http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Sumi2010.igem.org - User contributions [en]2024-03-29T04:53:47ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T09:23:04Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
<br />
== Synthesis system ==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
====Verification of the Part BBa_k419012====<br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
====Growth Curve of Synthesis System E. coli====<br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
==== Beta-carotene Productivity of Synthesis System E. coli====<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
====Estimation of the bacterial numbers of synthesis system E. coli====<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
====The productivity of beta-carotene produced by the synthesis system E. coli====<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
==Sensing system==<br />
===pSB1C3-nhaA-Lux I (The Part BBa_K419020)===<br />
====Verification of the Part BBa_K419020====<br />
The DNA fragment of nhaA promoter was amplified from the part BBa_K116002 by PCR method. The nhaA PCR products (274 bp) were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-nhaA plasmid successfully. Furthermore, the Lux I gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0161. The Lux I PCR products (585 bp) were digested with Xba I and Pst I, then ligated with the Spe I-Pst I treated pSB1C3-nhaAvector. After E. coli transformation and plasmid preparation, the part BBa_K419020 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the nhaA-Lux I DNA fragment (859 bp). The sensing system composite was transformed into E. coli for further experiments.<br><br />
[[Image:Synthesis10.jpg|300px]]<br />
<br />
===pSB1C3-nhaA-Lux R (The Part BBa_K419021)===<br />
The DNA fragment of nhaA promoter was amplified from the part BBa_K116002 by PCR method. The nhaA PCR products (274 bp) were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-nhaA plasmid successfully. Furthermore, the Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with Xba I and Pst I, then ligated with the Spe I-Pst I treated pSB1C3-nhaAvector. After E. coli transformation and plasmid preparation, the part BBa_K419021 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the nhaA-Lux R DNA fragment (1030 bp). The sensing system composite was transformed into E. coli for further experiments.<br><br />
<br />
[[Image:Synthesis9.jpg|300px]]<br />
<br />
=== pSB1C3-Lux R( The Part BBa_K419015 )===<br />
====Verification of the Part BBa_K419015====<br />
The Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with EcoR I and Pst I, then ligated with the EcoR I-Pst I treated pSB1C3. After E. coli transformation and plasmid preparation, the part BBa_K419015 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the Lux R DNA fragment. The sensing system composite was transformed into E. coli for further experiments.<br />
<br />
[[Image:Synthesis8.jpg|300px]]<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/File:Synthesis10.jpgFile:Synthesis10.jpg2010-12-04T09:22:37Z<p>Sumi: </p>
<hr />
<div></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T09:13:37Z<p>Sumi: /* pSB1C3-nhaA-Lux R (The Part BBa_K419021) */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
<br />
== Synthesis system ==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
====Verification of the Part BBa_k419012====<br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
====Growth Curve of Synthesis System E. coli====<br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
==== Beta-carotene Productivity of Synthesis System E. coli====<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
====Estimation of the bacterial numbers of synthesis system E. coli====<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
====The productivity of beta-carotene produced by the synthesis system E. coli====<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
==Sensing system==<br />
=== pSB1C3-Lux R( The Part BBa_K419015 )===<br />
====Verification of the Part BBa_K419015====<br />
The Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with EcoR I and Pst I, then ligated with the EcoR I-Pst I treated pSB1C3. After E. coli transformation and plasmid preparation, the part BBa_K419015 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the Lux R DNA fragment. The sensing system composite was transformed into E. coli for further experiments.<br />
<br />
[[Image:Synthesis8.jpg|300px]]<br />
<br />
===pSB1C3-nhaA-Lux R (The Part BBa_K419021)===<br />
The DNA fragment of nhaA promoter was amplified from the part BBa_K116002 by PCR method. The nhaA PCR products (274 bp) were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-nhaA plasmid successfully. Furthermore, the Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with Xba I and Pst I, then ligated with the Spe I-Pst I treated pSB1C3-nhaAvector. After E. coli transformation and plasmid preparation, the part BBa_K419021 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the nhaA-Lux R DNA fragment (1030 bp). The sensing system composite was transformed into E. coli for further experiments.<br><br />
<br />
[[Image:Synthesis9.jpg|300px]]<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T09:13:20Z<p>Sumi: /* pSB1C3-nhaA-Lux R (The Part BBa_K419021) */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
