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<div align="left" style="font-family: 'spr';font-size:40px;border-bottom:2px solid #584b4f;">June</div> | <div align="left" style="font-family: 'spr';font-size:40px;border-bottom:2px solid #584b4f;">June</div> | ||
</br> | </br> | ||
− | <div style="text-align:center; font-size: | + | <div style="text-align:center; font-size:30px; font-weight:bold;">6.30</div> |
Transformation</br> | Transformation</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 285: | Line 285: | ||
</br> | </br> | ||
− | 7.1 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">7.1</div> |
Liquid Culture</br> | Liquid Culture</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 331: | Line 331: | ||
</table> | </table> | ||
</br> | </br> | ||
− | 7.2</br> | + | <div style="text-align:center; font-size:30px; font-weight:bold;">7.2</div></br> |
miniprep</br> | miniprep</br> | ||
Participant: Shisheng Li</br> | Participant: Shisheng Li</br> | ||
Line 448: | Line 448: | ||
<div align="center"><img src="https://static.igem.org/mediawiki/2016/9/9a/NB_0702.jpeg" width="50%"></div> | <div align="center"><img src="https://static.igem.org/mediawiki/2016/9/9a/NB_0702.jpeg" width="50%"></div> | ||
− | 7.5 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">7.5</div> |
Construct of double oscillation plasmid Ptd103aiiA-luxS</br> | Construct of double oscillation plasmid Ptd103aiiA-luxS</br> | ||
Participant:Ran Jinshi, Zhu Hongya</br> | Participant:Ran Jinshi, Zhu Hongya</br> | ||
Line 459: | Line 459: | ||
• We screened the positive colony with colony PCR. After conformed, we inoculated it in 5ml LB and cultured it overnight to harvest the pTD103aiiA-luxS.</br></br> | • We screened the positive colony with colony PCR. After conformed, we inoculated it in 5ml LB and cultured it overnight to harvest the pTD103aiiA-luxS.</br></br> | ||
− | 7.8 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">7.8</div> |
Transformation</br> | Transformation</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 505: | Line 505: | ||
<tr> | <tr> | ||
</table> | </table> | ||
− | 7.9 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">7.9</div> |
Liquid Culture</br> | Liquid Culture</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 550: | Line 550: | ||
</table> | </table> | ||
− | 7.10 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">7.10</div> |
Miniprep</br> | Miniprep</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 668: | Line 668: | ||
The result of gel electrophoresis showed that we managed to get the pcr product of Cph8 and PompC but failed in getting double terminator. After we changed the annealing temperature, we finally made it. When sorting out data, we accidently deleted the picture of the gel.</br></br> | The result of gel electrophoresis showed that we managed to get the pcr product of Cph8 and PompC but failed in getting double terminator. After we changed the annealing temperature, we finally made it. When sorting out data, we accidently deleted the picture of the gel.</br></br> | ||
− | 6.15 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">6.15</div> |
Participant: Wang Zhanyu, Wan Han</br> | Participant: Wang Zhanyu, Wan Han</br> | ||
1.Get all the basic parts from Kit plate</br> | 1.Get all the basic parts from Kit plate</br> | ||
Line 717: | Line 717: | ||
2.Done the transformation</br> | 2.Done the transformation</br> | ||
− | 7.16</ | + | <div style="text-align:center; font-size:30px; font-weight:bold;">7.16</div></br> |
1.Measuring and modeling of promoter pluxR </br> | 1.Measuring and modeling of promoter pluxR </br> | ||
Participant:Ran Jinshi, Zhu Hongya</br> | Participant:Ran Jinshi, Zhu Hongya</br> | ||
Line 779: | Line 779: | ||
</table> | </table> | ||
