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<a href="#exp4"><h4> 8. Make a culture of DH5&alpha; pET43.1 to monitor the growth curve </h4></a></br>  

             <U> Aim:</U> Check the transformation and the digestion of the plasmids by electrophoresis on agarose gel.</br> Seed pET43.1 DH5&alpha; in LB + carbenicillin media to make stab culture. Indeed, the validity of transformants needs to be verified by checking if an insert is present in the transformed plasmid. Secondly, verified plasmid-DH5&alpha combinations need to be stored beyond the lifetime of plate colonies. </br></br>

            <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>

             <U>What we did in the lab:</U></br>Counting of colonies on petri dish: overnight grown plates at 37°C, supplemented with carbenicillin 50 &#181;g/ml (LB + CB50) or with chloramphenicol 34 &#181;g/ml (LB + CM34) were counted for the presence of individual distinct colonies. </br></br>LB + CB50</br></br>

       <td><strong><p>pET43.1a(+) DH5&alpha;</p></strong></td>

       <td>0</td>

       <td>2</td>

       <td>23</td>

       <td><strong> <p>pET43.1a(+) DH5&alpha;</br> (different batch)</p></strong></td>

       <td>Uncountable (Overgrown)</td>

       <td>2</td>

       <td><strong><p>pET43.1a(+) DH5&alpha;</br> (different batch)</p></strong></td>

       <td>Uncountable </br>(Overgrown)</td>

       <td>15</td>

</br>

<center>Table 3</center></br> LB + CM34</br></br>

       <td><strong><p>pSB1C3 DH5&alpha;</p></strong></td>

       <td>1</td>

       <td>78</td>

       <td><strong> <p>pSB1C3 DH5&alpha;</br> (different batch)</p></strong></td>

       <td>38 </td>

       <td> Uncountable (Overgrown) </td>

</table>

</br>

<center>Table 4</center></br>  

<U>What we did in the lab:</U></br>

           <U>Materials:</U>

&bull; Agarose <br>

&bull; Microwave oven /<br>

&bull; Precision balance </br>

 

</br>

           <U>Method:</U></br>

1. Buffer solution: we have 1.0X stock TAE (Tris Acetate EDTA) solution and we prepared 1.0X TAE gels with 0.5X TAE running buffer </br>

&bull; pSB1C3 plasmid (digested by SpeI/XbaI)<br>

       <td><strong><p>Sample </p></strong></td>

       <td>Molecular </br> weight</td>

       <td>pET43.1a(+)</br> uncut</td>

       <td></td>

       <td>pET43.1a(+)</br> B-H</td>

       <td></td>

       <td>pBS1C3</br> S-X</td>

       <td></td>

       <td>pBS1C3</br> uncut</td>

       <td><strong> </strong></td>

       <td>DNA (&micro;l)</td>

       <td> 5</td>

       <td> 2</td>

       <td></td>

       <td> 2</td>

       <td></td>

       <td> 4</td>

       <td></td>

       <td> 4</td>

       <td><strong> </strong></td>

       <td>H20</td>

       <td> 0</td>

       <td> 8</td>

       <td></td>

       <td>8</td>

       <td></td>

       <td>6</td>

       <td></td>

       <td>6</td>

       <td><strong> </strong></td>

       <td>6X</br>Loading</br>buffer</td>

       <td> 0</td>

       <td> 2</td>

       <td></td>

       <td>2</td>

       <td></td>

       <td>2</td>

       <td></td>

       <td>2</td>

</table>

</br>

<center>Table 5</center></br></br>   

<U>Results</U></br>

The digestion has worked since the plasmids digested have moved faster. </br></br>NB: the gel suffered from overheating. </br>

</br></br></br></br>

Figure 1: </br></br>

               <U> Aim:</U Repeat Midiprep for pET43.1a(+). </br>Since we need a lot of pET43.1a(+) we have to repeat the Midiprep.</br></br>

             <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>

               <U>What we did in the lab:</U></br>

               <U>Materials:</U>

&bull; Antibiotic: carbenicillin 100 mg/ml<br>

&bull; Cones for P10<br>

               <U>Method:</U></br>

1. Pick one colony and put it into 5 ml of LB medium + 2.5 &#181;l of carbenicillin stock, to obtain 50 &micro;g/&micro;l

               <U> Aim:</U> Use the preculture of June 13, 2016 to start the new culture.</br></br>