<br />
== Synthesis system ==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
====Verification of the Part BBa_k419012====<br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
====Growth Curve of Synthesis System E. coli====<br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
==== Beta-carotene Productivity of Synthesis System E. coli====<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
====Estimation of the bacterial numbers of synthesis system E. coli====<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
====The productivity of beta-carotene produced by the synthesis system E. coli====<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
==Sensing system==<br />
=== pSB1C3-Lux R( The Part BBa_K419015 )===<br />
====Verification of the Part BBa_K419015====<br />
The Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with EcoR I and Pst I, then ligated with the EcoR I-Pst I treated pSB1C3. After E. coli transformation and plasmid preparation, the part BBa_K419015 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the Lux R DNA fragment. The sensing system composite was transformed into E. coli for further experiments.<br />
<br />
[[Image:Synthesis8.jpg|300px]]<br />
<br />
===pSB1C3-nhaA-Lux R (The Part BBa_K419021)===<br />
The DNA fragment of nhaA promoter was amplified from the part BBa_K116002 by PCR method. The nhaA PCR products (274 bp) were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-nhaA plasmid successfully. Furthermore, the Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with Xba I and Pst I, then ligated with the Spe I-Pst I treated pSB1C3-nhaAvector. After E. coli transformation and plasmid preparation, the part BBa_K419021 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the nhaA-Lux R DNA fragment (1030 bp). The sensing system composite was transformed into E. coli for further experiments.<br />
[[Image:Synthesis9.jpg|300px]]<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T09:13:03Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
<br />
== Synthesis system ==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
====Verification of the Part BBa_k419012====<br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
====Growth Curve of Synthesis System E. coli====<br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
==== Beta-carotene Productivity of Synthesis System E. coli====<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
====Estimation of the bacterial numbers of synthesis system E. coli====<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
====The productivity of beta-carotene produced by the synthesis system E. coli====<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
==Sensing system==<br />
=== pSB1C3-Lux R( The Part BBa_K419015 )===<br />
====Verification of the Part BBa_K419015====<br />
The Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with EcoR I and Pst I, then ligated with the EcoR I-Pst I treated pSB1C3. After E. coli transformation and plasmid preparation, the part BBa_K419015 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the Lux R DNA fragment. The sensing system composite was transformed into E. coli for further experiments.<br />
<br />
[[Image:Synthesis8.jpg|300px]]<br />
<br />
===pSB1C3-nhaA-Lux R (The Part BBa_K419021)===<br />
The DNA fragment of nhaA promoter was amplified from the part BBa_K116002 by PCR method. The nhaA PCR products (274 bp) were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-nhaA plasmid successfully. Furthermore, the Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with Xba I and Pst I, then ligated with the Spe I-Pst I treated pSB1C3-nhaAvector. After E. coli transformation and plasmid preparation, the part BBa_K419021 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the nhaA-Lux R DNA fragment (1030 bp). The sensing system composite was transformed into E. coli for further experiments.<br />
[[Image:Synthesis9.jpg|500px]]<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/File:Synthesis9.jpgFile:Synthesis9.jpg2010-12-04T09:12:27Z<p>Sumi: </p>
<hr />
<div></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T09:05:30Z<p>Sumi: /* Verification of the Part BBa_K419015 */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
<br />
== Synthesis system ==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
====Verification of the Part BBa_k419012====<br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
====Growth Curve of Synthesis System E. coli====<br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
==== Beta-carotene Productivity of Synthesis System E. coli====<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
====Estimation of the bacterial numbers of synthesis system E. coli====<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
====The productivity of beta-carotene produced by the synthesis system E. coli====<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
==Sensing system==<br />
=== pSB1C3-Lux R( The Part BBa_K419015 )===<br />
====Verification of the Part BBa_K419015====<br />
The Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with EcoR I and Pst I, then ligated with the EcoR I-Pst I treated pSB1C3. After E. coli transformation and plasmid preparation, the part BBa_K419015 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the Lux R DNA fragment. The sensing system composite was transformed into E. coli for further experiments.<br />
