Difference between revisions of "Part:BBa I13600:Experience"

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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
how you used this part and how it worked out.
 
how you used this part and how it worked out.
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===Applications of BBa_I13600===
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2013 KIT-Kyoto iGEM team assessed influence of fluorescence proteins on the growth of E.coli.
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We used BBa_I13521, BBa_I13522 and BBa_I13600 in order to measure the growth.
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[[File:Kairyo2.png|500px]]
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We measured the turbidities of the transformants at hourly intervals and made the growth curves.
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We transferred the transformant prepared to 200 mL flasks.
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We measured the turbidities every 1 hour.
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The measurements were carried out for 12 hours.
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Liquid LB 50mL
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Sample 20μL
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Ampicillin 75μL
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37°C, 125rpm
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Control:HST08
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As the result, it was found that eCFP and mRFP have the effect of growth promotion relative to GFP.
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===Applications of BBa_I13600===
 
===Applications of BBa_I13600===
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This review comes from the old result system and indicates that this part worked in some test.
 
This review comes from the old result system and indicates that this part worked in some test.
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|}
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<!-- Aberdeen_Scotland 2009 user review -->
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{|width='80%' style='border:1px solid gray'
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<partinfo>BBa_I13600 AddReview 5</partinfo>
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<I> Aberdeen_Scotland 2009 </I>
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|width='60%' valign='top'|
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<html><p align="justify">
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The gel analysis and the plasmid miniprep worked quite nicely as expected. But later the construct was further tested and characterized by combining with the bio-brick BBa_K182005 which contained a tet repressor under the control of a CI operator. So, the CFP expression of BBa_I13600was repressed as expected when tested with the BBa_K182005. With the addition of Anhydrotetracycline (an analogue of tetracycline) the CFP expression was restored and this was confirmed performing the fluorescent microscopy assay.
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<br><br>
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Expression can be visualized very nicely under a fluorescent microscope. Even without having ideal filters. We used a GFP filter for the picture below.
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</p></html>
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[[Image:2 I13600 flu60aberdeen2009.jpg|center|400 px]]
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Transforming BBa_K182005 into these cells should stop the production of CFP, since it expresses TetR. If it works as anticipated, the K182005 Tet repressor should bind to the Tet Operator of I13600 which would stop the expression of CFP.
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<br>
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[[Image:1DanAberdeenigem2009fixed.png|center|400 px]]
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<br>
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Since K182005 is on an Ampicillin / chloramphenicol vector (pSB1AC3) and I13600 is on an Ampicillin vector, we decided to first transform I13600 into a cell and select for Ampicillin. We then checked for CFP expression and made the cells competent. After this we transformed K182005 into these cells and selected for Chloramphenicol.<br> <br>
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With both plasmids now in the cell we checked for fluorescence, in the presence and absence of anhydrotetracycline (AHT or ATc), as can be seen in the picture below. <br><br>
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[[Image:4 K182005-I13600 flu no AHTAberdeen2009.jpg|center|400 px]]
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<br>
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[[Image:16 K182005-I13600 no flu 1.8 AHTAberdeen2009.jpg|center|400 px]]
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<br>
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The above Pictures demonstrate that TetR effectivly binds to the I13600 Tet Operator.
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<!-- DON'T DELETE --><partinfo>BBa_I13600 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_I13600 EndReviews</partinfo>

Latest revision as of 12:41, 27 September 2013

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_I13600

2013 KIT-Kyoto iGEM team assessed influence of fluorescence proteins on the growth of E.coli. We used BBa_I13521, BBa_I13522 and BBa_I13600 in order to measure the growth.

Kairyo2.png

We measured the turbidities of the transformants at hourly intervals and made the growth curves. We transferred the transformant prepared to 200 mL flasks. We measured the turbidities every 1 hour. The measurements were carried out for 12 hours.

Liquid LB 50mL

Sample 20μL

Ampicillin 75μL

37°C, 125rpm

Control:HST08

As the result, it was found that eCFP and mRFP have the effect of growth promotion relative to GFP.


Applications of BBa_I13600

  • Grew up this Biobrick on a plate just to test its flourescence compared to the other constitutive flourescent proteins in the Registry as of July 2006. This has one of the weakest flourescent outputs. --Smelissali 19:39, 3 July 2006 (EDT)

User Reviews

UNIQ08aacf8d772a914d-partinfo-00000000-QINU

••••

Smelissali

Brick works, but the cyan flourescent protein is definitely not the brightest reporter you could use, and thus not my first choice. --Smelissali 19:38, 3 July 2006 (EDT)

•••••

Antiquity

This review comes from the old result system and indicates that this part worked in some test.

•••••

Aberdeen_Scotland 2009

The gel analysis and the plasmid miniprep worked quite nicely as expected. But later the construct was further tested and characterized by combining with the bio-brick BBa_K182005 which contained a tet repressor under the control of a CI operator. So, the CFP expression of BBa_I13600was repressed as expected when tested with the BBa_K182005. With the addition of Anhydrotetracycline (an analogue of tetracycline) the CFP expression was restored and this was confirmed performing the fluorescent microscopy assay.

Expression can be visualized very nicely under a fluorescent microscope. Even without having ideal filters. We used a GFP filter for the picture below.

2 I13600 flu60aberdeen2009.jpg

Transforming BBa_K182005 into these cells should stop the production of CFP, since it expresses TetR. If it works as anticipated, the K182005 Tet repressor should bind to the Tet Operator of I13600 which would stop the expression of CFP.

1DanAberdeenigem2009fixed.png


Since K182005 is on an Ampicillin / chloramphenicol vector (pSB1AC3) and I13600 is on an Ampicillin vector, we decided to first transform I13600 into a cell and select for Ampicillin. We then checked for CFP expression and made the cells competent. After this we transformed K182005 into these cells and selected for Chloramphenicol.

With both plasmids now in the cell we checked for fluorescence, in the presence and absence of anhydrotetracycline (AHT or ATc), as can be seen in the picture below.

4 K182005-I13600 flu no AHTAberdeen2009.jpg


16 K182005-I13600 no flu 1.8 AHTAberdeen2009.jpg


The above Pictures demonstrate that TetR effectivly binds to the I13600 Tet Operator.

UNIQ08aacf8d772a914d-partinfo-00000005-QINU