radio</a>.<br />
<br><br />
<br> <br />
One of the staff was at our synthetic biology fair. She was impressed by our idea and project, and hence arranged an interview with us. In the interview , we mainly discussed about synthetic biology, our university achievements and also our current project. Our advisor, Eric Yang also shared his personal iGEM experience with all the audience.</font> <br />
</div>><br> <br />
<br><br />
</html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:28:56Z<p>98712149: </p>
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<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
</div><br />
<br />
<style type="text/css"><br />
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</p><br />
</body><br />
<div align="center"><br />
<font color="#228B22" size=6><b>O</b></font><font size=3>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us. We have done education on synthetic biology via one of the most populat radio channel in Taiwan, <a href="http://radio.newdaai.tv/aod/index_in.php">DaAi Radio</a>.<br />
<br><br />
<br> <br />
One of the staff was at our synthetic biology fair. She was impressed by our idea and project, and hence arranged an interview with us. In the interview , we mainly discussed about synthetic biology, our university achievements and also our current project. Our advisor, Eric Yang also shared his personal iGEM experience with all the audience.</font> <br />
</div>><br> <br />
<br><br />
</html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:25:44Z<p>98712149: </p>
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<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
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</p><br />
</body><br />
<br />
<font color="#228B22" size=6><b>O</b></font><font size=3>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us. We have done education on synthetic biology via one of the most populat radio channel in Taiwan, <a href="http://radio.newdaai.tv/aod/index_in.php">DaAi Radio</a>.</font><br />
<br><br />
<br> <br />
One of the staff was at our synthetic biology fair. She was impressed by our idea and project, and hence arranged an interview with us. In the interview , we mainly discussed about synthetic biology, our university achievements and also our current project. Our advisor, Eric Yang also shared his personal iGEM experience with all the audience. <br />
<br> <br />
<br><br />
</html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:24:37Z<p>98712149: </p>
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<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
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<br />
<style type="text/css"><br />
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</object><br />
</div><br />
</p><br />
</body><br />
<br />
<font color="#228B22" size=6><b>O</b></font>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us. We have done education on synthetic biology via one of the most populat radio channel in Taiwan, <a href="http://radio.newdaai.tv/aod/index_in.php">DaAi Radio</a>.<br />
<br><br />
<br> <br />
One of the staff was at our synthetic biology fair. She was impressed by our idea and project, and hence arranged an interview with us. In the interview , we mainly discussed about synthetic biology, our university achievements and also our current project. Our advisor, Eric Yang also shared his personal iGEM experience with all the audience. <br />
<br> <br />
<br><br />
</html></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:24:18Z<p>98712149: </p>
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<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
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</div><br />
</p><br />
</body><br />
</html><br />
<font color="#228B22" size=6><b>O</b></font>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us. We have done education on synthetic biology via one of the most populat radio channel in Taiwan, <a href="http://radio.newdaai.tv/aod/index_in.php">DaAi Radio</a>.<br />
<br><br />
<br> <br />
One of the staff was at our synthetic biology fair. She was impressed by our idea and project, and hence arranged an interview with us. In the interview , we mainly discussed about synthetic biology, our university achievements and also our current project. Our advisor, Eric Yang also shared his personal iGEM experience with all the audience. <br />
<br> <br />
<br></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:23:02Z<p>98712149: </p>
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<div id="wrap"><br />
<div id="headerimage"><br />
<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
</div><br />
<br />
<style type="text/css"><br />
#headerimage{width:975px;margin-top:-18px;}<br />
#headerimage img{max-width:975px;}<br />
#top-section{height:0px;margin:0 auto !important;}<br />
.firstHeading{width:0;height:0;display:none;position:relative;top:0;left:0;margin:0;}<br />
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</div><br />
</p><br />
</body><br />
</html><br />
<font color="#228B22" size=6><b>O</b></font>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us. We have done education on synthetic biology via one of the most populat radio channel in Taiwan, <a href="http://radio.newdaai.tv/aod/index_in.php">DaAi Radio</a>.<br />
<br><br />
<br> <br />
One of the staff was at our synthetic biology fair. She was impressed by our idea and project, and hence arranged an interview with us. In the interview , we mainly discussed about synthetic biology, our university achievements and also our current project. Our advisor, Eric Yang also shared his personal iGEM experience with all the audience. <br />