− | 7.26</ | + | <div style="text-align:center; font-size:30px; font-weight:bold;">7.26</div></br> |
Find the T7ptag mutation site and design primers for point mutation. </br> | Find the T7ptag mutation site and design primers for point mutation. </br> | ||
Participant:Wang Zhanyu, Wan Han</br></br> | Participant:Wang Zhanyu, Wan Han</br></br> | ||
− | 7.27</ | + | <div style="text-align:center; font-size:30px; font-weight:bold;">7.27</div></br> |
Point mutation pcr </br> | Point mutation pcr </br> | ||
Participant:Wang Zhanyu, Wan Han</br></br> | Participant:Wang Zhanyu, Wan Han</br></br> | ||
Line 803: | Line 803: | ||
<div align="left" style="font-family: 'spr';font-size:40px;border-bottom:2px solid #584b4f;">August</div> | <div align="left" style="font-family: 'spr';font-size:40px;border-bottom:2px solid #584b4f;">August</div> | ||
</br> | </br> | ||
− | 8.2 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.2</div> |
Confirm other parts need in logic gate.</br> | Confirm other parts need in logic gate.</br> | ||
Participant:Wang Zhanyu, Wan Han</br> | Participant:Wang Zhanyu, Wan Han</br> | ||
Line 849: | Line 849: | ||
</table> | </table> | ||
</br> | </br> | ||
− | 8.3 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.3</div> |
pick colonies and shake </br> | pick colonies and shake </br> | ||
Participant:Participant:Wang Zhanyu, Wan Han</br> | Participant:Participant:Wang Zhanyu, Wan Han</br> | ||
Line 903: | Line 903: | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | 8.4</br> | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.4</div></br> |
Participant: Wang Zhanyu, Wan Han</br> | Participant: Wang Zhanyu, Wan Han</br> | ||
1. Extract the plasmid</br> | 1. Extract the plasmid</br> | ||
Line 976: | Line 976: | ||
3.Conduct electrophoresis in 1% agarose gel</br> | 3.Conduct electrophoresis in 1% agarose gel</br> | ||
After sequencing, the other parts are all confirmed.</br></br> | After sequencing, the other parts are all confirmed.</br></br> | ||
− | 8.5</br> | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.5</div></br> |
Doing overlap PCR, construct the logic gate plasmid.</br> | Doing overlap PCR, construct the logic gate plasmid.</br> | ||
Participant: Wang Zhanyu, Wan Han</br></br> | Participant: Wang Zhanyu, Wan Han</br></br> | ||
− | 8.7-8.9</br> | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.7-8.9</div></br> |
Finish the construction of logic gate’s input plasmid(~3500bp,marked with an arrow)</br> | Finish the construction of logic gate’s input plasmid(~3500bp,marked with an arrow)</br> | ||
Participant: Wang Zhanyu, Wan Han</br></br> | Participant: Wang Zhanyu, Wan Han</br></br> | ||
<div align="center"> | <div align="center"> | ||
<img src="https://static.igem.org/mediawiki/2016/9/97/NB_0807.jpeg" width="50%"></div></br> | <img src="https://static.igem.org/mediawiki/2016/9/97/NB_0807.jpeg" width="50%"></div></br> | ||
− | 8.9-8.11</br> | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.9-8.11</div></br> |
Finish the construction of logic gate’s output plasmid(~1100bp,marked with arrows) </br> | Finish the construction of logic gate’s output plasmid(~1100bp,marked with arrows) </br> | ||
Participant: Wang Zhanyu, Wan Han</br></br> | Participant: Wang Zhanyu, Wan Han</br></br> | ||
<div align="center"> | <div align="center"> | ||
<img src="https://static.igem.org/mediawiki/2016/9/9a/NB_0809.jpeg" width="50%"></div></br> | <img src="https://static.igem.org/mediawiki/2016/9/9a/NB_0809.jpeg" width="50%"></div></br> | ||
− | 8.11-8.14</br> | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.11-8.14</div></br> |
Using infusion cloning to integrate the input plasmid and output plasmid together. (~4500bp,marked with an arrow) </br> | Using infusion cloning to integrate the input plasmid and output plasmid together. (~4500bp,marked with an arrow) </br> | ||
