Team:CIDEB-UANL Mexico/labwork methods

From 2014hs.igem.org

(Difference between revisions)
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<p><h2> <b><a name="Experiments"></a><font size="6">Experiments</font> </b>- <a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_methods#"><font size="2">Return to the Top</font></a></h2></p>  
<p><h2> <b><a name="Experiments"></a><font size="6">Experiments</font> </b>- <a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_methods#"><font size="2">Return to the Top</font></a></h2></p>  
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<p>In this section, it is described all the experiments designed and performed in order to prove experimentally our modules. </p>
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<p>In this section, the experiments performed in order to test the effectivness of the predicted models for our proyect, will be described. </p>
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<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_methods#Capture" class="button2">Capture Module</a></p>
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<p>The tested  parts are the following:: </p>
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<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_methods#Capture" class="button2"> Capture Module</a></p>
<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_methods#AromaExp" class="button2">Aroma Module</a></p>
<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_methods#AromaExp" class="button2">Aroma Module</a></p>
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<p><a name="UVExp"></a><b>UV Experimentation</b></p>
<p><a name="UVExp"></a><b>UV Experimentation</b></p>
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<p>This experiment was designed in order to know if the UV promoter is working properly.</p>
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<p>This experiment was designed in order to test the UV promoter's efectivness.</p>
<p><b>Procedure:</b></p>
<p><b>Procedure:</b></p>
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<p><b>1.</b>  Inoculate by streak 2 Petri dishes with NhaS DNA in pSB1C3 Red bacteria</p>
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<p><b>1.</b>  2 Petri dishes were inoculated according to the &quot;streak method&quot; with transformed bacteria from a cultured petri dish, containing the NhaS and the RFP genes in their plasmids. (These colonies  looked red due to the RFP) </p>
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<p><b>2.</b>  Repeat step 1 but with NhaS DNA in pSB1C3 White bacteria</p>
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<p><b>2.</b>  The first step was repeated, this time with the transformed bacteria cultures containing  the NhaS gene only, without the RFP gene. (These colonies looked white due to the absence of the RFP) </p>
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<p><b>3.</b>  Let them grow during one day in the incubator at 37°C</p>
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<p><b>3.</b>  The bacteria was grown for one day in the incubator at 37°C</p>
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<p><b>4.</b>  Expose the four Petri dishes to UV irradiation (302nm) during 2 hours</p>
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<p><b>4.</b>  The four petri dishes were exposed to UV radiation (302nm) during a 2 hour period </p>
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<p><b>5.</b>   Take photos each 10 minutes and wait for results. (You can also take video during the 2 hours instead of the photos)</p>
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<p><b>5.</b> Pictures were taken of the bacteria cultures at 10 minute intervals for the 2 hour period </p>
<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_results#UV">Go to results</a></p>
<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_results#UV">Go to results</a></p>
<br>
<br>
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<p><a name="Viability"></a><h2><b>Viability in salt</b> - <a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_methods#"><font size="2">Return to the Top</font></a></h2></p>
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<p><a name="Viability"></a><h2><b>Viability test of the NhaS gene containing bacteria in salt</b> - <a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_methods#"><font size="2">Return to the Top</font></a></h2>
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<p>&nbsp;</p>
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<p>In the research on NhaS it was said that the gene gives certain resistance to salt, but the exact percentage of resistance is unknown. There were designed 3 experiments to know the viability in salt of the NhaS transformed bacteria, also in order to know the maximum concentration in which bacteria with the capture module can be exposed to later experiment with it and find out how much salt can capture.</p><p>
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<p>In theory, the NhaS gene gives the bacteria a certain resistance to salt, but the exact percentage of increase in the resistance is unknown. Three experiments were designed in order to test:</p>