<br> <br />
<br></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:20:51Z<p>98712149: </p>
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<div id="wrap"><br />
<div id="headerimage"><br />
<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
</div><br />
<br />
<style type="text/css"><br />
#headerimage{width:975px;margin-top:-18px;}<br />
#headerimage img{max-width:975px;}<br />
#top-section{height:0px;margin:0 auto !important;}<br />
.firstHeading{width:0;height:0;display:none;position:relative;top:0;left:0;margin:0;}<br />
img.bg{min-height: 100%;min-width: 1024px;width: 100%;height: auto;position: fixed;top: 0;left: 0;z-index:-1;}<br />
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<font color="#228B22" size=6><b>O</b></font>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us. We have done education on synthetic biology via one of the most populat radio channel in Taiwan, <a STYLE="text-decoration:none" href_new="http://radio.newdaai.tv/aod/index_in.php">DaAi Radio</a>.<br />
<br><br />
<br> <br />
One of the staff was at our synthetic biology fair. She was impressed by our idea and project, and hence arranged an interview with us. In the interview , we mainly discussed about synthetic biology, our university achievements and also our current project. Our advisor, Eric Yang also shared his personal iGEM experience with all the audience. <br />
<br> <br />
<br></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:16:16Z<p>98712149: </p>
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<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
</div><br />
<br />
<style type="text/css"><br />
#headerimage{width:975px;margin-top:-18px;}<br />
#headerimage img{max-width:975px;}<br />
#top-section{height:0px;margin:0 auto !important;}<br />
.firstHeading{width:0;height:0;display:none;position:relative;top:0;left:0;margin:0;}<br />
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<font color="#228B22" size=6><b>O</b></font>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us. We have done education on synthetic biology via one of the most populat radio channel DaAi Radio.<br />
<br><br />
<br> <br />
One of the staff was at our synthetic biology fair. She was impressed by our idea and project, and hence arranged an interview with us. In the interview , we mainly discussed about synthetic biology, our university achievements and also our current project. Our advisor, Eric Yang also shared his personal iGEM experience with all the audience. <br />
<br> <br />
<br><br />
http://radio.newdaai.tv/aod/index_in.php</div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:15:47Z<p>98712149: </p>
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<font color="#228B22" size=6><b>O</b></font>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us. We have done education on synthetic biology via one of the most populat radio channel DaAi Radio.<br />
<br> <br />
One of the staff was at our synthetic biology fair. She was impressed by our idea and project, and hence arranged an interview with us. In the interview , we mainly discussed about synthetic biology, our university achievements and also our current project. Our advisor, Eric Yang also shared his personal iGEM experience with all the audience. <br />
<br> <br />
http://radio.newdaai.tv/aod/index_in.php</div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Humanpractice/mediaTeam:TzuChiU Formosa/Humanpractice/media2011-10-06T01:14:00Z<p>98712149: </p>
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<font size=6>O</font>n the journey of promoting synthetic biology, we're so lucky to have DaAi Radio for helping us. We have done education on synthetic biology via one of the most populat radio channel DaAi Radio.<br />
<br />
One of the staff was at our synthetic biology fair. She was impressed by our idea and project, and hence arranged an interview with us. In the interview , we mainly discussed about synthetic biology, our university achievements and also our current project. Our advisor, Eric Yang also shared his personal iGEM experience with all the audience. <br />
<br />
http://radio.newdaai.tv/aod/index_in.php</div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Notebook/photopaperTeam:TzuChiU Formosa/Notebook/photopaper2011-10-06T01:03:59Z<p>98712149: </p>
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<font color="#228B22" size=6>Photopaper</font><br />
<br><br><font color="#000080" size=4>Meeting Minutes</font><br />
<Hr Align="left" width="100%" size=2><br />
<font color="#000000" size=3><b>2011.02.24</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Team organization[[File:001.jpg|right|500px|caption]]<br />
*Brain storming<br />
**paper made by bacteria with add-ons such as colors, fragrance, etc.<br />
**"light up" the plants for replacing lamp posts.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.04</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Team advisory <br />
*Brain storming<br />
**Add-ons: colorful cellulose which produced by bacteria such as beta-carotene<br />
**information exchange with iGEM 2009 Cambridge team<br />
<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.14</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Task Allocation<br />
*Brain storming<br />