Participant: Wang Zhanyu, Wan Han</br></br> | Participant: Wang Zhanyu, Wan Han</br></br> | ||
Line 1,004: | Line 1,004: | ||
For details, see our protocol in the experiment part. | For details, see our protocol in the experiment part. | ||
− | 8.20</br> | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.20</div></br> |
Co-transformation</br> | Co-transformation</br> | ||
Participant: Li Shisheng</br></br> | Participant: Li Shisheng</br></br> | ||
Line 1,042: | Line 1,042: | ||
</table> | </table> | ||
</br> | </br> | ||
− | 8.21 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.21</div> |
Liquid Culture</br> | Liquid Culture</br> | ||
Participant:Participant:Wang Zhanyu, Wan Han</br> | Participant:Participant:Wang Zhanyu, Wan Han</br> | ||
Line 1,138: | Line 1,138: | ||
</table> | </table> | ||
</br> | </br> | ||
− | 8.22 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.22</div> |
Fluorescence intensity measuring</br> | Fluorescence intensity measuring</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 1,284: | Line 1,284: | ||
</br> | </br> | ||
− | 8.23 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.23</div> |
Verifying the single oscillation circuit in microfluidics device</br> | Verifying the single oscillation circuit in microfluidics device</br> | ||
Participant:Ran Jinshi, Zhu Hongya</br> | Participant:Ran Jinshi, Zhu Hongya</br> | ||
Line 1,295: | Line 1,295: | ||
</br> | </br> | ||
− | 8.25 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.25</div> |
Liquid Culture</br> | Liquid Culture</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 1,392: | Line 1,392: | ||
</br> | </br> | ||
− | 8.26 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.26</div> |
Fluorescence intensity measuring</br> | Fluorescence intensity measuring</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 1,572: | Line 1,572: | ||
</br> | </br> | ||
− | 8.27</br> | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.27</div></br> |
PCR</br> | PCR</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 1,645: | Line 1,645: | ||
<div align="center"> | <div align="center"> | ||
<img src="https://static.igem.org/mediawiki/2016/c/c6/NB_0827.jpeg" width="50%"></div></br> | <img src="https://static.igem.org/mediawiki/2016/c/c6/NB_0827.jpeg" width="50%"></div></br> | ||
− | 8.28 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.28</div> |
Validate the logic gate ‘s response time. For details, see our protocol in the experiment part.</br> | Validate the logic gate ‘s response time. For details, see our protocol in the experiment part.</br> | ||
Participant:Wang Zhanyu, Wan Han</br></br> | Participant:Wang Zhanyu, Wan Han</br></br> | ||
− | 8.30 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">8.30</div> |
Verifying the double oscillation circuit</br> | Verifying the double oscillation circuit</br> | ||
Participant:Ran Jinshi, Zhu Hongya</br> | Participant:Ran Jinshi, Zhu Hongya</br> | ||
Line 1,661: | Line 1,661: | ||
<div align="left" style="font-family: 'spr';font-size:40px;border-bottom:2px solid #584b4f;">September</div> | <div align="left" style="font-family: 'spr';font-size:40px;border-bottom:2px solid #584b4f;">September</div> | ||
</br> | </br> | ||
− | 9.2 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">9.2</div> |
Transformation</br> | Transformation</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 1,734: | Line 1,734: | ||
<div align="center"> | <div align="center"> | ||
<img src="https://static.igem.org/mediawiki/2016/f/f1/NB_0902.jpeg" width="50%"></div></br> | <img src="https://static.igem.org/mediawiki/2016/f/f1/NB_0902.jpeg" width="50%"></div></br> | ||
− | 9.4 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">9.4</div> |