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As in the ligation on NhaS and pSB1C3 was obtain two types of bacteria (red and white) it us uncertain if the ligation occurred properly and why some are red and some are white, through the next three experiments it can be determined which type has the gene or if both of them have it.</p>
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<ul>
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</br>
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  <li> The viability of the NhaS transformed bacteria to survive in a salty environment</li>
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<p><b>Experiment 1</b></p>
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  <li>The maximum ammount of salt in the medium which can be withstanded by the transformed bacteria</li>
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  <li>The transformed bacteria's hability to capture sodium from the medium  </li>
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  </ul>
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<p>In the ligation process of the NhaS gene, two different kinds of plasmids were obtained due to a possible mutation. The ones containing the RFP producing gene, and the ones that did not. Because it was uncertain the presence of the NhaS gene in the non-RFP producing bacteria these experiments were designed and performed in order to determine which one of the groups contain the NhaS gene. </p>
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<p>&nbsp;</p>
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<p><b>Experiment #1</b></p>
Procedure:  
Procedure:  
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<p><b>1.</b> Put in 27 test tubes 9 milliliters of different concentrations of NaCl (0.85%, 0.90%, 1.00%, 1.50%, 2%, </p><p> 2.50%, 3.50%, 5.00% and 10.00%) to finish with 3 test tubes of each concentration.
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<p><b>1.</b> Twenty seven, 9 milliliters test tubes were filled with different concentrations of NaCl (0.85%, 0.90%, 1.00%, 1.50%, 2%, 2<b> .</b>50%, 3.50%, 5.00% and 10.00%) with 3 test tubes per concentration.</p>
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</p><p><b>2.</b> Separate the different groups of bacteria: white NhaS, red NhaS and bacteria without Nhas gene (Control)
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<p>2. The different groups of bacteria were separated: white NhaS, red NhaS and bacteria without Nhas gene (Control)
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</p><p><b>3.</b> Separate the 27 test tubes into 3 groups (for each type of bacteria) so that each group has 9 different salt concentrations without repeated.
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</p><p><b>4.</b> Put 1000 micro liters (1 uL) of red NhaS bacteria in each of the 9 corresponding test tubes to obtain a ratio of 1:10. Perform this step over a distance of no more than 30 cm of a Bunsen burner to avoid sample contamination
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</p><p><b>5.</b> Repeat step 4 but with White NhaS bacteria.
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</p><p><b>6.</b> Repeat step 4 but with the Control bacteria. At the end there will be 9 test tubes of red bacteria, 9 of white ones and 9 with the control.
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</p><p><b>7.</b> Separating two petri dishes (LB medium must contain chloramphenicol) for each concentration of NaCl with appropriate bacteria.
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</p><p><b>8.</b> Select one of 9 test tubes with NaCl and bacteria
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</p><p><b>9.</b> Introduce 1 milliliter of the contents of the test tube into a petri dish and repeat it in two petri dishes. Perform this step over a distance of not more than 30 cm of a Bunsen burner to avoid sample contamination.
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</p><p><b>10.</b> Put a glass inoculation spreader into a glass full of alcohol and remove it burning it with a Bunsen burner so that alcohol is removed from the spreader
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</p><p><b>11. </b>Wait 5 - 10 seconds
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</p><p><b>12. </b>Spread the content of the test tube into the petri dish. Perform this step over a distance of no more than 30cm of a Bunsen burner to avoid contamination
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</p><p><b>13. </b>Repeat step 7 to 12 with all test tubes
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</p><p><b>14.</b> Incubate the 54 petri dishes (9 concentrations of duplicates for each different bacteria) at 37 ºC during a day.
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</p>
</p>
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<p><b>3.</b> The 27 test tube were separated into 3 groups (for each type of bacteria), so that each group had 9 different salt concentrations.</p>
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<p><b>4.</b> In a sterile environment, no more than 30cm away from the bunsen burner, 1000 micro liters (1 mL) of RFP producing NhaS bacteria were added into each of the 9 corresponding test tubes, to obtain a ratio of 1:10. </p>
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<p><b>5.</b> The previous step was repeated with non RFP producing NhaS bacteria and with the non-NhaS producing control bacteria. </p>