**Avatar - "light up" the plants by transfecting symbiotic bacteria which cloned into fluorescence gene<br />
**Eco-friendly warmer - biotic thermal pad<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.23</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
**Project : paperia <br />
**Option 1 : Culture bacteria which has pigment gene<br />
**Option 2 : Cellulose-producing bacteria secrete pigment into the medium<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.24</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Exp. procedure: <br />
** cloning of cellulose gene’s CDS<br />
** the product should operate within E. coli.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.06.22</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Due to some unforseen reason, the team decided to change their project.<br />
* New project: Biojenny <br />
-economical and humane way to produce paper in large quantities.<br><br />
-yeast to be our host<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.01</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Freeze > grin > genome DNA isolation > Cloning = silk protein gene<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.09</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* the connections between 3 silk proteins : Fibl Fibh P25<br />
* major proteins : H-chain, L-chain, P25<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.15</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Due to limited resources and techniques required, the team decided to switch back to the previous project. Paper making !<br />
* However it would be modified to be more innovative and creative.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.18</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Latest project : Photo paper<br />
* cyanobacteria is designed as the host, cellulose made up of the glucose produced by cyanobacteria could be one of the main attraction of the project.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.23</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* system modification to overcome the problems arises during preliminary round<br />
* Biobricks from Tokyo 2010 team will be utilized<br />
**regulator promoter in order to regulate the secretion of cellulose to solve the aggregation of the bacteria<br />
<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.09.15</b></font><br />
<br />
'''Genome miniprep'''<br><br />
Gluconacetobacter hansenii<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.09.18</b></font><br />
<br />
'''Gel/PCR DNA extraction'''<br><br />
Gluconacetobacter hansenii<br />
<br />
<br />
<br />
<font size=4 color="#000080"><b>Protocols</b></font><br />
<Hr Align="left" width="100%" size=3><br />
<br />
<br />
<br>[[File:1-20110907.jpg|lefe|650px|caption]][[File:Catt.gif|right|250px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:2-20110908-13.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:3-20110910-11.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:4-20110912-20.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:5-20110921.jpg|lefe|550px|caption]][[File:Cats.jpg|right|404px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:6-20110922.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:7-20110923.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:8-20110924.jpg|lefe|550px|caption]][[File:Cats2.gif |right|404px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:9-20110925.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:10-20110926.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:11-20110927.jpg|lefe|550px|caption]][[File:Cats3.jpg|right|404px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:12-20110928.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:13-20110929.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.10.01</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>modelling test</b></font><br />
<br><br />
<br><br />
<br></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Notebook/photopaperTeam:TzuChiU Formosa/Notebook/photopaper2011-10-06T01:02:10Z<p>98712149: </p>
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<div id="wrap"><br />
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<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
</div><br />
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<style type="text/css"><br />
#headerimage{width:975px;margin-top:-18px;}<br />
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.firstHeading{width:0;height:0;display:none;position:relative;top:0;left:0;margin:0;}<br />
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<font color="#228B22" size=6>Photopaper</font><br />
<br><br><font color="#000080" size=4>Meeting Minutes</font><br />
<Hr Align="left" width="100%" size=2><br />
<font color="#000000" size=3><b>2011.02.24</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Team organization[[File:001.jpg|right|500px|caption]]<br />
*Brain storming<br />
**paper made by bacteria with add-ons such as colors, fragrance, etc.<br />
**"light up" the plants for replacing lamp posts.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.04</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Team advisory <br />
*Brain storming<br />
**Add-ons: colorful cellulose which produced by bacteria such as beta-carotene<br />
**information exchange with iGEM 2009 Cambridge team<br />
<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.14</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Task Allocation<br />
*Brain storming<br />