Transformation</br> | Transformation</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 1,807: | Line 1,807: | ||
<div align="center"> | <div align="center"> | ||
<img src="https://static.igem.org/mediawiki/2016/e/e5/NB_0904.jpeg" width="50%"></div></br> | <img src="https://static.igem.org/mediawiki/2016/e/e5/NB_0904.jpeg" width="50%"></div></br> | ||
− | 9.7 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">9.7</div> |
Transformation</br> | Transformation</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> | ||
Line 1,880: | Line 1,880: | ||
<div align="center"> | <div align="center"> | ||
<img src="https://static.igem.org/mediawiki/2016/d/d8/NB_0907.jpeg" width="50%"></div></br> | <img src="https://static.igem.org/mediawiki/2016/d/d8/NB_0907.jpeg" width="50%"></div></br> | ||
− | 9.9 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">9.9</div> |
Validate the plasmid combining logic gate and red light-induced system: PompC+Plac | Validate the plasmid combining logic gate and red light-induced system: PompC+Plac | ||
(3600bp) | (3600bp) | ||
Line 1,888: | Line 1,888: | ||
<img src="https://static.igem.org/mediawiki/2016/8/8c/NB_0909.png" width="50%"></div></br> | <img src="https://static.igem.org/mediawiki/2016/8/8c/NB_0909.png" width="50%"></div></br> | ||
− | 9.17 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">9.17</div> |
Validate the plasmid combining logic gate and green light-induced system: PcpcG2+Plac | Validate the plasmid combining logic gate and green light-induced system: PcpcG2+Plac | ||
(3700bp) | (3700bp) | ||
Line 1,901: | Line 1,901: | ||
<div align="left" style="font-family: 'spr';font-size:40px;border-bottom:2px solid #584b4f;">October</div> | <div align="left" style="font-family: 'spr';font-size:40px;border-bottom:2px solid #584b4f;">October</div> | ||
</br> | </br> | ||
− | 10.10 | + | <div style="text-align:center; font-size:30px; font-weight:bold;">10.10</div> |
PCR</br> | PCR</br> | ||
Participant: Li Shisheng</br> | Participant: Li Shisheng</br> |
Revision as of 09:40, 18 October 2016
June
6.30
Transformation
Participant: Li Shisheng
Transformation | ||||||
Gene of interest | Vector | Ice | 42°C | Ice | 37°C+LB | Plate | BBa_I15008 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |
---|---|---|---|---|---|---|
BBa_I15009 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |
July
7.1
Liquid Culture
Participant: Li Shisheng
Liquid Culture | ||
Name | Medium | Volume/ml | BBa_I15008(pSB1C3) colony_1 | LB | 10 |
---|---|---|
BBa_I15008(pSB1C3) colony_2 | LB | 10 |
BBa_I15008(pSB1C3) colony_3 | LB | 10 |
BBa_I15009(pSB1C3) colony_1 | LB | 10 |
BBa_I15009(pSB1C3) colony_2 | LB | 10 |
BBa_I15009(pSB1C3) colony_3 | LB | 10 |
7.2
miniprep
Participant: Shisheng Li
Miniprep | ||
Name | 260/280 | C ng/μl | BBa_I15008(pSB1C3) colony_1 | 1.90 | 34.6 |
---|---|---|
BBa_I15008(pSB1C3) colony_2 | 1.87 | 33.2 |
BBa_I15008(pSB1C3) colony_3 | 1.88 | 58.3 |
BBa_I15009(pSB1C3) colony_1 | 1.75 | 33.0 |
BBa_I15009(pSB1C3) colony_2 | 1.85 | 45.1 |
BBa_I15009(pSB1C3) colony_3 | 1.93 | 49.0 |
PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
---|---|---|---|---|---|
taq polymerase | 0.5 | Pre Denature | 94 | 180 | / |
tempalte | 0.5 | Denature | 95 | 30 | 30 |
10X Taq buffer | 5 | Annealing | 55 | 30 | 30 |
primer-forward(10μM) | 2 | Extension | 72 | 45/45 | 30 |
primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
dNTP(2.5mM) | 4 | storage | 4 | / | / |
ddH2O | 36 | / | / | / | / |
7.5
Construct of double oscillation plasmid Ptd103aiiA-luxS
Participant:Ran Jinshi, Zhu Hongya
• We got luxS gene from 15M, plate 6, distribution kit 2016.
• We amplified luxS with PCR and added sequences homo with pTD103aiiA to it.
• We amplified the backbone pTD103aiiA, and added sequence homo with luxS.
• Using one-step cloning, we assembled them together.