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<p><b>6.</b>   Two petri dishes, per each concentration, per each bacteria groups were filled with prepared LB medium containing chloramphenicol.</p>
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<p><b>7.</b>One of the nine test tubes with NaCl and bacteria was selected.</p>
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<p><b>8.</b> One milliliter of the contents of the test tube was introduced into the three petri dishes. This step was performed  at a distance of not more than 30 cm of a Bunsen burner to avoid sample contamination.</p>
 +
<p><b>9.</b> A glass inoculation was placed in the spreader into a glass full of alcohol and remove it burning it with a Bunsen burner so that alcohol is removed from the spreader and  5 - 10 seconds were waited before the next step.</p>
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<p><b>11. </b>Spread the content of the test tube into the petri dish. Perform this step over a distance of no more than 30cm of a Bunsen burner to avoid any contamination
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</p>
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<p><b>12. </b>The previous series of steps were repeated with all of the tubes </p>
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<p><b>13.</b> The 54 petri dishes were incubated (9 concentrations of duplicates for each group of bacteria) at a temperature of 37 ºC during 24 hours.</p>
<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_results#Salt1">Go to results</a></p>
<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_results#Salt1">Go to results</a></p>
<br>
<br>
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<p><b>Experiment 2</b></p>
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<p><b>Experiment #2</b></p>
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<p>Procedure: </p><p>
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<p>Procedure: </p>
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<b>1. </b>Prepare 5 concentrations of NaCl in mQ water (1.0%, 2.5%, 5.0%, 10.0%, 15.0%) in different flasks. </p><p><b>
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<p>
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2. </b>Get the three groups of bacteria (white, red and without Nhas gene [control]) in different test tubes. </p><p><b>
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<b>1. </b>Five concentrations of NaCl in mQ water (1.0%, 2.5%, 5.0%, 10.0%, 15.0%) were prepared in separated flasks. </p>
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3. </b>In a petri dish (with LB agar and Chloramphenicol), introduce 1 milliliter of the 1.0% concentration of NaCl, repeat it for a total of two petri dishes. Perform this step over a distance of no more than 30cm of a Bunsen burner to avoid contamination</p><p><b>
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<p><b>
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4. </b>In both both petri dishes introduce 200 microliters of nhaS transformed red bacteria, and distribute the content with a sterile glass inoculation spreader in a distance of less than 30 cm of a Bunsen burner to avoid contamination. </p><p><b>
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2. </b>The three groups of bacteria (RFP+NhaS, NhaS and the contro) were separated in different test tubes. </p>
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5. </b>Repeat step 3 and 4, four times but with the other four concentrations: 2.5%, 5.0%, 10.0%, 15.0%.</p><p><b>
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<p><b>
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6. </b>Repeat steps 3 through 5 two more times, but each time with a different group of bacteria, the white ones and the control. </p><p><b>
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3. </b>In a petri dish with LB agar and Chloramphenicol,1 milliliter   of the 1.0% concentration of NaCl was introduced; this process was repeated in the three petri dishes and in a sterile environment 30 cm near the bunsen burner in order to avoid contamination.</p>
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7. </b>Inoculate 3 petri dishes (must contain only LB agar) with the bacteria without the gene Nhas (control), introducing 200 microliters of it and spreading it with a sterile glass inoculation spreader. </p><p><b>
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<p><b>
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8. </b>Incubate all the 33 total petri dishes at 37º C during a day. </p>
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4. </b>In both both petri dishes, 200 microliters of NhaS transformed red bacteria were introduced, and distributed the content with a sterile glass inoculation spreader in a sterile environment. </p>
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<p><b>
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5. </b>The steps 3 and 4 were repeated four times, but with the other four concentrations (2.5%, 5.0%, 10.0%, 15.0%.). </p>
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<p><b>
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6. </b>The s teps 3 through 5 were repeated two more times, with a the remaining groups of bacteria.</p>
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<p><b>