**Avatar - "light up" the plants by transfecting symbiotic bacteria which cloned into fluorescence gene<br />
**Eco-friendly warmer - biotic thermal pad<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.23</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
**Project : paperia <br />
**Option 1 : Culture bacteria which has pigment gene<br />
**Option 2 : Cellulose-producing bacteria secrete pigment into the medium<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.24</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Exp. procedure: <br />
** cloning of cellulose gene’s CDS<br />
** the product should operate within E. coli.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.06.22</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Due to some unforseen reason, the team decided to change their project.<br />
* New project: Biojenny <br />
-economical and humane way to produce paper in large quantities.<br><br />
-yeast to be our host<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.01</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Freeze > grin > genome DNA isolation > Cloning = silk protein gene<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.09</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* the connections between 3 silk proteins : Fibl Fibh P25<br />
* major proteins : H-chain, L-chain, P25<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.15</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Due to limited resources and techniques required, the team decided to switch back to the previous project. Paper making !<br />
* However it would be modified to be more innovative and creative.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.18</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Latest project : Photo paper<br />
* cyanobacteria is designed as the host, cellulose made up of the glucose produced by cyanobacteria could be one of the main attraction of the project.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.23</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* system modification to overcome the problems arises during preliminary round<br />
* Biobricks from Tokyo 2010 team will be utilized<br />
**regulator promoter in order to regulate the secretion of cellulose to solve the aggregation of the bacteria<br />
<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.09.15</b></font><br />
<br />
'''Genome miniprep'''<br><br />
Gluconacetobacter hansenii<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.09.18</b></font><br />
<br />
'''Gel/PCR DNA extraction'''<br><br />
Gluconacetobacter hansenii<br />
<br />
<br />
<br />
<font size=4 color="#000080"><b>Protocols</b></font><br />
<Hr Align="left" width="100%" size=3><br />
<br />
<br />
<br>[[File:1-20110907.jpg|lefe|650px|caption]][[File:Catt.gif|right|250px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:2-20110908-13.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:3-20110910-11.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:4-20110912-20.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:5-20110921.jpg|lefe|550px|caption]][[File:Cats.jpg|right|404px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:6-20110922.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:7-20110923.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:8-20110924.jpg|lefe|550px|caption]][[File:Cats2.gif |right|404px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:9-20110925.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:10-20110926.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:11-20110927.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:12-20110928.jpg|lefe|550px|caption]][[File:Cats3.jpg|right|404px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:13-20110929.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.10.01</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>modelling test</b></font><br />
<br><br />
<br><br />
<br></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Notebook/photopaperTeam:TzuChiU Formosa/Notebook/photopaper2011-10-06T01:01:35Z<p>98712149: </p>
<hr />
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<br />
<img src="https://static.igem.org/mediawiki/2011/a/a3/BACK.jpg" class="bg" height="100%" width="100%"><br />
<div id="wrap"><br />
<div id="headerimage"><br />
<img src ="https://static.igem.org/mediawiki/2011/4/47/2011banner.gif" height="150" width="960"><br />
</div><br />
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<style type="text/css"><br />
#headerimage{width:975px;margin-top:-18px;}<br />
#headerimage img{max-width:975px;}<br />
#top-section{height:0px;margin:0 auto !important;}<br />
.firstHeading{width:0;height:0;display:none;position:relative;top:0;left:0;margin:0;}<br />
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<br />
</html><br />
<font color="#228B22" size=6>Photopaper</font><br />
<br><br><font color="#000080" size=4>Meeting Minutes</font><br />
<Hr Align="left" width="100%" size=2><br />
<font color="#000000" size=3><b>2011.02.24</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Team organization[[File:001.jpg|right|500px|caption]]<br />
*Brain storming<br />
**paper made by bacteria with add-ons such as colors, fragrance, etc.<br />
**"light up" the plants for replacing lamp posts.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.04</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Team advisory <br />
*Brain storming<br />
**Add-ons: colorful cellulose which produced by bacteria such as beta-carotene<br />
**information exchange with iGEM 2009 Cambridge team<br />
<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.14</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Task Allocation<br />
*Brain storming<br />