• We transformed the new constructed plasmid into DH5alpha and cultured it overnight.
• We screened the positive colony with colony PCR. After conformed, we inoculated it in 5ml LB and cultured it overnight to harvest the pTD103aiiA-luxS.
7.8
Transformation
Participant: Li Shisheng
Transformation | ||||||
Gene of interest | Vector | Ice | 42°C | Ice | 37°C+LB | Plate | cph8 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |
---|---|---|---|---|---|---|
double terminator | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |
PompC | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |
7.9
Liquid Culture
Participant: Li Shisheng
Liquid Culture | ||
Name | Medium | Volume/ml | Cph8 colony_1 | LB | 10 |
---|---|---|
Cph8 colony_2 | LB | 10 |
double terminator colony_1 | LB | 10 |
double terminator colony_2 | LB | 10 |
PompC colony_1 | LB | 10 |
PompC colony_2 | LB | 10 |
7.10
Miniprep
Participant: Li Shisheng
Miniprep | ||
Name | 260/280 | C ng/μl | Cph8 colony_1 | 1.90 | 53.3 |
---|---|---|
Cph8 colony_2 | 1.87 | 74.1 |
double terminator colony_1 | 1.84 | 44.2 |
double terminator colony_2 | 1.61 | 50.8 |
PompC colony_1 | 1.87 | 43.8 |
PompC colony_2 | 2.12 | 37.6 |
PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
---|---|---|---|---|---|
taq polymerase | 0.5 | Pre Denature | 94 | 180 | / |
tempalte | 0.5 | Denature | 95 | 30 | 30 |
10X Taq buffer | 5 | Annealing | 55 | 30 | 30 |
primer-forward(10μM) | 2 | Extension | 72 | 180/45/45 | 30 |
primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
dNTP(2.5mM) | 4 | storage | 4 | / | / |
ddH2O | 36 | / | / | / | / |
6.15
Participant: Wang Zhanyu, Wan Han
1.Get all the basic parts from Kit plate
supD-tRNA ,T7RNAP &PT7
Transformation | ||||||
Gene of interest | Vector | Ice | 42°C | Ice | 37°C+LB | Plate | BBa_K228001 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |
---|---|---|---|---|---|---|
BBa_K228000 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |
BBa_K1321338 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |
7.16
1.Measuring and modeling of promoter pluxR
Participant:Ran Jinshi, Zhu Hongya
For better description of single oscillation system, we measured the response intensity of pluxR to AHL. First, we mutated pTD103luxI-sfGFP with knocking gene luxI out with PCR. We named the new plasmid pTD103sfGFP.
We transformed MG1655 strain of E•coil with pTD103sfGFP, inoculated positive colony in 5ml LB with antibiotic 100μg/ml kanamycin and cultured it over night.
We inoculated over night culture with a 1:1000 dilution in 1ml LB and added AHL according a concentration range:10-6~10-11M when the bacteria solution reached an A600nm 0.1.
Cultured it in 37℃ for 8h. We spun it down and concentrated in PBS (pH=7.2), then measured the fluorescence intensity.
2.pick colonies and shake
Participant:Participant:Wang Zhanyu, Wan Han
Liquid Culture | ||
Name | Medium | Volume/ml | BBa_K28001(pSB1C3) colony_1 | LB | 10 |
---|---|---|
BBa_K28001(pSB1C3) colony_2 | LB | 10 |
BBa_K28001(pSB1C3) colony_3 | LB | 10 |
BBa_K28000(pSB1C3) colony_1 | LB | 10 |
BBa_K28000(pSB1C3) colony_2 | LB | 10 |
BBa_K1321338(pSB1C3) colony_1 | LB | 10 |
BBa_K1321338(pSB1C3) colony_2 | LB | 10 |
BBa_K1321338(pSB1C3) colony_3 | LB | 10 |
7.26
Find the T7ptag mutation site and design primers for point mutation.
Participant:Wang Zhanyu, Wan Han
7.27
Point mutation pcr
Participant:Wang Zhanyu, Wan Han
August
8.2
Confirm other parts need in logic gate.