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7. </b> Three petri dishes with LB agar were inoculated with the control bacteria, introducing 200 microliters of it and spreading it with a sterile glass inoculation spreader. </p>
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<p><b>
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8. </b>The 33 total petri dishes were cultivated at 37º C for 24 hours. </p>
<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_results#Salt2">Go to results</a></p>
<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_results#Salt2">Go to results</a></p>
<br>
<br>
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<p><b>Experiment 3</b></p>
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<p><b>Experiment #3</b></p>
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<p>Procedure: </p><p><b>
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<p>Procedure: </p>
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1. </b>Prepare in flasks of 100mL different concentrations of NaCl in mQ water (1.0%, 2.5%, 5.0%, 10.0%, 15.0%) having a finale volume of 20mL, 3 flasks of each concentration</p><p><b>
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<p><b>
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2. </b>Put 200 uL of NhaS transformed white bacteria in the flasks with each of the five different concentrations Perform this step over a distance of no more than 30 cm of a Bunsen burner to avoid sample contamination. </p><p><b>
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1. </b>In flasks of 100mL, different concentrations of NaCl in mQ water were preparated(1.0%, 2.5%, 5.0%, 10.0%, 15.0%), having a finale volume of 20mL, and were divided into 3 flasks of each concentration</p>
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3. </b>Repeat step 2 but with NhaS transformed red bacteria. </p><p><b>
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<p><b>
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4. </b>Repeat step 2 bur with the control bacteria. At the end there will be 15 flasks. five of each type of bacteria. </p><p><b>
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2. </b> 200 uL of NhaS transformed white bacteria solution was placed in the flasks with each of the five different concentrations in a sterile environment, no more than 30 cm away from a Bunsen burner, to avoid sample contamination. </p>
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5. </b>Incubate the 15 flasks at 37 ºC during a day. </p>
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<p><b>
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<p>Go to results</p>
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3. </b>The previous step was repeated with the other two groups of bacteria. At the end there will be 15 flasks. of bacteria (five of each type). </p>
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<p><b>4. </b>The 15 flasks were incubated at 37 ºC during a day. </p>
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<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_results#Salt2">Go to results</a></p>
<p><a name="AromaExp"></a><h2><b>Aroma module - </b><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_methods#"><font size="2">Return to the Top</font></a></h2></p>
<p><a name="AromaExp"></a><h2><b>Aroma module - </b><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_methods#"><font size="2">Return to the Top</font></a></h2></p>
<b>Qualitative experiment</b>
<b>Qualitative experiment</b>
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<p>In our project, one of the modules consists in the production of an ester with aroma (Winter Green). In order for the gene to work how it is supposed to work, the bacteria must be in an environment with a temperature higher than 32º C. This is because the gene has a constitutive promoter, so in order to control the aroma production, we used temperature. But even if the gene is expressed, it will not have any smell. The reason is because Winter Green odor is produced when the BSTM1 protein comes in contact with salicylic acid. In order to know if the riboswitch is working and at which concentration of salicylic acid smells more, it was performed the following experiment. </p>
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<p>In the project, one of the modules (the aroma module) consisted in the production of a scented ester in (Winter Green). In order for the gene to work how it was supposed to work, the bacteria must be in an environment with a temperature higher than 32º C. This is because the gene has a constitutive promoter. In order to control the aroma production, temperature was used. But even if the gene is expressed, it did not had any smell. The reason was because Winter Green odor is produced when the BSTM1 protein comes in contact with salicylic acid. In order to know if the riboswitch is working and at which concentration of salicylic acid smells the strongest, the following experiment was performed . </p>
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<p>Procedure:</p>
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<p><strong>Procedure:</strong></p>
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<p><b>First part:</b></p><p><b>
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<p><b>First part:</b></p>
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1. </b>Prepare 12 Petri dishes with an LB medium and the Chloramphenicol antibiotic.</p><p><b>
