Team:Grenoble-EMSE-LSU/Documentation/Notebook/July
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<h1>July</h1> | <h1>July</h1> | ||
- | + | <h2>Week 1 (1-5)</h2> | |
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- | </ | + | |
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<h3>Monday</h3> | <h3>Monday</h3> | ||
<p>- Present our project to the American Summer School (poster) at Maison Minatec in Grenoble. </br> | <p>- Present our project to the American Summer School (poster) at Maison Minatec in Grenoble. </br> | ||
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- Try to preculture cells with pJT122 (Voigt 1) and pPLPCB(S)(Voigt 3) but didn’t grow, so re-transform BW with pJT122, pJT106B(Voigt 2) and pPLPCB(S).</br></p> | - Try to preculture cells with pJT122 (Voigt 1) and pPLPCB(S)(Voigt 3) but didn’t grow, so re-transform BW with pJT122, pJT106B(Voigt 2) and pPLPCB(S).</br></p> | ||
<h3>Tuesday</h3> | <h3>Tuesday</h3> | ||
+ | |||
+ | <p>- Extraction of KillerRed from the pBabe-KillerRed eukaryotic vector by PCR, using the following primers, enabling the addition of BamHI and KpnI at the 5’ and 3’ ends of the KillerRed gene, respectively. The restriction sites are highlighted in yellow.</br> | ||
+ | |||
+ | Name: KR_left_pQE | ||
+ | TAATTCCGGATCCATGGGTTCAGAGGGCGGC (Tm=67∞C for 31 bases) | ||
+ | Restriction Site: (BamH1) | ||
+ | |||
+ | Name: KR_right_pQE | ||
+ | CAGGTACCTTAATCCTCGTCGCTACCGATGG (Tm=66∞C for 31 bases) | ||
+ | Restriction Site: (Kpn1) | ||
+ | |||
+ | Protocol available here. | ||
+ | |||
+ | 10 µL of PCR products were subsequently analyzed by gel electrophoresis (1.2 % agarose, 30 min, 135 V). The results of the migration, on which a 700 bp DNA band is clearly visible, demonstrate the success of the amplification of KillerRed. | ||
+ | |||
+ | The rest of the PCR product was purified using the QIAprep® PCR Purification Kit (#28104, Qiagen, Venlo, Netherlands) and titrated using the nanodrop spectrophotometer available in the lab (final concentration : 95.8 ng/µL). Results of migration and titration are available in the figure below. | ||
+ | |||
<p>- Use luster terminals to avoid the use of crocodile clip and have longer cable for the highpower LED.</br> | <p>- Use luster terminals to avoid the use of crocodile clip and have longer cable for the highpower LED.</br> | ||
- Test the system where the LED is put in the incubateur and measure the temperature of the surface of a Petri dish exposed to the light of the LED that is 7cm above the dish - conclusion: no overwarming - this is a very good point! If the cells expressing KillerRed died, it would not be because of the overwarming of illumination but because of the ROS emission!</br> | - Test the system where the LED is put in the incubateur and measure the temperature of the surface of a Petri dish exposed to the light of the LED that is 7cm above the dish - conclusion: no overwarming - this is a very good point! If the cells expressing KillerRed died, it would not be because of the overwarming of illumination but because of the ROS emission!</br> | ||
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<p>- The transformations work more or less for all of them but there is only one colony for the transformed cells with 3 plasmids</br> | <p>- The transformations work more or less for all of them but there is only one colony for the transformed cells with 3 plasmids</br> | ||
- Put three clones of each Petri dish in 3 mL of LB medium to make them grow overnight with the appropriate antibiotics</p> | - Put three clones of each Petri dish in 3 mL of LB medium to make them grow overnight with the appropriate antibiotics</p> | ||
- | + | <p><strong>pLac-RBS-sspB</strong> | |
+ | <br>Culture and miniprep pLac, RBS, sspB and BackBone BioBricks | ||
+ | <br>Problems with RBS (Elowitz), I could not miniprem it, it seems like it was a problem of size. | ||
+ | <br>Miniprep sspB: 64,1ng/µL, Miniprep pLac: 172,3ng/µL, Miniprep backbone: 226,8ng/µL | ||
+ | <br>Test of restriction enzyme on pLac | ||
+ | </p> | ||
+ | |||
+ | |||
+ | <h2>Week 2 (8-12)</h2> | ||
<h3>Monday</h3> | <h3>Monday</h3> | ||
<p>- Draw a first draft of the Printed Circuit Board (PCB) for the photodiode</br> | <p>- Draw a first draft of the Printed Circuit Board (PCB) for the photodiode</br> | ||
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<p>- Think of a way to put GFP under the PcpcG2 promotor in pJT122 instead of LacZ- put restriction site (BglI) at the beginning and at the end of KR ? (because BglI cut LacZ twice at the beginning and at the end of the gene) | <p>- Think of a way to put GFP under the PcpcG2 promotor in pJT122 instead of LacZ- put restriction site (BglI) at the beginning and at the end of KR ? (because BglI cut LacZ twice at the beginning and at the end of the gene) | ||