**Avatar - "light up" the plants by transfecting symbiotic bacteria which cloned into fluorescence gene<br />
**Eco-friendly warmer - biotic thermal pad<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.23</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
**Project : paperia <br />
**Option 1 : Culture bacteria which has pigment gene<br />
**Option 2 : Cellulose-producing bacteria secrete pigment into the medium<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.24</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Exp. procedure: <br />
** cloning of cellulose gene’s CDS<br />
** the product should operate within E. coli.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.06.22</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Due to some unforseen reason, the team decided to change their project.<br />
* New project: Biojenny <br />
-economical and humane way to produce paper in large quantities.<br><br />
-yeast to be our host<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.01</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Freeze > grin > genome DNA isolation > Cloning = silk protein gene<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.09</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* the connections between 3 silk proteins : Fibl Fibh P25<br />
* major proteins : H-chain, L-chain, P25<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.15</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Due to limited resources and techniques required, the team decided to switch back to the previous project. Paper making !<br />
* However it would be modified to be more innovative and creative.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.18</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Latest project : Photo paper<br />
* cyanobacteria is designed as the host, cellulose made up of the glucose produced by cyanobacteria could be one of the main attraction of the project.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.23</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* system modification to overcome the problems arises during preliminary round<br />
* Biobricks from Tokyo 2010 team will be utilized<br />
**regulator promoter in order to regulate the secretion of cellulose to solve the aggregation of the bacteria<br />
<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.09.15</b></font><br />
<br />
'''Genome miniprep'''<br><br />
Gluconacetobacter hansenii<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.09.18</b></font><br />
<br />
'''Gel/PCR DNA extraction'''<br><br />
Gluconacetobacter hansenii<br />
<br />
<br />
<br />
<font size=4 color="#000080"><b>Protocols</b></font><br />
<Hr Align="left" width="100%" size=3><br />
<br />
<br />
<br>[[File:1-20110907.jpg|lefe|650px|caption]][[File:Catt.gif|right|250px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:2-20110908-13.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:3-20110910-11.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:4-20110912-20.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:5-20110921.jpg|lefe|550px|caption]][[File:Cats.jpg|right|404px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:6-20110922.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:7-20110923.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:8-20110924.jpg|lefe|550px|caption]][[File:Cats2.gif |right|404px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:9-20110925.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:10-20110926.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:11-20110927.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:12-20110928.jpg|lefe|650px|caption]][[File:Cats3.jpg|right|404px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:13-20110929.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.10.01</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>modelling test</b></font><br />
<br><br />
<br><br />
<br></div>98712149http://2011.igem.org/Team:TzuChiU_Formosa/Notebook/photopaperTeam:TzuChiU Formosa/Notebook/photopaper2011-10-06T00:56:36Z<p>98712149: </p>
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<font color="#228B22" size=6>Photopaper</font><br />
<br><br><font color="#000080" size=4>Meeting Minutes</font><br />
<Hr Align="left" width="100%" size=2><br />
<font color="#000000" size=3><b>2011.02.24</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Team organization[[File:001.jpg|right|500px|caption]]<br />
*Brain storming<br />
**paper made by bacteria with add-ons such as colors, fragrance, etc.<br />
**"light up" the plants for replacing lamp posts.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.04</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Team advisory <br />
*Brain storming<br />
**Add-ons: colorful cellulose which produced by bacteria such as beta-carotene<br />
**information exchange with iGEM 2009 Cambridge team<br />
<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.14</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
*Task Allocation<br />
*Brain storming<br />
**Avatar - "light up" the plants by transfecting symbiotic bacteria which cloned into fluorescence gene<br />
**Eco-friendly warmer - biotic thermal pad<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.23</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
**Project : paperia <br />
**Option 1 : Culture bacteria which has pigment gene<br />
**Option 2 : Cellulose-producing bacteria secrete pigment into the medium<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.03.24</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Exp. procedure: <br />
** cloning of cellulose gene’s CDS<br />
** the product should operate within E. coli.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.06.22</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Due to some unforseen reason, the team decided to change their project.<br />
* New project: Biojenny <br />