<br />
[[Image:Synthesis8.jpg|300px]]<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T09:05:11Z<p>Sumi: /* Verification of the Part BBa_K419015 */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
<br />
== Synthesis system ==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
====Verification of the Part BBa_k419012====<br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
====Growth Curve of Synthesis System E. coli====<br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
==== Beta-carotene Productivity of Synthesis System E. coli====<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
====Estimation of the bacterial numbers of synthesis system E. coli====<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
====The productivity of beta-carotene produced by the synthesis system E. coli====<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
==Sensing system==<br />
=== pSB1C3-Lux R( The Part BBa_K419015 )===<br />
====Verification of the Part BBa_K419015====<br />
The Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with EcoR I and Pst I, then ligated with the EcoR I-Pst I treated pSB1C3. After E. coli transformation and plasmid preparation, the part BBa_K419015 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the Lux R DNA fragment. The sensing system composite was transformed into E. coli for further experiments.<br />
<br />
[[Image:Synthesis8.jpg|500px]]<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/File:Synthesis8.jpgFile:Synthesis8.jpg2010-12-04T09:04:40Z<p>Sumi: </p>
<hr />
<div></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T09:01:12Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
<br />
== Synthesis system ==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
====Verification of the Part BBa_k419012====<br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
====Growth Curve of Synthesis System E. coli====<br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
==== Beta-carotene Productivity of Synthesis System E. coli====<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
====Estimation of the bacterial numbers of synthesis system E. coli====<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
====The productivity of beta-carotene produced by the synthesis system E. coli====<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
==Sensing system==<br />
=== pSB1C3-Lux R( The Part BBa_K419015 )===<br />
====Verification of the Part BBa_K419015====<br />
The Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with EcoR I and Pst I, then ligated with the EcoR I-Pst I treated pSB1C3. After E. coli transformation and plasmid preparation, the part BBa_K419015 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the Lux R DNA fragment. The sensing system composite was transformed into E. coli for further experiments.<br />
<br />
<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:59:49Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==<br />
<br />
== Synthesis system ==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
====Verification of the Part BBa_k419012====<br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
====Growth Curve of Synthesis System E. coli====<br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
==== Beta-carotene Productivity of Synthesis System E. coli====<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
====Estimation of the bacterial numbers of synthesis system E. coli====<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
====The productivity of beta-carotene produced by the synthesis system E. coli====<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
==Sensing system==<br />
=== pSB1C3-Lux R( The Part BBa_K419015 )===<br />
====Verification of the Part BBa_K419015====<br />
The Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with EcoR I and Pst I, then ligated with the EcoR I-Pst I treated pSB1C3. After E. coli transformation and plasmid preparation, the part BBa_K419015 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the Lux R DNA fragment. The sensing system composite was transformed into E. coli for further experiments.<br />
<br />
<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:59:28Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==<br />
<br />
== Synthesis system < "師" div style="FONT-SIZE: 160%">==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
====Verification of the Part BBa_k419012====<br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
====Growth Curve of Synthesis System E. coli====<br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
==== Beta-carotene Productivity of Synthesis System E. coli====<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
====Estimation of the bacterial numbers of synthesis system E. coli====<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
====The productivity of beta-carotene produced by the synthesis system E. coli====<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
==Sensing system==<br />
=== pSB1C3-Lux R( The Part BBa_K419015 )===<br />
====Verification of the Part BBa_K419015====<br />
The Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with EcoR I and Pst I, then ligated with the EcoR I-Pst I treated pSB1C3. After E. coli transformation and plasmid preparation, the part BBa_K419015 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the Lux R DNA fragment. The sensing system composite was transformed into E. coli for further experiments.<br />
<br />
<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:55:09Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
====Verification of the Part BBa_k419012====<br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
====Growth Curve of Synthesis System E. coli====<br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
==== Beta-carotene Productivity of Synthesis System E. coli====<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
====Estimation of the bacterial numbers of synthesis system E. coli====<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
====The productivity of beta-carotene produced by the synthesis system E. coli====<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
==Sensing system==<br />
=== pSB1C3-Lux R( The Part BBa_K419015 )===<br />
====Verification of the Part BBa_K419015====<br />
The Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with EcoR I and Pst I, then ligated with the EcoR I-Pst I treated pSB1C3. After E. coli transformation and plasmid preparation, the part BBa_K419015 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the Lux R DNA fragment. The sensing system composite was transformed into E. coli for further experiments.<br />
<br />
<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:53:11Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
===The productivity of beta-carotene produced by the synthesis system E. coli===<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
==Sensing system==<br />
=== pSB1C3-Lux R( The Part BBa_K419015 )===<br />
====Verification of the Part BBa_K419015====<br />
The Lux R gene with ribosome binding site (RBS) DNA was amplified from the part BBa_C0062. The Lux R PCR products (756 bp) were digested with EcoR I and Pst I, then ligated with the EcoR I-Pst I treated pSB1C3. After E. coli transformation and plasmid preparation, the part BBa_K419015 for sensing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the Lux R DNA fragment. The sensing system composite was transformed into E. coli for further experiments.<br />
<br />
<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:48:55Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
===The productivity of beta-carotene produced by the synthesis system E. coli===<br />
Based on the standard concentration curve of beta-carotene as mention above, we calculate the productivity of beta-carotene produced by our synthesis system E. coli. <br><br />
<br><br />
y= 0.1865x + 0.0014 <br><br />
y: absorbance at 450 nm, x: concentration of beta-carotene (μg)<br><br />
<br><br />
For example, we choose two time point to measure the productivity of beta-carotene<br><br />
At 6 h culture, y= 0.204 x1 = 1.086 (μg)<br><br />
At 8 h culture, y= 0.248 x2 = 1.322 (μg)<br><br />
<br><br />
At 6 h culture, total bacterial numbers = 7x108<br><br />
Productivity of beta-carotene per cell<br><br />
1.086 / 7 x108 = 1.551 x10-9<br><br />
At 8 h culture, total bacterial numbers = 1.018x109 <br><br />
Productivity of beta-carotene per cell<br><br />
1.322 / 1.018x109 = 1.299 x10-9<br><br />
<br><br />
Average productivity per cell: <br><br />
(1.551 x10-9 + 1.299 x10-9) / 2 = 1.425 x10-9 (μg) [0.001425 pg]<br><br />
<br><br />
The aliquot of 8 h culture (1 ml) was used to determine the concentration of beta-carotene. The maximum productivity of beta-carotene is estimated approximately 1.322 mg/L.<br><br />
<br />
<br />
<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:43:29Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|900px]]<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:43:02Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|700px]]<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:42:44Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|500px]]<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:42:19Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|180]]<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:41:45Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br><br />
The level of beta-carotene was calculated by the concentration standard curve of beta-carotene. The commercial available beta-carotene was purchased and established the concentration curve of beta-carotene as shown in the following Figure.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis7.jpg|500]]<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/File:Synthesis7.jpgFile:Synthesis7.jpg2010-12-04T08:41:18Z<p>Sumi: </p>
<hr />
<div></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:35:58Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]<br />
<br />
<br />
<br />
<br />
==Releasing system==<br />
=== LuxPR-lysis device(The Part BBa_k419003)===<br />
Verification of the Part BBa_k419003<br><br />
