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Team:BostonU/Collaborations
From 2014.igem.org
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They gave us two copies of the same construct, one in a chloramphenicol resistant backbone and the other in an ampicillin resistant backbone. The device has <a href="http://parts.igem.org/Part:BBa_K1423005"> BclA-YFP </a>, which is a cell surface targeted protein to express YFP on the cell surface. We also ran one of our own single color YFP controls, which is listed at "Internal YFP" as this is not targeted to the cell surface. It should be noted that this control is in a kanamycin backbone. | They gave us two copies of the same construct, one in a chloramphenicol resistant backbone and the other in an ampicillin resistant backbone. The device has <a href="http://parts.igem.org/Part:BBa_K1423005"> BclA-YFP </a>, which is a cell surface targeted protein to express YFP on the cell surface. We also ran one of our own single color YFP controls, which is listed at "Internal YFP" as this is not targeted to the cell surface. It should be noted that this control is in a kanamycin backbone. | ||
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| + | The results our shown below. For details on how we converted the arbitrary YFP units to MEFLs, please see the <a href="https://2014.igem.org/Team:BostonU/Software#flow">TASBE Tools</a> page for information about controls required to do that conversion. | ||
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| + | <td scope="col"><img src="https://static.igem.org/mediawiki/2014/c/c8/WPI_Team_Logo.png" width="220px" alt="WPIlogo" style="float:right"></td></tr> | ||
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<center><img src="https://static.igem.org/mediawiki/2014/0/0e/Bar_graphs_YFP.png" width="45%"></center> | <center><img src="https://static.igem.org/mediawiki/2014/0/0e/Bar_graphs_YFP.png" width="45%"></center> | ||
<center><capt>Fluorescent measurements of three devices expressing YFP. The BclA-YFP parts were provided by WPI while the Internal YFP is one of the control parts provided by Boston University.</capt></center> | <center><capt>Fluorescent measurements of three devices expressing YFP. The BclA-YFP parts were provided by WPI while the Internal YFP is one of the control parts provided by Boston University.</capt></center> | ||
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The pTet-pBad-RFP or pBad-pTet-RFP expressing E. coli strains were cultured in LB with kanamycin plus the additives indicated overnight at 37°C overnight with shaking. 20µL samples of each culture were dropped onto microscope slides and allowed to air dry. Slides were then fixed in methanol for 10 minutes, dried, and wet mounted with a coverslip in a 50% glycerol solution. Slides were imaged on a Zeiss AxioVert microscope with an 100x oil immersion lens, and imaged with a Zeiss AxioCam MRm camera. Images were acquired using Zen software, and processed with Adobe Photoshop. All images were exposed for 2 seconds. Brightness and contrast adjustments made during processing were applied equally to all panels within the figure. <br><br> | The pTet-pBad-RFP or pBad-pTet-RFP expressing E. coli strains were cultured in LB with kanamycin plus the additives indicated overnight at 37°C overnight with shaking. 20µL samples of each culture were dropped onto microscope slides and allowed to air dry. Slides were then fixed in methanol for 10 minutes, dried, and wet mounted with a coverslip in a 50% glycerol solution. Slides were imaged on a Zeiss AxioVert microscope with an 100x oil immersion lens, and imaged with a Zeiss AxioCam MRm camera. Images were acquired using Zen software, and processed with Adobe Photoshop. All images were exposed for 2 seconds. Brightness and contrast adjustments made during processing were applied equally to all panels within the figure. <br><br> | ||
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| - | <center><img src="https://static.igem.org/mediawiki/2014/a/af/BTWPICollab.png" | + | <center><img src="https://static.igem.org/mediawiki/2014/4/47/TBWPICollab.png" height="300px"></center> |
| - | <capt><center>Micrographs showing the expression of RFP and GFP at different small molecule concentrations for pBAD+pTet+RFP </center></capt>< | + | </td> |
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| + | <center><img src="https://static.igem.org/mediawiki/2014/a/af/BTWPICollab.png" height="300px"></center> | ||
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| + | <capt><center>Micrographs showing the expression of RFP and GFP at different small molecule concentrations for pTet+pBAD+RFP </center></capt></td> | ||
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| + | <capt><center>Micrographs showing the expression of RFP and GFP at different small molecule concentrations for pBAD+pTet+RFP </center></capt></td></tr> | ||
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Arabinose is clearly inducing RFP induction but aTc induction is weak at best. This may have been due to a mix up in the media we gave them. Likewise, the presence of GFP in some of the cells suggests we may have gave them a mixed culture since we didn't see GFP for the same constructs when we ran flow cytometry analysis. The change in RFP under arabinose suggests that there are cells with the correct construct growing in the culture. For a more detailed result analysis, click<a href="https://2014.igem.org/Team:BostonU/Data"> here </a>.<br><br> | Arabinose is clearly inducing RFP induction but aTc induction is weak at best. This may have been due to a mix up in the media we gave them. Likewise, the presence of GFP in some of the cells suggests we may have gave them a mixed culture since we didn't see GFP for the same constructs when we ran flow cytometry analysis. The change in RFP under arabinose suggests that there are cells with the correct construct growing in the culture. For a more detailed result analysis, click<a href="https://2014.igem.org/Team:BostonU/Data"> here </a>.<br><br> | ||