-economical and humane way to produce paper in large quantities.<br><br />
-yeast to be our host<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.01</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Freeze > grin > genome DNA isolation > Cloning = silk protein gene<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.09</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* the connections between 3 silk proteins : Fibl Fibh P25<br />
* major proteins : H-chain, L-chain, P25<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.15</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Due to limited resources and techniques required, the team decided to switch back to the previous project. Paper making !<br />
* However it would be modified to be more innovative and creative.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.18</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* Latest project : Photo paper<br />
* cyanobacteria is designed as the host, cellulose made up of the glucose produced by cyanobacteria could be one of the main attraction of the project.<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.07.23</b></font><br />
<br><font size=2><b>Discussion:</b></font><br />
<br />
* system modification to overcome the problems arises during preliminary round<br />
* Biobricks from Tokyo 2010 team will be utilized<br />
**regulator promoter in order to regulate the secretion of cellulose to solve the aggregation of the bacteria<br />
<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.09.15</b></font><br />
<br />
'''Genome miniprep'''<br><br />
Gluconacetobacter hansenii<br />
<br />
<br />
<br />
<br><font color="#000000" size=3><b>2011.09.18</b></font><br />
<br />
'''Gel/PCR DNA extraction'''<br><br />
Gluconacetobacter hansenii<br />
<br />
<br />
<br />
<font size=4 color="#000080"><b>Protocols</b></font><br />
<Hr Align="left" width="100%" size=3><br />
<br />
<br />
<br>[[File:1-20110907.jpg|lefe|650px|caption]][[File:Catt.gif|right|250px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:2-20110908-13.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:3-20110910-11.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:4-20110912-20.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:5-20110921.jpg|lefe|550px|caption]][[File:Cats.jpg|right|404px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:6-20110922.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:7-20110923.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:8-20110924.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:9-20110925.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:10-20110926.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:11-20110927.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:12-20110928.jpg|lefe|650px|caption]]<br />
<br><br />
<br><br />
<br><br />
<br>[[File:13-20110929.jpg|lefe|650px|caption]]<br />
<br />
<br><font color="#000000" size=3><b>2011.09.08-13</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Plasmid miniprep kit</b></font><br />
*PSB1C3 plasmid[[File:Cats.jpg|right|404px|caption]]<br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>Raise Rhodobacter rubrum</b></font><br />
:: 1.50ml LB+500μl CHLORAMPHENICOL<br />
:: 2.37℃, overnight (14-16hrs)<br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.09</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Raise Gluconacetobacter hansenii</b></font><br />
:: 1.50ml LB+500μl CHLORAMPHENICOL<br />
:: 2.37℃, overnight (14-16hrs)<br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.10-11</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Digestion check of DNA</b></font><br />
<br><br />
[pSB1C3/EcoRI]<br />
<br>DNA 500ng<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>EcoRⅠ 1μl<br><br />
<br>ddH2O 29μl<br><br />
<br>_______________________________<br />
<br><br />
<br>total 50μl<br><br />
<br><br />
<br><br />
[pSB1C3/PstⅠ]<br />
<br>DNA 500ng<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>PstⅠ 1μl<br><br />
<br>ddH2O 29μl<br> <br />
<br>_________________________________<br />
<br><br />
<br>total 50μl<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>Digestion of DNA</b></font><br />
<br><br />
[pSB1C3/EcoRⅠ+PstⅠ]<br />
<br>DNA 500ng<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>EcoRⅠ 1μl<br><br />
<br>pstⅠ 1μl<br><br />
<br>ddH2O 28μl<br> <br />
<br>__________________________________<br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 30 mins<br />
<br><br />
<br><br />
[pSB1C3/XbaⅠ+SpeⅠ]<br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>XbaⅠ 1μl<br><br />
<br>SpeⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>____________________________________<br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 2 hrs<br />
<br><br />
<br><br />
[pSB1C3/SpeⅠ+PstⅠ]<br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>SpeⅠ 1μl<br><br />
<br>pstⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>____________________________________<br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 2 hrs<br />
<br><br />
<br><br />
[pSB1C3/EcoRⅠ+XbaⅠ]<br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>EcoRⅠ 1μl<br><br />
<br>XbaⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>____________________________________<br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 2 hrs<br />
<br><br />
<br><br />
[pSB1A3/EcoRⅠ+PstⅠ]<br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>EcoRⅠ 1μl<br><br />
<br>PstⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>____________________________________<br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 2 hrs<br />