The DNA fragment of LuxR responsive promoter, LuxPR, was amplified from the part BBa_R0062 by PCR method. The LuxPR PCR products were purified and digested with EcoRI and PstI, then ligated with EcoRI-PstI cut pSB1C3, the iGEM 2010 standard backbone. We got the pSB1C3-LuxPR plasmid successfully. In addition, the lysis device DNA which contained holing and lysozyme genes was amplified from the part BBa_K112021. The lysis device PCR products were digested with Xba I and Pst I and ligated with the Spe I-Pst I treated pSB1C3-LuxPR vector. After transformation and plasmid preparation, the part BBa_k419003 for releasing system in our Neutrient Synthesizer was verified by EcoRI and PstI enzyme digestion in 1% agarose gel electrophoresis. The arrow (red) indicates the lysis device DNA fragment. The releasing system composite was transformed into DH5 E. coli for further experiment.<br><br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===pLuxPR-Lysis device( Demonstration of Releasing System )===<br />
A single colony of releasing system E. coli was inoculated in the LB/chloramphenicol medium at 37℃. After 8 h of culture, we found that the LuxPR was auto-activated and lysis device was expressed causing the lysis of E. coli host. In our observation, we found that the autolysis of the host bacterial was almost completed after 8 hours. The result indicated that other factors may be involved in the activation of the luxR responsive promoter.<br><br />
[[Image:Synthesis6.jpg|500px]]</div>Sumihttp://2010.igem.org/File:Synthesis6.jpgFile:Synthesis6.jpg2010-12-04T08:35:01Z<p>Sumi: </p>
<hr />
<div></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:27:53Z<p>Sumi: /* constitutive promoter-CrtEBIY (The Part BBa_k419012 ) */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis5.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]</div>Sumihttp://2010.igem.org/File:Synthesis5.jpgFile:Synthesis5.jpg2010-12-04T08:27:29Z<p>Sumi: </p>
<hr />
<div></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:21:51Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5α), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T08:16:56Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
★<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 μg/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]</div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T07:52:30Z<p>Sumi: /* Estimation of the bacterial numbers of synthesis system E. coli */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br><br />
<br />
[[Image:Synthesis4.jpg|500px]]</div>Sumihttp://2010.igem.org/File:Synthesis4.jpgFile:Synthesis4.jpg2010-12-04T07:52:08Z<p>Sumi: </p>
<hr />
<div></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T07:50:13Z<p>Sumi: /* Beta-carotene Productivity of Synthesis System E. coli */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
[[Image:Synthesis3.jpg|500px]]<br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br></div>Sumihttp://2010.igem.org/File:Synthesis3.jpgFile:Synthesis3.jpg2010-12-04T07:49:51Z<p>Sumi: </p>
<hr />
<div></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T07:46:06Z<p>Sumi: /* constitutive promoter-CrtEBIY (Demonstration of Synthesis System) */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
[[Image:Synthesis2 1.jpg|500px]]<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br></div>Sumihttp://2010.igem.org/File:Synthesis2_1.jpgFile:Synthesis2 1.jpg2010-12-04T07:45:42Z<p>Sumi: </p>
<hr />
<div></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T07:42:09Z<p>Sumi: /* constitutive promoter-CrtEBIY (The Part BBa_k419012 ) */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis1.jpg|500px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T07:41:57Z<p>Sumi: /* constitutive promoter-CrtEBIY (The Part BBa_k419012 ) */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis1.jpg|300px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T07:41:46Z<p>Sumi: /* constitutive promoter-CrtEBIY (The Part BBa_k419012 ) */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis1.jpg|180px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br></div>Sumihttp://2010.igem.org/File:Synthesis1.jpgFile:Synthesis1.jpg2010-12-04T07:41:25Z<p>Sumi: </p>
<hr />
<div></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T07:34:06Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis2.jpg|100px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T07:33:51Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis2.jpg|180px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T07:33:19Z<p>Sumi: /* constitutive promoter-CrtEBIY (The Part BBa_k419012 ) */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis2.jpg|250px]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T07:32:49Z<p>Sumi: /* constitutive promoter-CrtEBIY (The Part BBa_k419012 ) */</p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
[[Image:Synthesis2.jpg]]<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br></div>Sumihttp://2010.igem.org/File:Synthesis2.jpgFile:Synthesis2.jpg2010-12-04T07:32:10Z<p>Sumi: </p>
<hr />
<div></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ModelingTeam:TzuChiU Formosa/Modeling2010-12-04T07:28:44Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
[[Image:MODO1.jpg|180px]]<br />
[[Image:MODO2.jpg|180px]]<br />
[[Image:MODO3.jpg|180px]]<br />
[[Image:MODO4.jpg|180px]]<br />
<br />
==Synthesis system==<br />
===constitutive promoter-CrtEBIY (The Part BBa_k419012 )===<br />
Verification of the Part BBa_k419012 <br><br />