Participant:Wang Zhanyu, Wan Han
Transformation | ||||||
Gene of interest | Vector | Ice | 42°C | Ice | 37°C+LB | Plate | BBa_R0010 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |
---|---|---|---|---|---|---|
BBa_I13453 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |
BBa_895006 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |
8.3
pick colonies and shake
Participant:Participant:Wang Zhanyu, Wan Han
Liquid Culture | ||
Name | Medium | Volume/ml | BBa_R0010(pSB1C3) colony_1 | LB | 10 |
---|---|---|
BBa_R0010(pSB1C3) colony_2 | LB | 10 |
BBa_R0010(pSB1C3) colony_3 | LB | 10 |
BBa_I13453(pSB1C3) colony_1 | LB | 10 |
BBa_I13453(pSB1C3) colony_2 | LB | 10 |
BBa_K895006(pSB1C3) colony_1 | LB | 10 |
BBa_K895006(pSB1C3) colony_2 | LB | 10 |
BBa_K895006(pSB1C3) colony_3 | LB | 10 |
8.4
Participant: Wang Zhanyu, Wan Han
1. Extract the plasmid
2.PCR
PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
---|---|---|---|---|---|
taq polymerase | 0.5 | Pre Denature | 94 | 180 | / |
tempalte | 0.5 | Denature | 95 | 30 | 30 |
10X Taq buffer | 5 | Annealing | 55 | 30 | 30 |
primer-forward(10μM) | 2 | Extension | 72 | 45/45 | 30 |
primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
dNTP(2.5mM) | 4 | storage | 4 | / | / |
ddH2O | 36 | / | / | / | / |
8.5
Doing overlap PCR, construct the logic gate plasmid.
Participant: Wang Zhanyu, Wan Han
8.7-8.9
Finish the construction of logic gate’s input plasmid(~3500bp,marked with an arrow)
Participant: Wang Zhanyu, Wan Han
8.9-8.11
Finish the construction of logic gate’s output plasmid(~1100bp,marked with arrows)
Participant: Wang Zhanyu, Wan Han
8.11-8.14
Using infusion cloning to integrate the input plasmid and output plasmid together. (~4500bp,marked with an arrow)
Participant: Wang Zhanyu, Wan Han
8.20
Co-transformation
Participant: Li Shisheng
Co-Transformation | ||||||
Gene of interest | Vector | Ice | 42°C | Ice | 37°C+LB | Plate | Two green-TCS plasmids | / | 30min | 90s | 5min | 1h/1ml | LBCM |
---|---|---|---|---|---|---|
Two red-TCS plasmids | / | 30min | 90s | 5min | 1h/1ml | LBCM |
8.21
Liquid Culture
Participant:Participant:Wang Zhanyu, Wan Han
Liquid Culture | ||
Name | Medium | Volume/ml | Green TCS_1 | LB | 5 |
---|---|---|
Green TCS_2 | LB | 5 |
Green TCS_3 | LB | 5 |
Green TCS_4 | LB | 5 |
Green TCS_5 | LB | 5 |
Green TCS_6 | LB | 5 |
Green TCS_7 | LB | 5 |
Green TCS_8 | LB | 5 | Red TCS_1 | LB | 5 |
Red TCS_2 | LB | 5 |
Red TCS_3 | LB | 5 |
Red TCS_4 | LB | 5 |
Red TCS_5 | LB | 5 |
Red TCS_6 | LB | 5 |
Red TCS_7 | LB | 5 |
Red TCS_8 | LB | 5 |
8.22
Fluorescence intensity measuring
Participant: Li Shisheng
Green TCS | ||||
RFU | Control | Dark/th> | green light | red light | 1 | 9074 | 31813 | 39406 | 32336 |
---|---|---|---|---|
2 | 9022 | 31512 | 39105 | 32491 |
3 | 9063 | 31980 | 38535 | 32000 |
4 | 9072 | 32396 | 39816 | 32261 |
5 | 9130 | 32203 | 39201 | 33136 |
6 | 9063 | 32463 | 39829 | 33519 |
7 | 9329 | 32370 | 39991 | 33775 |
8 | 9192 | 32724 | 39886 | 33168 |
Red light TCS | |||
RFU | Control | Dark/th> | red light | 1 | 369 | 3716 | 3241 |
---|---|---|---|
2 | 248 | 3719 | 3332 |
3 | 384 | 3743 | 3263 |
4 | 404 | 3761 | 3449 |
5 | 408 | 3663 | 3179 |
6 | 409 | 3761 | 3416 |
7 | 350 | 3761 | 3416 |
8 | 357 | 3584 | 3178 |
8.23
Verifying the single oscillation circuit in microfluidics device
Participant:Ran Jinshi, Zhu Hongya
• We co-transformed the MG1655 strain with pTD103aiiA and pTD103luxI-sfGFP.