             <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>

               <U>What we did in the lab:</U></br>

               <U>Materials:</U></br>

&bull; LB autoclaved 500 ml<br>

&bull; swing bucket centrifuge (JOUAN GR4i)</br>

 

</br>

               <U>Method:</U></br>

1. Get back preculture performed on the June 13, 2016, put it in the swing bucket centrifuge during 7 min (3500 RPM) and remove the supernatant. </br>

2. Add 5ml of fresh LB, resuspend the pellet and centrifuge again at 3500 RPM during 7 min. </br>

3. Repeat one more time step 2</br>

4. Resuspend the pellet into 5 mL of fresh LB+ 2.5 &micro;l of carbenicillin 100 mg/ml</br>

5. Grow in the shaking incubator (T= 37 °C / 130 RPM) Overnight</br>

               <U> Aim:</U> to make a stab culture we need to capture the bacteria growing at the exponential phase </br> In order to do that we need to take optical density at 600_nm (OD600_nm).</br></br>

             <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>

               <U>What we did in the lab:</U></br>

               <U>Materials:</U></br>

&bull; Microbiology equipment</br>

 

</br>

               <U>Method:</U></br> Before starting the culture we need to have molten LB-agar so we need to place the solid LB agar on a hot plate with stirring. </br>

1. During growth of the bacterial culture, take one ml aliquot and measure the absorbance at OD600_nm every hour for the first three hours, then every 20 min onwards. We recorded OD600_nm for this specific experiment after 3 hours.</br>

</br></br>

 

       <td><strong><p>12h37 </p></strong></td>

       <td></td>

       <td>0.013</td>

       <td>0.015</td>

       <td><strong> <p>13h07</p></strong></td>

       <td>0.023</td>

       <td> 0.027</td>

       <td><strong> <p>13h37</p></strong></td>

       <td>0.063</td>

       <td> 0.059 </td>

       <td><strong> <p>14h07</p></strong></td>

       <td>0.110 </td>

       <td> 0.105</td>

       <td><strong> <p>14h27</p></strong></td>

       <td>0.172 </td>

       <td> 0.170</td>

       <td><strong> <p>14h47</p></strong></td>

       <td>0.265 </td>

       <td> 0.256</td>

       <td><strong> <p>15h07</p></strong></td>

       <td>0.384 </td>

       <td> 0.377 </td>

       <td><strong> <p>15h27</p></strong></td>

       <td>0.491 </td>

       <td> 0.482</td>

       <td><strong> <p>15h47</p></strong></td>

       <td>0.567 </td>

       <td> 0.557</td>

       <td><strong> <p>16h06</p></strong></td>

       <td>0.697</td>

       <td> 0.608</td>

       <td><strong> <p>16h29</p></strong></td>

       <td>0.816</td>

       <td> 0.755</td>

       <td><strong> <p>17h03</p></strong></td>

       <td>0.954 </td>

       <td> 0.909 </td>

       <td><strong> <p>17h20</p></strong></td>

       <td>0.954</td>

       <td> 0.909</td>

       <td><strong> <p>17h40</p></strong></td>

       <td>1.061</td>

       <td> 1.029</td>

       <td><strong> <p>18h00</p></strong></td>

       <td>1.092</td>

       <td> 1.082</td>

       <td><strong> <p>18h00</p></strong></td>

       <td>1.122</td>

       <td> 1.132</td>

</br>

<center>Table 6</center></br> </br>

<U>Growth curve :</U>

 

</br> Figure 2: Growth curve for DH5&alpha;-pET43.1a(+) by measurement of  OD600_nm. O-time point corresponds to 12h37</br>

             <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>

<U> What we did on the lab:</U></br>

<U> Method:</U></br>

1. 20 ml of warm LB + agar is placed in a 50 ml Falcon and 20 &micro;l of carbenicillin 50 mg/ml added, swirl to mix</br>

2. Aliquot 1 ml of LB + agar in 2 ml sterile tubes (10 tubes per each culture, so 20 tubes total) </br>

3. When the mixture solidified, soak a toothpick in bacterium culture and stab the LB agar several times</br>

4. Grow overnight at 37 °C and next day place it at 4°C</br>

             <U> Aim:</U> we received our gene block synthesized DNA constructs in lyophilized form. In order to ligate them in our plasmid, we need to resuspend them in buffer. </br> Add TE (Tris 10 mM pH 8.0 EDTA 1 mM) buffer in each tubes and refer to the next table for volumes