We use constitutive promoter to drive the CrtEBIY composite for beta-carotene synthesis. The CrtEBIY composite (BBa_K274210) was obtained from the distribution part of iGEM 2009 Cambrige and digested with restriction enzymes, EcoR I and Pst I, then ligated to EcoR I and Pst I sites of pSB1C3, the iGEM 2010 standard backbone. We verified our composite for synthesis system by EcoRI and PstI digestion and the agarose gel electrophoresis result was shown. The arrow (red) indicates the CrtEBIY with constitutive promoter (R001) DNA fragment. The synthesis system composite was transformed into DH5 E. coli for further experiment.<br><br />
<br />
<br />
===constitutive promoter-CrtEBIY (Demonstration of Synthesis System)=== <br />
Growth Curve of Synthesis System E. coli <br><br />
To verify the beta-carotene production by our synthesis system E. coli (DH5), which contains the part BBa_k419012 (beta-carotene producing genes CrtEBIY with constitutive promoter) in the iGEM 2010 standard backbone pSB1C3, the bacterial culture of our synthesis system was performed and the growth curve was determined in the experiment.<br />
Single colony of our synthesis system or control (without beta-carotene genes) bacteria was inoculated into LB medium containing 34 g/ml chloramphenical , then incubated at 37℃ overnight. The next day, the absorbance of the overnight cultures was measured at 600 nm and 0.1 OD of diluted bacterial culture (1 ml) was inoculated into 40 ml fresh LB/chloramphenical broth, then grow the culture at 37℃ incubator. We aliquot the bacterial culture (1 ml) every 2 hours and measured the bacterial growth by spectrophotometer at 600nm. Finally, we determine and draw the bacterial growth curve of our synthesis system E. coli. The Figure shows the growth curve of our synthesis system or control E. coli. The result shows that both control (green curve) and synthesis system (synthesizer, orange curve) E. coli reached to plateau at 8 h and grown equally well. We prove that the growth of synthesis system (containing part BBa_k274200) is well in the same growth condition compared with control.<br><br />
<br />
<br />
=== Beta-carotene Productivity of Synthesis System E. coli===<br />
The level of beta-carotene production was measured every two hours. The beta-carotene produced by the synthesis system E. coli (1 ml) was extracted by acetone, then the absorbance was determined at 450nm by spectrophotometer. Figure shows the absorbance of beta-carotene. The result shows that that the level of the beta-carotene in our synthesis system contained part BBa_K419012 (synthesizer, orange curve) was elevated at 4 hour and reached a maximum level at 8 h. No beta-carotene was produced in the control E. coli (green curve). <br><br />
<br />
===Estimation of the bacterial numbers of synthesis system E. coli===<br />
Figure shows that the calculation of the total number of the synthesis system E. coli. The bacterial cultures were series diluted and plated on LB agar contained cploramphenical. As shown in figure, we count the number of colony at 106x and 107x dilution of 6h bacterial culture. We calculate the average bacterial numbers at 6 h culture is 7x108.<br></div>Sumihttp://2010.igem.org/AcknowledgementAcknowledgement2010-12-04T07:16:05Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
[[Image:TCUA.png|500px]]<br />
[[Image:TCUA1.png|500px]]<br />
[[Image:TCUA2.png|500px]]<br />
<br />
<br />
<br />
<br />
<font size="5">== Contribution ==</font><br />
<br><br />
<br><br />
<font size="3"><br />
Experiments: Guan Nan Peng, I Huang , Shih-Shan Huang, Tung Chin Yang, Wei Hsuan Sun , Yi Ru Ho, Ying Yu Huang<br><br />
<br><br />
Modeling: Tung Chin Yang, Wei Hsuan Sun<br><br />
<br><br />
Safety: I Huang<br><br />
<br><br />
Human Practice: I Huang, Tung Chin Yang<br><br />
<br><br />
Wiki: Chang Chau Chieh<br><br />
<br><br />
Poster and Slides: Wei Hsuan Sun , Yi Ru Ho, Ying Yu Huang<br><br />
<br><br />
Advisor: Ni Chun Chung, Nathan Hew<br><br />
<br><br />
Instructor: JH Chen, Jui Hung Yen, Peng Yeong Woon, Ingrid Y.-C. Liu<br />
<br><br />
Collabaration<br><br />
This iGEM 2010, as the Tzu Chi_U Formosa iGEM team, we understand that the creativity of experiment design were crazy and outstanding. We have to go farer and higher to not limit our project, but there are much more people outside the world. One of the criteria for GOLD is the collaborations with other universities participating in iGEM competition. For this year we did collaboration with multiple teams which to improve the project and increased the member’s background and skills.<br />
<br />
<br />
<br />
We have responded Matt Coombes the University of Edinburgh team conducted a survey about perception of iGEM better. For the competition, improved whole project is very important for the later iGEM. As the survey we think that the most decisive question is what will affect you in the future. We sure the iGEM affect us a lot by the process of the competition. No matter how the outcome of experiment are, fail or success, the whole spirit of iGEM is how we learn. <br />
<br />
<br />
METU_TURKEY_SOFTWARE team did a great effort on develop new standards for Parts Registry and user-friendly Part Registry Forms and Parts Entry pages. I’m sure that with the hard working by the METU_TURKEY_SOFTWARE team’s survey the iGEM Part Registry Forms and Parts Entry pages will be friendlier than ever, that why we participated in this survey.<br />
<br />
<br />