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<p><b>
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2. </b>Add 3 mL of a solution containing salicylic acid and mQ water to 4 Petri dishes, with the concentration of salicylic acid being of 10 mM. </p><p>
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1. </b>12 Petri dishes with an LB medium and the Chloramphenicol antibiotic were prepared.</p>
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*Repeat step noº 2, two more times changing the concentration of 10 mM of salicylic acid to 20 mM and 30 mM. </p><p><b>
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<p><b>
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3. </b>Prepare 6 Petri dishes with LB medium. </p><p><b>
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2.</b>3 mL of a solution containing salicylic acid and mQ water was added to four Petri dishes, with the concentration of salicylic acid being of 10 mM. </p>
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4. </b>Add 3 mL of a solution containing salicylic acid and mQ water to 2 Petri dishes, with the concentration of salicylic acid being of 10 mM. </p><p>
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<p>
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*Repeat step noº 4 two more times changing the concentration of 10 mM of salicylic acid to 20 mM and 30 mM. </p><p><b>
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*The step noº 2, was repeated two more times changing the concentration of 10 mM of salicylic acid to 20 mM and 30 mM. </p>
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5. </b>To all the Petri dishes, add 200 µL of bacteria. </p><p><b>
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<p><b>
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6. </b>Incubate 2 Petri dishes containing Chloramphenicol from each of the three concentrations and 1 Petri dish without Chloramphenicol from each concentration at 29 ºC for one day. </p><p><b>
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3. </b>6 Petri dishes with LB medium were prepared. </p>
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7. </b>Incubate 2 Petri dishes containing Chloramphenicol from each of the three concentrations and 1 Petri dish without Chloramphenicol from each concentration at 35 ºC for one day. </p><p>
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<p><b>
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4. </b>3 mL of a solution containing salicylic acid and mQ water was added into 2 Petri dishes, with the concentration of salicylic acid being of 10 mM. </p>
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<p>
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*The step noº 4 was repeated two more times changing the concentration of 10 mM of salicylic acid to 20 mM and 30 mM. </p>
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<p><b>
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5. </b> 200 µL of bacteriawas added into all of the petri dishes. </p>
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<p><b>
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6. </b>2 Petri dishes containing Chloramphenicol from each of the three concentrations and 1 Petri dish without Chloramphenicol from each concentration were incubated at 29 ºC for one day. </p>
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<p><b>
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7. </b>2 Petri dishes containing Chloramphenicol from each of the three concentrations and 1 Petri dish without Chloramphenicol from each concentration were incubated at 35 ºC for one day. </p>
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<p>
<b>Second part: </b><b>
<b>Second part: </b><b>
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1. </b>Choose random people to smell the bacteria and take video of they reaction: </p><p>
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1. </b>Random people were chosen to smell the bacteria and a video was taken of their experience: </p>
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<p>
- A group of bacteria with the aroma module at a temperature below 32 ºC. </p><p>
- A group of bacteria with the aroma module at a temperature below 32 ºC. </p><p>
- A group of bacteria with the aroma module at a temperature above 32 ºC. </p><p>
- A group of bacteria with the aroma module at a temperature above 32 ºC. </p><p>
- A controlled group of bacteria without the gene at a temperature below 32 ºC. </p><p>
- A controlled group of bacteria without the gene at a temperature below 32 ºC. </p><p>
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- A controlled group of bacteria without the gene at a temperature above 32 ºC. </p><p><b>
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- A controlled group of bacteria without the gene at a temperature above 32 ºC. </p>
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2.  </b>Ask people to describe what they are smelling</p>
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<p><b>
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2.  </b>The people were asked to describe what they were smelling </p>
<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_results#AromaRe1">Go to results</a></p>
<p><a href="https://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_results#AromaRe1">Go to results</a></p>