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| - | <h3> | + | <h3> Interlab Study Collaboration with Tufts iGEM </a> and MIT iGEM teams</h3> |
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| - | In August, researchers from the <a href="https://2014.igem.org/Team:Tufts"> Tufts iGEM </a> team contacted us and described that they had been having trouble transforming some parts, which were required for the <a href="https://2014.igem.org/Tracks/Measurement/Interlab_study"> | + | In August, researchers from the <a href="https://2014.igem.org/Team:Tufts"> Tufts iGEM </a> team contacted us and described that they had been having trouble transforming some parts, which were required for the <a href="https://2014.igem.org/Tracks/Measurement/Interlab_study"> Interlab Study </a>, into cells. The parts that they needed were - |
<ul><li> BBa_I20260 | <ul><li> BBa_I20260 | ||
<li>BBa_E0240 | <li>BBa_E0240 | ||
</ul><br> | </ul><br> | ||
| - | We located those parts, members from the Tufts team picked | + | We quickly located those parts in our freezer, aliquoted a few microliters of the plasmid out for them, and members from the Tufts team picked up the DNA so they could continue working towards finishing the Interlab Study. |
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| - | During the 3rd NEGEM Meetup in October, <a href="https://2014.igem.org/Team:MIT"> MIT iGEM </a> also expressed interest in taking part in the | + | During the 3rd NEGEM Meetup in October, <a href="https://2014.igem.org/Team:MIT"> MIT iGEM </a> also expressed interest in taking part in the Interlab Study. We promptly streaked all required constructs on agar plates for them, which they could then use to make cultures and test on their Flow Cytometer. |
<br><br> | <br><br> | ||
| - | Our results for the study can be found on our <a href="https://2014.igem.org/Team:BostonU/Interlab"> | + | Our results for the study can be found on our <a href="https://2014.igem.org/Team:BostonU/Interlab"> Interlab Study </a> page. |
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Latest revision as of 03:06, 18 October 2014
WPI Worcester Polytechnic Institute | |
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This year, the BU iGEM team collaborated with the WPI-Worcester iGEM team. The collaboration became possible at the NEGEM meet up in June, where six New England iGEM teams presented their project ideas to the rest. During one of our small group discussions, a team member from WPI talked about how lucky we, the BU team, were to get access to a flow cytometer for single cell fluorescence measurement. Right then, the two teams decided to collaborate: we would test some of their constructs on our flow cytometer and they would view cells expressing our devices with their confocal microscope. They gave us two copies of the same construct, one in a chloramphenicol resistant backbone and the other in an ampicillin resistant backbone. The device has BclA-YFP , which is a cell surface targeted protein to express YFP on the cell surface. We also ran one of our own single color YFP controls, which is listed at "Internal YFP" as this is not targeted to the cell surface. It should be noted that this control is in a kanamycin backbone. The results our shown below. For details on how we converted the arbitrary YFP units to MEFLs, please see the TASBE Tools page for information about controls required to do that conversion. |
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 The WPI team took micrographs of cells containing tandem promoter- RFP testing constructs. We gave them media with two concentrations of arabinose (1 mM and 100 mM) and aTp (100 ng/mL and 1000 ng/mL). The used the following methodology for microscopy: The pTet-pBad-RFP or pBad-pTet-RFP expressing E. coli strains were cultured in LB with kanamycin plus the additives indicated overnight at 37°C overnight with shaking. 20µL samples of each culture were dropped onto microscope slides and allowed to air dry. Slides were then fixed in methanol for 10 minutes, dried, and wet mounted with a coverslip in a 50% glycerol solution. Slides were imaged on a Zeiss AxioVert microscope with an 100x oil immersion lens, and imaged with a Zeiss AxioCam MRm camera. Images were acquired using Zen software, and processed with Adobe Photoshop. All images were exposed for 2 seconds. Brightness and contrast adjustments made during processing were applied equally to all panels within the figure. | |
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Arabinose is clearly inducing RFP induction but aTc induction is weak at best. This may have been due to a mix up in the media we gave them. Likewise, the presence of GFP in some of the cells suggests we may have gave them a mixed culture since we didn't see GFP for the same constructs when we ran flow cytometry analysis. The change in RFP under arabinose suggests that there are cells with the correct construct growing in the culture. For a more detailed result analysis, click here . |
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Interlab Study Collaboration with Tufts iGEM and MIT iGEM teams
In August, researchers from the Tufts iGEM team contacted us and described that they had been having trouble transforming some parts, which were required for the Interlab Study , into cells. The parts that they needed were -
We quickly located those parts in our freezer, aliquoted a few microliters of the plasmid out for them, and members from the Tufts team picked up the DNA so they could continue working towards finishing the Interlab Study. During the 3rd NEGEM Meetup in October, MIT iGEM also expressed interest in taking part in the Interlab Study. We promptly streaked all required constructs on agar plates for them, which they could then use to make cultures and test on their Flow Cytometer. Our results for the study can be found on our Interlab Study page. |