We have been working very closely with NYMU_Taipei since Year 2009. We had several conference calls and technical supports with the team last year. We have celebration dinner together last year after the jamboree and we will do the same this year. In addition to what we have done last year, both teams organized the iGEM workshop Asia at Tzu Chi University. We have very strong bond and connection with NYMU_Taipei officially and personally. We share, we help and we support each others. This is the spirit of iGEMers.<br />
<br />
</font></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/HumanPracticeTeam:TzuChiU Formosa/HumanPractice2010-12-04T07:15:39Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=utf-8" /><br />
<title>無標題文件</title><br />
</head><br />
<br />
<body><br />
<table width="960" border="0"><br />
<tr><br />
<td bgcolor="#ff9900"><h2>Overview</h2></td><br />
</tr><br />
<tr><br />
<td height="50" valign="top"><blockquote><br />
<h3>Over the past decade, synthetic biology has emerged as a new star within the fields of life science and engineering which brings biology, chemistry, genetics as well as engineering even closer to form a promising field of scientific research.<br />
<br />
Conventional biologist sees biology as how a nature (organism) works in macro, and what supports a nature (organism) in micro. While synthetic biologist intended to build and introduce a man-made system into living organisms to fulfill their needs. To achieve their goal, synthetic biologists require extra tools such as mathematics and statistics to strengthen their success-very often during the process. They have to introduce new “'''parts'''” as “'''systems'''” into the living organism.<br />
<br />
Engineers see biology as technology. Engineers and biologists have been cooperating long in biotechnology field to design computer or machine. Machine helps biologist to achieve a better accuracy in measurement, further reaching the boundaries in medicinal field, providing finer resolution images for researchers and etc. In synthetic biology, engineers helped to design “circuit board” and “chipsets” in biological systems, to produce energy (biofuel), food (GMO products), drug (insulin), ''de novo'' DNA synthesis and etc. And there is more emerging applications in this field, and some of them come from our iGEMers.<br />
<br />
Despite synthetic biology all began with the idea to bring benefits for human in agriculture, industry, science and medicine, some conservatives are shouting for a stop to those who are “playing God”. Some are afraid that genetic modification or alteration will give rise to harmful impact in a long run. And thus, safety, security and ethic issues are very important among synthetic biologist when it comes to HUMAN PRACTICE.<br />
</h3><br />
</blockquote></td><br />
</tr><br />
<br />
<tr><br />
<td bgcolor="#ff9900"><h2>Our project</h2></td><br />
</tr><br />
<tr><br />
<td height="50" valign="top"><blockquote><br />
<h3>Our current project, “'''Nutrient Synthesizer'''” aimed to resolve the problem of land unpredictble werather and food supply shortage in underdeveloped countries. The importance of this project could be foreseen in the near future because the weather has become more unpredictable (water shortage, extreme heat or cold and etc.) and overwhelming population. “Nutrient Synthesizer” is created to solve nutrients deficiency problem. Thus, we hope that Nutrient Synthesizer can synthesize nutrients and intake by human body rapidly. <br />
<br />
Before proceeding to human practice, we have the responsibility to verify the safety problem which may cause by microorganisms or the created synthetic life form. We hereby introduce a “Vector” or “Carrier system” which can be easily manipulated and cultured. In our current research, we are using ''E.Coli'' as our nutrient production machine. However, in the future before proceeding to human practice, we plan to introduce the same parts into a vector system which is not harmful and which is able to coexist with human. For instance, lactobacillus in human intestine is a good carrier system in our project.<br />
</h3><br />
</blockquote></td><br />
</tr><br />
<br />
<br />
<tr><br />
<td height="31" valign="top" bgcolor="#ff9900"><h2>Education</h2></td><br />
</tr><br />
<tr><br />
<td height="50" valign="top"><blockquote><br />
<h3>Since synthetic biology will be the main stream of science and everyday knowledge, it will be very important for us to start educating the next generation of people about the pros and cons about synthetic biology. Teaching them how to judge wisely like what the biologist did for the past decades on animal studies are very crucial. <br />
</h3><br />
</blockquote></td><br />
</tr><br />
<br />
<br />
</table><br />
</body><br />
</html></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/SafetyTeam:TzuChiU Formosa/Safety2010-12-04T07:14:45Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=utf-8" /><br />
<title>無標題文件</title><br />
</head><br />
<br />
<body><br />
<blockquote><br />
<h2>Safety Q&amp;A</h2><br />
<h3>For iGEM 2010 we are asked to detail how we approached any issues of biological safety </h3><br />
<h3>associated with their projects (<a href="https://2010.igem.org/Team:TzuChiU_Formosa/Nutrient_synthesizer">see here</a>). Here are the questions and our answers:</h3><br />