Revision as of 05:00, 17 June 2014

iGEM CIDEB 2014 - Project

Construction plan and Protocols - Return to the Top

How do we plan to get the construction of our modules? Here is shown all the construction plan that we followed with all the aspects that we considered.

Protocols and Construction Plan PDF

It appears your Web browser is not configured to display PDF files. No worries, just click here to download the PDF file.


Experiments - Return to the Top

In this section, the experiments performed in order to test the effectivness of the predicted models for our proyect, will be described.

The tested parts are the following::

Capture Module

Aroma Module

Capture Module - Return to the Top

UV Experimentation

This experiment was designed in order to test the UV promoter's efectivness.

Procedure:

1. 2 Petri dishes were inoculated according to the "streak method" with transformed bacteria from a cultured petri dish, containing the NhaS and the RFP genes in their plasmids. (These colonies looked red due to the RFP)

2. The first step was repeated, this time with the transformed bacteria cultures containing the NhaS gene only, without the RFP gene. (These colonies looked white due to the absence of the RFP)

3. The bacteria was grown for one day in the incubator at 37°C

4. The four petri dishes were exposed to UV radiation (302nm) during a 2 hour period

5. Pictures were taken of the bacteria cultures at 10 minute intervals for the 2 hour period

Go to results


Viability test of the NhaS gene containing bacteria in salt - Return to the Top

 

In theory, the NhaS gene gives the bacteria a certain resistance to salt, but the exact percentage of increase in the resistance is unknown. Three experiments were designed in order to test:

  • The viability of the NhaS transformed bacteria to survive in a salty environment
  • The maximum ammount of salt in the medium which can be withstanded by the transformed bacteria
  • The transformed bacteria's hability to capture sodium from the medium

In the ligation process of the NhaS gene, two different kinds of plasmids were obtained due to a possible mutation. The ones containing the RFP producing gene, and the ones that did not. Because it was uncertain the presence of the NhaS gene in the non-RFP producing bacteria these experiments were designed and performed in order to determine which one of the groups contain the NhaS gene.

 

Experiment #1

Procedure:

1. Twenty seven, 9 milliliters test tubes were filled with different concentrations of NaCl (0.85%, 0.90%, 1.00%, 1.50%, 2%, 2 .50%, 3.50%, 5.00% and 10.00%) with 3 test tubes per concentration.

2. The different groups of bacteria were separated: white NhaS, red NhaS and bacteria without Nhas gene (Control)

3. The 27 test tube were separated into 3 groups (for each type of bacteria), so that each group had 9 different salt concentrations.

4. In a sterile environment, no more than 30cm away from the bunsen burner, 1000 micro liters (1 mL) of RFP producing NhaS bacteria were added into each of the 9 corresponding test tubes, to obtain a ratio of 1:10.

5. The previous step was repeated with non RFP producing NhaS bacteria and with the non-NhaS producing control bacteria.

6. Two petri dishes, per each concentration, per each bacteria groups were filled with prepared LB medium containing chloramphenicol.

7.One of the nine test tubes with NaCl and bacteria was selected.

8. One milliliter of the contents of the test tube was introduced into the three petri dishes. This step was performed at a distance of not more than 30 cm of a Bunsen burner to avoid sample contamination.

9. A glass inoculation was placed in the spreader into a glass full of alcohol and remove it burning it with a Bunsen burner so that alcohol is removed from the spreader and 5 - 10 seconds were waited before the next step.

11. Spread the content of the test tube into the petri dish. Perform this step over a distance of no more than 30cm of a Bunsen burner to avoid any contamination

12. The previous series of steps were repeated with all of the tubes

13. The 54 petri dishes were incubated (9 concentrations of duplicates for each group of bacteria) at a temperature of 37 ºC during 24 hours.