<br><br />
<br><br />
[pSB1A3/EcoRⅠ+SpeⅠ]<br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>EcoRⅠ 1μl<br><br />
<br>SpeⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>____________________________________<br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 2 hrs<br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>electroelution Purification</b></font><br />
<br><br />
*[pSB1C3/EcoRⅠ+PstⅠ]<br />
*[pSB1C3/XbaⅠ+SpeⅠ]<br />
*[pSB1C3/SpeⅠ+PstⅠ]<br />
*[pSB1C3/EcoRⅠ+XbaⅠ]<br />
*[pSB1A3/EcoRⅠ+PstⅠ]<br />
*[pSB1A3/EcoRⅠ+SpeⅠ]<br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.12-20</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>PCR</b></font><br />
[[File:Cats2.gif |right|404px|caption]]<br />
<br><br />
*[acsAB] <br />
*[acsCD] <br />
*[CCcax-Ccp] <br />
<br>template DNA 1μl<br><br />
<br>5×Buffer 4μl<br><br />
<br>2.5μM dNTP 1.6μl<br><br />
<br>10μM F 1μl<br><br />
<br>10μM R 1μl<br><br />
<br>Taq 0.2μl<br><br />
<br>ddH2O 8.8μl<br><br />
<br>_______________________________<br />
<br><br />
<br>total 20μl<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>electroelution Purification</b></font><br />
<br><br />
*[acsAB]<br />
*[acsCD]<br />
*[CMCax-Ccp] <br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.21</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Digestion of DNA</b></font><br />
<br><br />
[acsAB/ XbaⅠ+SpeⅠ]<br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>EcoRⅠ 1μl<br><br />
<br>pstⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>____________________________<br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 16 hr<br />
<br><br />
<br><br />
[acsCD/XbaⅠ+SpeⅠ]<br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>XbaⅠ 1μl<br><br />
<br>SpeⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>__________________________________<br />
<br><br />
total 50μl<br><br />
<br>→37℃ for 16 hr<br />
<br><br />
<br><br />
[CMCax/SpeⅠ+AlwNⅠ]<br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>SpeⅠ 1μl<br><br />
<br>AlwNⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>__________________________________<br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 16 hr<br />
<br><br />
<br><br />
[Ccp/AlwNⅠ+PstⅠ]<br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>AlwNⅠ 1μl<br><br />
<br>PstⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>__________________________________<br />
<br><br />
total 50μl<br><br />
<br>→37℃ for 16 hr<br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>electroelution Purification</b></font><br />
<br><br />
*[acsAB/XbaⅠ+SpeⅠ]<br />
*[acsCD/XbaⅠ+SpeⅠ]<br />
*[CMCax/SpeⅠ+AlwNⅠ]<br />
*[Ccp/AlwNⅠ+PstⅠ]<br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.22</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Ligation of DNA</b></font><br />
<br><br />
[pSB1C3-acsAB][[File:Cats3.jpg|right|404px|caption]]<br />
<br>Vector 3μl<br><br />
<br>Insert 14μl<br><br />
<br>ligase buffer 2μl<br><br />
<br>ligase 1μl<br><br />
<br>ddH2O -μl<br><br />
<br>________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br>→16℃ for 16 hr <br />
<br><br />
<br><br />
[pSB1A3-acsCD]<br />
<br>Vector 3μl<br><br />
<br>Insert 14μl<br><br />
<br>ligase buffer 2μl<br><br />
<br>ligase 1μl<br><br />
<br>ddH2O -μl<br><br />
<br>_________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br>→16℃ for 16 hr<br />
<br><br />
<br><br />
[pSB1C3-acsCD]<br />
<br>Vector 3μl<br><br />
<br>Insert 14μl<br><br />
<br>ligase buffer 2μl<br><br />
<br>ligase 1μl<br><br />
<br>ddH2O -μl<br><br />
<br>_________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br>→16℃ for 16 hr<br />
<br><br />
<br><br />
[pSB1C3-CMCax-Ccp]<br />
<br>Vector 3μl<br><br />
<br>Insert 14μl<br><br />
<br>ligase buffer 2μl<br><br />
<br>ligase 1μl<br><br />
<br>ddH2O -μl<br><br />
<br>_________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br>→16℃ for 16 hr<br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.23</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>PCR</b></font><br />
<br><br />
<br><br />
[R0011 promoter]<br />
<br>R0011 promoter 1μl<br><br />
<br>5×Buffer 4μl<br><br />
<br>2.5μM dNTP 1.6μl<br><br />
<br>Taq 0.2μl<br><br />
<br>ddH2O 13.2μl<br><br />
<br>_____________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>electroelution Purification</b></font><br />
<br><br />
*[R0011 promoter]<br />
<br><font color="#000000" size=2><b>Transformation of DNA</b></font><br />
<br><br />
*[pSB1C3-acsAB]<br />
:: Transform into E.coli<br />
:: LB+CHLORAMPHENICOL<br />
:: →37℃ for 14 hr <br />
<br><br />
<br><br />
*[pSB1C3-acsCD]<br />
:: Transform into E.coli<br />
:: LB+CHLORAMPHENICOL<br />
:: →37℃ for 14 hr <br />
<br><br />
<br><br />
*[pSB1A3-acsCD]<br />
:: Transform into E.coli<br />
:: LB+Ampicillin<br />
:: →37℃ for 14 hr <br />
<br><br />
<br><br />
*[pSB1C3-CMCax-Ccp]<br />
:: Transform into E.coli<br />
:: LB+CHLORAMPHENICOL<br />
:: →37℃ for 14 hr <br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>Digestion of DNA</b></font><br />
<br><br />
<br><br />
[R0011 promoter/EcoRⅠ+XbaⅠ] <br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>EcoRⅠ 1μl<br><br />
<br>XbaⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>____________________________ <br />
<br>total 50μl<br><br />
<br>→37℃ for 16 hr<br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>electroelution Purification</b></font><br />
<br><br />
*[R0011 promoter/EcoRⅠ+XbaⅠ] <br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.24</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Plasmid miniprep kit</b></font><br />
<br><br />
*[pSB1C3-acsAB]<br />
*[pSB1C3-acsCD]<br />
*[pSB1A3-acsCD]<br />
