Difference between revisions of "Part:BBa K117002:Experience"

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===Applications of BBa_K117002===
 
===Applications of BBa_K117002===
 
This promoter is activated indirectly by AI-2 to promote whatever downstream gene ligated behind it.
 
This promoter is activated indirectly by AI-2 to promote whatever downstream gene ligated behind it.
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The 2015 Genspace iGEM Team replaced this part with a working version of pLsrA by submitting part BBa_K1799000. We have shown that it works in the device BBa_K1799022.
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===Characterisation:===
 
===Characterisation:===
 
'''For information on characterisation of this new part, please visit [https://parts.igem.org/Part:BBa_K117010:Experience Part K117010 Experience] and [https://parts.igem.org/Part:BBa_K117008:Experience Part K117008 Experience]'''
 
'''For information on characterisation of this new part, please visit [https://parts.igem.org/Part:BBa_K117010:Experience Part K117010 Experience] and [https://parts.igem.org/Part:BBa_K117008:Experience Part K117008 Experience]'''
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'''For information on the characterization of the new Genspace 2015 version of this part visit [https://parts.igem.org/Part:BBa_K1799000 BBa_K1799000]'''
 
===User Reviews===
 
===User Reviews===
 
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{|width='80%' style='border:1px solid gray'
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|-
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|width='10%'|
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<partinfo>BBa_K117002 AddReview 1</partinfo>
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<I>Tokyo Tech 2011</I>
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|width='60%' valign='top'|
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[[Image:PlsrA2.png|thumb|center|600px|Fluorescence intensity of promoter lsrA-gfp((BBa_K117002)-gfp) was almost the same as promoterless-gfp(negative control).<br>Strain used in this assay lacks <i>lsrR</i>.<br>This work is done by Takuya Tsubaki.]]
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This part(BBa_K117002) does not work properly. To confirm this, we introduced a <i>gfp</i> gene(BBa_J54103) downstream of the promoter. As a consequence, fluorescence intensity of lsrA promoter-<i>gfp</i>((BBa_K117002)-<i>gfp</i>) was almost the same as promoterless-<i>gfp</i>(negative control), showing that lsrA promoter(BBa_K117002) does not work properly. In spite of no LsrR repression, gene transcription does not take place sufficiently.
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We improved this part.Our new lsrA promoter([https://parts.igem.org/Part:BBa_K649104:Experience BBa_K649100]) works.
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If you want to know why our part works, click [http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5. here] and visit our team's wiki.
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[sample]
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Ptet-<i>gfp</i> on pSB1A2(JD22597)
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Promoterless-<i>gfp</i> on pSB6A1(JD22597)
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PlsrA-<i>gfp</i> on pSB1A2(BBa_K649104)(JD22597)
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PlsrA-<i>gfp</i> on pSB1A2(BBa_K117002-<i>gfp</i>)(JD22597)
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JD22597 is a strain lacking <i>lsrR</i>.
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[Method]
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1.Overnight cultures of reporter strains grown at 37 °C in LB medium containing appropriate antibiotics were diluted 1:100 into 3 ml of LB medium and were incubated at 37 °C as fresh cultures.
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2. After their OD590 reached 0.15, the fresh cultures were diluted 1:100.
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3. After 4-hour incubation at 37 °C, 1 ml of each culture was moved to 1.6ml tube and its fluorescence intensity was measured with a flow cytometer.
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|};

Latest revision as of 03:55, 19 September 2015

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_K117002

This promoter is activated indirectly by AI-2 to promote whatever downstream gene ligated behind it.

The 2015 Genspace iGEM Team replaced this part with a working version of pLsrA by submitting part BBa_K1799000. We have shown that it works in the device BBa_K1799022.

Characterisation:

For information on characterisation of this new part, please visit Part K117010 Experience and Part K117008 Experience

For information on the characterization of the new Genspace 2015 version of this part visit BBa_K1799000

User Reviews

UNIQ4e8fece5fd8f69b3-partinfo-00000000-QINU UNIQ4e8fece5fd8f69b3-partinfo-00000001-QINU

•

Tokyo Tech 2011

Fluorescence intensity of promoter lsrA-gfp((BBa_K117002)-gfp) was almost the same as promoterless-gfp(negative control).
Strain used in this assay lacks lsrR.
This work is done by Takuya Tsubaki.

This part(BBa_K117002) does not work properly. To confirm this, we introduced a gfp gene(BBa_J54103) downstream of the promoter. As a consequence, fluorescence intensity of lsrA promoter-gfp((BBa_K117002)-gfp) was almost the same as promoterless-gfp(negative control), showing that lsrA promoter(BBa_K117002) does not work properly. In spite of no LsrR repression, gene transcription does not take place sufficiently.


We improved this part.Our new lsrA promoter(BBa_K649100) works.


If you want to know why our part works, click [http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#5. here] and visit our team's wiki.

[sample]

Ptet-gfp on pSB1A2(JD22597)

Promoterless-gfp on pSB6A1(JD22597)

PlsrA-gfp on pSB1A2(BBa_K649104)(JD22597)

PlsrA-gfp on pSB1A2(BBa_K117002-gfp)(JD22597)

JD22597 is a strain lacking lsrR.

[Method]

1.Overnight cultures of reporter strains grown at 37 °C in LB medium containing appropriate antibiotics were diluted 1:100 into 3 ml of LB medium and were incubated at 37 °C as fresh cultures.

2. After their OD590 reached 0.15, the fresh cultures were diluted 1:100.

3. After 4-hour incubation at 37 °C, 1 ml of each culture was moved to 1.6ml tube and its fluorescence intensity was measured with a flow cytometer.



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