• We screened the positive colony and inoculated in 50ml LB with antibiotic 100μg/ml ampicillin (Amp) and 50 mgml21 kanamycin (Kan). We spun it down and concentrated it in 5ml of fresh media with surfactant concentration of0.075% Tween20 when the bacteria solution reach an A600nm of 0.05~0.1.
• We loaded the sample from the cell port while keeping the media port at sufficiently higher pressure than the waste port below to prevent contamination. We manually applied pressure pulses to line to induce a momentary flow change. The flow was then reversed and allow for cell to receive fresh media with 0.075% Tween20 which prevented cells from adhering to the main channels and waste ports.
• We took pictures of microfluidics in fluorescence microscope every 5 mins and analyzed the fluorescent density with imageJ.
8.25
Liquid Culture
Participant: Li Shisheng
Liquid Culture | ||
Name | Medium | Volume/ml | Green TCS_1 | LB | 5 |
---|---|---|
Green TCS_2 | LB | 5 |
Green TCS_3 | LB | 5 |
Green TCS_4 | LB | 5 |
Green TCS_5 | LB | 5 |
Green TCS_6 | LB | 5 |
Green TCS_7 | LB | 5 |
Green TCS_8 | LB | 5 | Red TCS_1 | LB | 5 |
Red TCS_2 | LB | 5 |
Red TCS_3 | LB | 5 |
Red TCS_4 | LB | 5 |
Red TCS_5 | LB | 5 |
Red TCS_6 | LB | 5 |
Red TCS_7 | LB | 5 |
Red TCS_8 | LB | 5 |
8.26
Fluorescence intensity measuring
Participant: Li Shisheng
Delay of Red TCS | ||||||
RFU | 0h | 1h | 2h | 3h | 4h | 5h | 1 | 411 | 431 | 439 | 489 | 739 | 1681 |
---|---|---|---|---|---|---|
2 | 438 | 489 | 403 | 471 | 667 | 1690 |
3 | 431 | 503 | 541 | 599 | 820 | 1756 |
4 | 384 | 489 | 456 | 419 | 833 | 1681 |
5 | 506 | 434 | 434 | 530 | 815 | 1553 |
6 | 330 | 364 | 426 | 481 | 684 | 1501 |
7 | 361 | 391 | 482 | 552 | 739 | 1482 |
8 | 395 | 460 | 426 | 574 | 775 | 1641 |
Green TCS | ||||||
RFU | 0h | 1h | 2h | 3h | 4h | 5h | 1 | 411 | 516 | 736 | 917 | 1680 | 4200 |
---|---|---|---|---|---|---|
2 | 438 | 554 | 715 | 907 | 1752 | 4190 |
3 | 431 | 534 | 723 | 923 | 1712 | 4210 |
4 | 384 | 702 | 805 | 839 | 1692 | 4148 |
5 | 506 | 590 | 687 | 878 | 1563 | 4045 |
6 | 330 | 577 | 639 | 891 | 1580 | 3968 |
7 | 361 | 588 | 617 | 860 | 1559 | 3881 |
8 | 395 | 569 | 692 | 992 | 1541 | 3849 |
8.27
PCR
Participant: Li Shisheng
PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
---|---|---|---|---|---|
super fidelity DNA polymerase | 1 | Pre Denature | 95 | 180 | / |
tempalte | 0.5 | Denature | 95 | 15 | 30 |
2X buffer | 25 | Annealing | 55 | 15 | 30 |
primer-forward(10μM) | 2 | Extension | 72 | 80 | 30 |
primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
dNTP(10mM) | 1 | storage | 4 | / | / |
ddH2O | 18.5 | / | / | / | / |
8.28
Validate the logic gate ‘s response time. For details, see our protocol in the experiment part.