       <td><strong><p>A1 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

     <td><strong><p>A2 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

     <td><strong><p>C1 </p></strong></td>

       <td>1000</td>

       <td>10</td>

       <td>100</td>

     <td><strong><p>C2 </p></strong></td>

       <td>1000</td>

       <td>10</td>

       <td>100</td>

   <td><strong><p>D1 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

   <td><strong><p>D2 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

   <td><strong><p>E1 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

   <td><strong><p>E2 </p></strong></td>

       <td>500</td>

       <td>10</td>

       <td>50</td>

   <td><strong><p>B2 </p></strong></td>

       <td>1000</td>

       <td>10</td>

       <td>100</td>

</table>

</br>

<center>Table 7</center></br> </br>

             <U> Aim:</U> We want to have only the DNA backbone of pET43.1a(+) and pSB1C3, not the inserts that lie between the restriction sites. Spe I/ Xba I or BamH I/Hind III respectively, so we have to extract the plasmid backbone on out of an agarose gel. Furthermore, to clean the pET43.1a(+) and pSB1C3 backbones we use the gel extraction kit of QIAGEN.  Afterwards we ligated our plasmid with the inserts. In this experiment we use inserts C1 and C2

             <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>

             <U>What we did in the lab:</U></br>

             <U>Material:</U></br>

&bull; We use the NEB extraction kit. Monarch DNA Gel Extraction Kit (/#T1020G) </br></br>

<U>Method:</U></br>

2. Add 4 volumes of gel dissolving buffer (NT1) to the gel slice. For NT1 volumes, refer to the Table 8: . </br>

       <td><strong><p>pET43.1a(+)</p></strong></td>

       <td>m1</td>

       <td>0.9</td>

       <td>1.3</td>

       <td>329.6</td>

       <td><strong><p>pET43.1a(+)</p></strong></td>

       <td>m2</td>

       <td>0.9</td>

       <td>1.3</td>

       <td>265.3</td>

     <td><strong><p>pSB1C3 </p></strong></td>

       <td>m1</td>

       <td>0.9</td>

       <td>1.2</td>

       <td>251.1</td>

     <td><strong><p> pSB1C3 </p></strong></td>

       <td>m2</td>

       <td>1.0</td>

       <td>1.2</td>

       <td>185.6</td>

<center>Table 8</center></br> </br>

3. Incubate the samples between 37-55°C, vortexing periodically until the gel slice is completely dissolved (/ ~ 10 min) </br>

4. Load sample onto the column. Close the cap, spin for 1 min then discard flow through</br>

5. Re insert column into collection tube. Add 200 &micro;µl DNA wash buffer and spin for one minute. Discard flow-through</br>

6. Repeat step 5</br>

7. Transfer column to a clean 1.5 ml microfuge tube</br>

8. Add 6 &micro;l of DNA Elution buffer to the center of the matric. Wait for 1 min and spin for 1 min to elute DNA</br>

 

             <U> Aim:</U> after digestion of the plasmid backbone we now proceed with the digestion of the inserts C1 and C2.  </br></br>

             <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>

             <U>What we did in the lab:</U></br>

            <U>Material:</U></br></br>

<U>Method:</U></br>

Add all reagents in 1 ml Eppendorf </br></br>

- Digest during 2 h at 37 °C </br>

- For the reagent volumes, refer to the Table 9. Total volume = 50 &micro;l</br></br>

       <td><strong><p>DNA (&micro;l)</p></strong></td>

       <td>20</td>

       <td>20</td>

       <td><strong><p>BamH I (&micro;l)</p></strong></td>

       <td>1</td>

       <td>1</td>

    </tr>

     <td><strong><p>Hind III (&micro;l)</ </p></strong></td>

             <td>2</td>

       <td>2</td

     <td><strong><p> H₂0 (&micro;l)</ </p></strong></td>

       <td>22</td>

       <td>22</td>

</tr>

     <td><strong><p> CutSmart(&micro;l)</ </p></strong></td>

       <td>5</td>

       <td>5</td>

</br>

<center>Table 9</center></br> </br>

 

             <U> Aim:</U</br></br>

 

            <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>

             <U> Aim:</U</br></br>

 

            <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>

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