Go to results


Experiment #2

Procedure:

1. Five concentrations of NaCl in mQ water (1.0%, 2.5%, 5.0%, 10.0%, 15.0%) were prepared in separated flasks.

2. The three groups of bacteria (RFP+NhaS, NhaS and the contro) were separated in different test tubes.

3. In a petri dish with LB agar and Chloramphenicol,1 milliliter of the 1.0% concentration of NaCl was introduced; this process was repeated in the three petri dishes and in a sterile environment 30 cm near the bunsen burner in order to avoid contamination.

4. In both both petri dishes, 200 microliters of NhaS transformed red bacteria were introduced, and distributed the content with a sterile glass inoculation spreader in a sterile environment.

5. The steps 3 and 4 were repeated four times, but with the other four concentrations (2.5%, 5.0%, 10.0%, 15.0%.).

6. The s teps 3 through 5 were repeated two more times, with a the remaining groups of bacteria.

7. Three petri dishes with LB agar were inoculated with the control bacteria, introducing 200 microliters of it and spreading it with a sterile glass inoculation spreader.

8. The 33 total petri dishes were cultivated at 37º C for 24 hours.

Go to results


Experiment #3

Procedure:

1. In flasks of 100mL, different concentrations of NaCl in mQ water were preparated(1.0%, 2.5%, 5.0%, 10.0%, 15.0%), having a finale volume of 20mL, and were divided into 3 flasks of each concentration

2. 200 uL of NhaS transformed white bacteria solution was placed in the flasks with each of the five different concentrations in a sterile environment, no more than 30 cm away from a Bunsen burner, to avoid sample contamination.

3. The previous step was repeated with the other two groups of bacteria. At the end there will be 15 flasks. of bacteria (five of each type).

4. The 15 flasks were incubated at 37 ºC during a day.

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Aroma module - Return to the Top

Qualitative experiment

In the project, one of the modules (the aroma module) consisted in the production of a scented ester in (Winter Green). In order for the gene to work how it was supposed to work, the bacteria must be in an environment with a temperature higher than 32º C. This is because the gene has a constitutive promoter. In order to control the aroma production, temperature was used. But even if the gene is expressed, it did not had any smell. The reason was because Winter Green odor is produced when the BSTM1 protein comes in contact with salicylic acid. In order to know if the riboswitch is working and at which concentration of salicylic acid smells the strongest, the following experiment was performed .

Procedure:

First part:

1. 12 Petri dishes with an LB medium and the Chloramphenicol antibiotic were prepared.

2.3 mL of a solution containing salicylic acid and mQ water was added to four Petri dishes, with the concentration of salicylic acid being of 10 mM.

*The step noº 2, was repeated two more times changing the concentration of 10 mM of salicylic acid to 20 mM and 30 mM.

3. 6 Petri dishes with LB medium were prepared.

4. 3 mL of a solution containing salicylic acid and mQ water was added into 2 Petri dishes, with the concentration of salicylic acid being of 10 mM.

*The step noº 4 was repeated two more times changing the concentration of 10 mM of salicylic acid to 20 mM and 30 mM.

5. 200 µL of bacteriawas added into all of the petri dishes.

6. 2 Petri dishes containing Chloramphenicol from each of the three concentrations and 1 Petri dish without Chloramphenicol from each concentration were incubated at 29 ºC for one day.

7. 2 Petri dishes containing Chloramphenicol from each of the three concentrations and 1 Petri dish without Chloramphenicol from each concentration were incubated at 35 ºC for one day.

Second part: 1. Random people were chosen to smell the bacteria and a video was taken of their experience:

- A group of bacteria with the aroma module at a temperature below 32 ºC.

- A group of bacteria with the aroma module at a temperature above 32 ºC.

- A controlled group of bacteria without the gene at a temperature below 32 ºC.

- A controlled group of bacteria without the gene at a temperature above 32 ºC.

2. The people were asked to describe what they were smelling

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