- Think of the design of our device</p> | - Think of the design of our device</p> | ||
- | + | <h3>Friday</h3> | |
+ | <p><strong>pLac-RBS-sspB</strong> | ||
+ | <br> We realised that given that pLac was already used it was better to use pBad.Also as RBS is only 15 base pair long it was decided that it will be added to sspb via PCR. | ||
+ | <br>Culture and miniprep pBAD</p> | ||
<p></p> | <p></p> | ||
- | <h2>Week 3</h2> | + | <h2>Week 3 (15-19)</h2> |
<h3>Monday</h3> | <h3>Monday</h3> | ||
<p>- Put Voigt 1&3 in liquid culture with 4x Xgal ON</p> | <p>- Put Voigt 1&3 in liquid culture with 4x Xgal ON</p> | ||
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- Think of a way to put KR and RFP under red sensitive promoter - restriction/ligation seems to be easier here</p> | - Think of a way to put KR and RFP under red sensitive promoter - restriction/ligation seems to be easier here</p> | ||
<h3>Friday</h3> | <h3>Friday</h3> | ||
- | <p>- Print the 2 PCBs and drill holes into them and weld the different component on them.</br> | + | <p>- Print the 2 PCBs and drill holes into them and weld the different component on them.</br></br></p> |
+ | <p align="center"><img src="https://static.igem.org/mediawiki/2013/2/24/Welding_PCB.png" alt="Welding_stuff_on_PCB" /> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2013/f/f9/Drilling_PCB.png" alt="drilling_on_PCB" /></br></br></p> | ||
+ | <p> | ||
- Test the PCB that control the intensity of the light with a function generator and the oscilloscope, it works well.</br> | - Test the PCB that control the intensity of the light with a function generator and the oscilloscope, it works well.</br> | ||
- Notice that the connector on the photodiode PCB is not welded on the right side</br></p> | - Notice that the connector on the photodiode PCB is not welded on the right side</br></p> | ||
- | <h2>Week 4</h2> | + | |
+ | <p><strong>pBad-RBS-sspB</strong> | ||
+ | <br>new culture and miniprep of sspb : 173 ng/µL </p> | ||
+ | <h2>Week 4 (22-26)</h2> | ||
<h3>Monday</h3> | <h3>Monday</h3> | ||
<p>- Weld the connector on the photodiode PCB on the right side</br> | <p>- Weld the connector on the photodiode PCB on the right side</br> | ||
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<p>- The solution of the condensator works but since it is not draw on the main PCB, new holes must be drilled on the existing wires. It works with a 100nF condensator, there is no more noise and Arduino stops freezing =).</br> | <p>- The solution of the condensator works but since it is not draw on the main PCB, new holes must be drilled on the existing wires. It works with a 100nF condensator, there is no more noise and Arduino stops freezing =).</br> | ||
- Bolt Arduino on the main PCB.</br> | - Bolt Arduino on the main PCB.</br> | ||
- | - Put E. Coli with KR and luciferase in pre-culture to test if the photodiode measure low bioluminescence and fluorescence.</br> | + | - Put <em>E. Coli</em> with KR and luciferase in pre-culture to test if the photodiode measure low bioluminescence and fluorescence.</br> |
</p> | </p> | ||
<h3>Wednesday</h3> | <h3>Wednesday</h3> | ||
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<h3>Friday</h3> | <h3>Friday</h3> | ||
<p>- Have a <strong>big and long meeting with the advisor</strong></br></p> | <p>- Have a <strong>big and long meeting with the advisor</strong></br></p> | ||
- | <h3> | + | <h3>Monday</h3> |
<p>- Try to measure fluorecence by changing filters or adding blue filter (as a low pass filter) but it does not improve the measurement.</br> | <p>- Try to measure fluorecence by changing filters or adding blue filter (as a low pass filter) but it does not improve the measurement.</br> | ||
</p> | </p> | ||
<h3>Tuesday</h3> | <h3>Tuesday</h3> | ||
- | <p>- | + | <p>- Receive a cube from a microscope with two excitation filters -interference filters- (green and blue), two colored filter as emission filters (red and yellow) and two dichroic mirrors. There is also a lens with a 30mm focal. Change a little bit the setup with the new components, it looks like this:</br> |
We cut a 50mL tube and paint it in black and wrap the lamp with aluminium to avoid the photodiode to be disturbed by the lamp.</br></br></p> | We cut a 50mL tube and paint it in black and wrap the lamp with aluminium to avoid the photodiode to be disturbed by the lamp.</br></br></p> | ||
- | <p align="center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fluo_measure.png" alt="fluo_measure" /></p> | + | <p align="center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fluo_measure.png" alt="fluo_measure" /></br></br></p> |
<table align="center" style="border-collapse:collapse;font-size: 14px;"> | <table align="center" style="border-collapse:collapse;font-size: 14px;"> |
Latest revision as of 03:36, 5 October 2013