Participant:Wang Zhanyu, Wan Han
8.30
Verifying the double oscillation circuit
Participant:Ran Jinshi, Zhu Hongya
• We co-transform the MG1655 strain with pTD103 aiiA-luxS、pTD103 luxI-sfGFP and pSB1C3 plsr-YFP.
• We screened the positive colony and inoculated it in 50ml LB with antibiotic 100μg/ml ampicillin (Amp) and 50 mg/ml kanamycin (Kan)and Chloromycetin(C). We spun it down and concentrated it in 5ml LB when the bacteria solution reached an A600nm of 0.05~0.1.
• We inoculated it with a 1:1000 dilution in 100ml fresh media. When the bacteria solution reached A600nm 0.2~0.3, we sampled in every 10 min and measured the fluorescence intensity.
September
9.2
Transformation
Participant: Li Shisheng
PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
---|---|---|---|---|---|
super fidelity DNA polymerase | 1 | Pre Denature | 95 | 180 | / |
tempalte | 0.5 | Denature | 95 | 15 | 30 |
2X buffer | 25 | Annealing | 55 | 15 | 30 |
primer-forward(10μM) | 2 | Extension | 72 | 80 | 30 |
primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
dNTP(10mM) | 1 | storage | 4 | / | / |
ddH2O | 18.5 | / | / | / | / |
9.4
Transformation
Participant: Li Shisheng
PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
---|---|---|---|---|---|
super fidelity DNA polymerase | 1 | Pre Denature | 95 | 180 | / |
tempalte | 0.5 | Denature | 95 | 15 | 30 |
2X buffer | 25 | Annealing | 55 | 15 | 30 |
primer-forward(10μM) | 2 | Extension | 72 | 80 | 30 |
primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
dNTP(10mM) | 1 | storage | 4 | / | / |
ddH2O | 18.5 | / | / | / | / |
9.7
Transformation
Participant: Li Shisheng
PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
---|---|---|---|---|---|
super fidelity DNA polymerase | 1 | Pre Denature | 95 | 180 | / |
tempalte | 0.5 | Denature | 95 | 15 | 30 |
2X buffer | 25 | Annealing | 55 | 15 | 30 |
primer-forward(10μM) | 2 | Extension | 72 | 80 | 30 |
primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
dNTP(10mM) | 1 | storage | 4 | / | / |
ddH2O | 18.5 | / | / | / | / |
9.9
Validate the plasmid combining logic gate and red light-induced system: PompC+Plac
(3600bp)
Participant:Wang Zhanyu, Wan Han
9.17
Validate the plasmid combining logic gate and green light-induced system: PcpcG2+Plac
(3700bp)
Participant:Wang Zhanyu, Wan Han
October
10.10
PCR
Participant: Li Shisheng
PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
---|---|---|---|---|---|
super fidelity DNA polymerase | 1 | Pre Denature | 95 | 180 | / |
tempalte | 0.5 | Denature | 95 | 15 | 30 |
2X buffer | 25 | Annealing | 55 | 15 | 30 |
primer-forward(10μM) | 2 | Extension | 72 | 80 | 30 |
primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
dNTP(10mM) | 1 | storage | 4 | / | / |
ddH2O | 18.5 | / | / | / | / |
Contact Us
Room 413,Biology lab center, Zijingang Campus
Zhejiang University, YuHangTang Road NO.866
Hangzhou, China
iGEM ZJU-China 2016 Team
first.last@example.com
first.last@example.com