</blockquote><br />
<table width="960" border="0"><br />
<tr><br />
<td height="26" valign="top" bgcolor="#FFDDAC"><blockquote><br />
<h2><strong>Q1. Would any of your project ideas raise safety issues in terms of: <br /><br />
researcher safety, public safety, or: environmental safety? </strong></h2><br />
</blockquote></td><br />
</tr><br />
<tr><br />
<td height="169" valign="top"><blockquote><br />
<h3>We only used <em>E. coli</em> DH5α in all our experiments when required. DH5α is not pathogenic to humans nor animals and will not survive in the environment. The inserted sequences encode only plant enzymes which is necessary to made β-carotene and are highly unlikely to confer any harmful phenotype on the disabled <em>E.coli</em> host nor animals. Standard good lab practices were followed strictly in handling all bacterial samples, including wearing protective groves, working in lamina flow, autoclaving contaminated waste, bleaching discarded liquid cultures, and washing hands before and after experiments.</h3><br />
<p>&nbsp;</p><br />
</blockquote></td><br />
</tr><br />
<tr><br />
<td bgcolor="#FFDDAD"><blockquote><br />
<h2><strong>Q2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, <br /><br />
did you document these issues in the Registry? <br /><br />
how did you manage to handle the safety issue? <br /><br />
how could other teams learn from your experience?</strong></h2><br />
</blockquote></td><br />
</tr><br />
<tr><br />
<td height="50" valign="top"><blockquote><br />
<h3>No.</h3><br />
</blockquote></td><br />
</tr><br />
<tr><br />
<td height="28" valign="top" bgcolor="#FFDDAD"><blockquote><br />
<h2><strong>Q3. Is there a local biosafety group, committee, or review board at your institution? <br /><br />
If yes, what does your local biosafety group think about your project? <br /><br />
If no, which specific biosafety rules or guidelines do you have to consider in your country?</strong></h2><br />
</blockquote></td><br />
</tr><br />
<tr><br />
<td height="156" valign="top"><blockquote><br />
<h3>Yes. All bacterial or plasmid used have to obtain an approval from University's environmental and biosafety committees. Environmental and biosafety officers made regular random visit to each laboratory to ensure all laboratories are comply with environmental and biosafety regulations. So far our project has received positive responds from the committees.</h3><br />
</blockquote></td><br />
</tr><br />
<tr><br />
<td height="33" valign="top" bgcolor="#FFDDAD"><blockquote><br />
<h2><strong>Q4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering? </strong></h2><br />
</blockquote></td><br />
</tr><br />
<tr><br />
<td height="104" valign="top"><blockquote><br />
<h3>Perhaps parts, devices or systems can be divided into two categories such as prokaryotes and eukaryotes and regulate under standard environmental and biosafety regulations.</h3><br />
</blockquote></td><br />
</tr><br />
</table><br />
</body><br />
</html></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/PartsTeam:TzuChiU Formosa/Parts2010-12-04T07:14:23Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br><br />
<br />
===Parts===<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>iGEM010 TzuChiU_Formosa</groupparts></div>Sumihttp://2010.igem.org/Team:TzuChiU_FormosaTeam:TzuChiU Formosa2010-12-04T07:14:06Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="600" width="960" data="https://static.igem.org/mediawiki/2010/8/84/Homepage.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/8/84/Homepage.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/TeamTeam:TzuChiU Formosa/Team2010-12-04T07:13:26Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="800" width="960" data="https://static.igem.org/mediawiki/2010/2/29/Teamfinal2.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/2/29/Teamfinal2.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/ProjectTeam:TzuChiU Formosa/Project2010-12-04T07:13:00Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<br />
<br />
==Brainstorming==<br />
<html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="700" width="960" data="https://static.igem.org/mediawiki/2010/2/2e/Brainstorming_fINAL.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/2/2e/Brainstorming_fINAL.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html></div>Sumihttp://2010.igem.org/Team:TzuChiU_Formosa/PoseidonTeam:TzuChiU Formosa/Poseidon2010-12-04T07:12:41Z<p>Sumi: </p>
<hr />
<div><html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="319.6" width="960" data="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/c/c4/Barnew.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="900" width="960" data="https://static.igem.org/mediawiki/2010/6/6c/Project_Poseidon1.swf" align="left" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/6/6c/Project_Poseidon1.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html><br />
<br />
<html><br />
<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="700" width="960" data="https://static.igem.org/mediawiki/2010/9/98/Poseidon_SAVE_THE_WORLD.swf" style="float: left"><br />
<param name="movie" value="https://static.igem.org/mediawiki/2010/9/98/Poseidon_SAVE_THE_WORLD.swf"/><br />
<param name="quality" value="low" /><br />
</object><br />
</p><br />
</body><br />
</html></div>Sumi