*[pSB1C3-CMCax-Ccp]<br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>Ligation of DNA</b></font><br />
<br><br />
[pSB1C3-R0011]<br />
<br>Vector 3μl<br><br />
<br>Insert 14μl<br><br />
<br>ligase buffer 2μl<br><br />
<br>ligase 1μl<br><br />
<br>ddH2O -μl<br><br />
<br>________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br>→16℃ for 16 hr <br />
<br><br />
<br><br />
[R0011-acsAB]<br />
<br>Vector 3μl<br><br />
<br>Insert 14μl<br><br />
<br>ligase buffer 2μl<br><br />
<br>ligase 1μl<br><br />
<br>ddH2O -μl<br><br />
<br>________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br>→16℃ for 16 hr <br />
<br><br />
<br><br />
[R0011-acsCD]<br />
<br>Vector 3μl<br><br />
<br>Insert 14μl<br><br />
<br>ligase buffer 2μl<br><br />
<br>ligase 1μl<br><br />
<br>ddH2O -μl<br><br />
<br>________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br>→16℃ for 16 hr <br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.24</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Digestion of DNA</b></font><br />
<br><br />
[R0011-acsAB/EcoRⅠ+SpeⅠ] <br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>EcoRⅠ 1μl<br><br />
<br>SpeⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>____________________________ <br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 16 hr<br />
<br><br />
<br><br />
[R0011-acsCD/EcoRⅠ+SpeⅠ] <br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>EcoRⅠ 1μl<br><br />
<br>SpeⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>____________________________ <br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 16 hr<br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>electroelution Purification</b></font><br />
<br><br />
*[R0011-acsAB/EcoRⅠ+SpeⅠ]<br />
<br><br />
*[R0011-acsCD/EcoRⅠ+SpeⅠ] <br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.25</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Ligation of DNA</b></font><br />
<br><br />
[pSB1C3-R0011-acsAB]<br />
<br>Vector 3μl<br><br />
<br>Insert 14μl<br><br />
<br>ligase buffer 2μl<br><br />
<br>ligase 1μl<br><br />
<br>ddH2O -μl<br><br />
<br>________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br>→16℃ for 16 hr <br />
<br><br />
<br><br />
[pSB1C3-R0011-acsCD]<br />
<br>Vector 3μl<br><br />
<br>Insert 14μl<br><br />
<br>ligase buffer 2μl<br><br />
<br>ligase 1μl<br><br />
<br>ddH2O -μl<br><br />
<br>________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br>→16℃ for 16 hr <br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.26</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Transformation of DNA</b></font><br />
<br><br />
*[pSB1C3-R0011-acsAB]<br />
:: Transform into E.coli<br />
:: LB+CHLORAMPHENICOL<br />
:: →37℃ for 14 hr <br />
<br><br />
<br><br />
*[pSB1C3-R0011-acsCD]<br />
:: Transform into E.coli<br />
:: LB+CHLORAMPHENICOL<br />
:: →37℃ for 14 hr <br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>Digestion of DNA</b></font><br />
<br><br />
[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ] <br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>SpeⅠ 1μl<br><br />
<br>PstⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>____________________________ <br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 16 hr<br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>electroelution Purification</b></font><br />
*[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ] <br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.27</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Plasmid miniprep kit</b></font><br />
<br><br />
*[pSB1C3-R0011-acsAB] <br />
*[pSB1C3-R0011-acsCD]<br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>Ligation of DNA</b></font><br />
<br><br />
[pSB1C3-R0011-acsCD-CMCax-Ccp]<br />
<br>Vector 3μl<br><br />
<br>Insert 14μl<br><br />
<br>ligase buffer 2μl<br><br />
<br>ligase 1μl<br><br />
<br>ddH2O -μl<br><br />
<br>________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br>→16℃ for 16 hr <br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>Digestion of DNA</b></font><br />
<br><br />
<br><br />
[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ] <br />
<br>DNA 10μl<br><br />
<br>10×buffer 5μl<br><br />
<br>BSA 5μl<br><br />
<br>PstⅠ 1μl<br><br />
<br>EcoRⅠ 1μl<br><br />
<br>ddH2O 28μl<br><br />
<br>____________________________ <br />
<br><br />
<br>total 50μl<br><br />
<br>→37℃ for 16 hr<br />
<br><br />
<br><br />
<br><font color="#000000" size=2><b>electroelution Purification</b></font><br />
*[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ] <br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.28</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Ligation of DNA</b></font><br />
<br><br />
<br><br />
[pSB1A3-R0011-acsCD-CMCax-Ccp]<br />
<br>Vector 3μl<br><br />
<br>Insert 14μl<br><br />
<br>ligase buffer 2μl<br><br />
<br>ligase 1μl<br><br />
<br>ddH2O -μl<br><br />
<br>________________________________<br />
<br><br />
<br>total 20μl<br><br />
<br>→16℃ for 16 hr <br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.09.29</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>Transformation of DNA</b></font><br />
<br><br />
*[pSB1C3-R0011-acsAB]<br />
*[pSB1A3-R0011-acsAB-CMCax-Ccp]<br />
:: Transform into E.coli<br />
:: LB+Ampiclin+CHLORAMPHENICOL<br />
:: →37℃ for 14 hr <br />
<br><br />
<br><br />
<br><br />
<br><font color="#000000" size=3><b>2011.10.01</b></font><br />
<br><br />
<br><font color="#000000" size=2><b>modelling test</b></font><br />
<br><br />
<br><br />
<br></div>98712149http://2011.igem.org/File:13-20110929.jpgFile:13-20110929.jpg2011-10-06T00:56:19Z<p>98712149: </p>
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<div></div>98712149