Team:Groningen/project characterisation mu lasr

From 2011.igem.org

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==LasR Autoinducing Loops==
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{{HeaderGroningen2011}}
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Our second memory unit consists of a LasR gene(BBa_) driven the LasB promoter(BBa_). LasR only activates transcription in the presence of HSL, this was added to the medium in a concentration of 100nM.  
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=LasR autoinducing loop family=
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Our second memory unit consists of a LasR gene(Bba_C0179) driven the LasB promoter(Bba_R0079). LasR only activates transcription in the presence of HSL, this was added to the medium in a concentration of 100nM.
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==Leakage of the LasR autoinducing loops==
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To estimate the autoactivation of the LasR loop through a leaky promoter we measured fluorescence of cells carrying an pSB1K3 plasmid with the autoinducing loop biobrick (different variants) and the plasmid pSB1A3 with an input(PLacI controlling LasR) as well as the output (PlasB controlling RFP-LVA). The measurements were done in a presence of 100nM PAI.
{| class="wikitable"
{| class="wikitable"
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! colspan="2" | cI autoinducing loop 1 (BBa_K00) after 4 hours of incubation
 
|-
|-
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| [[File:ci1ui.png]]
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| || no-tag|| DAS || LVA ||   
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Figure 1. A dotplot of uninduced cells carrying the pSB1K3 plasmid with the biobrick BBa_ and the plasmid pSB1A3 with as well an input(PBAD controlling cI) as well as the output (PRM controlling GFP-LVA).
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{| class="wikitable"
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!  
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! Number
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! % of total
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! Mean fluorescence
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|-
|-
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| Visible
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| RBS 0,07 || <gallery>File:lasR9.png | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K607031 BBa_K607031] </gallery> || <gallery>File:lasR5.png | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K607027 BBa_K607027] </gallery>
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| 50000
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  ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607023 BBa_K607023]
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| 100
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|rowspan="4"| [[File:legenda.png]]
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| ---
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|-
|-
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| Fluorescent || 9180 || 18,36 || 11419
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| RBS 0,3 || <gallery>File:lasR10.png| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K607032 BBa_K607032]</gallery> || <gallery>File:lasR6.png| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K607028 BBa_K607028]</gallery> || <gallery>File:lasR2.png| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K607024 BBa_K607024]</gallery>
|-
|-
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| Non-Fluorescent|| 34424 || 68 || 5974
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| RBS 0,6 || <gallery> File:lasR11.png|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607033 BBa_K607033] </gallery>
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|}
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|| <gallery>File:lasr7.png| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K607029 BBa_K607029]</gallery> || <gallery>File:lasr3.png| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K607025 BBa_K607025]</gallery>
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|-
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| colspan=2 | Two populations are visible on the dotplot. We seperated both populations on the histogram to show that the major peak of nonfluorescent cells overlaps the minor peak of fluorescenting cells.
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| RBS 1 || <gallery>File:lasr12.png| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K607034 BBa_K607034]</gallery> || <gallery>File:lasr8.png| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K607030 BBa_K607030]</gallery> || <gallery>File:lasR4.png| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K607026 BBa_K607026]</gallery>
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|}
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{| class="wikitable"
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! colspan="2" | cI autoinducing loop 6 (BBa_K00) after 4 hours of incubation
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|-
|-
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| [[File:ci6ui.png]]
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|colspan="4"| '''Figure 1.''' Flow cytometer data obtained at different time-points during exponential growth of cells carrying different LasR autoinducing loops in presence of 100 nM HSL.
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Figure 2. A dotplot of uninduced cells carrying the pSB1K3 plasmid with the biobrick BBa_ and the plasmid pSB1A3 with as well an input(PBAD controlling cI) as well as the output (PRM controlling GFP-LVA).
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|  
|  
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{| class="wikitable"
 
-
! 
 
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! Number
 
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! % of total
 
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! Mean fluorescence
 
-
|-
 
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| Visible
 
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| 50000
 
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| 100
 
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| ---
 
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|-
 
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| Fluorescent || 9180 || 18,36 || 11419
 
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|-
 
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| Non-Fluorescent|| 34424 || 68 || 5974
 
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|}
 
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|-
 
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| colspan=2 | Only one population is visible on the dotplot. The histogram was obtained in the same fashion as in figure 1 to show that there are cultures with activated cI loops and cultures without.
 
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|}
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{{FooterGroningen2011}}

Latest revision as of 23:35, 21 September 2011


LasR autoinducing loop family

Our second memory unit consists of a LasR gene(Bba_C0179) driven the LasB promoter(Bba_R0079). LasR only activates transcription in the presence of HSL, this was added to the medium in a concentration of 100nM.

Leakage of the LasR autoinducing loops

To estimate the autoactivation of the LasR loop through a leaky promoter we measured fluorescence of cells carrying an pSB1K3 plasmid with the autoinducing loop biobrick (different variants) and the plasmid pSB1A3 with an input(PLacI controlling LasR) as well as the output (PlasB controlling RFP-LVA). The measurements were done in a presence of 100nM PAI.

no-tag DAS LVA
RBS 0,07

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607031 BBa_K607031]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607027 BBa_K607027]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607023 BBa_K607023] Legenda.png
RBS 0,3

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607032 BBa_K607032]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607028 BBa_K607028]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607024 BBa_K607024]

RBS 0,6

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607033 BBa_K607033]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607029 BBa_K607029]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607025 BBa_K607025]

RBS 1

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607034 BBa_K607034]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607030 BBa_K607030]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607026 BBa_K607026]

Figure 1. Flow cytometer data obtained at different time-points during exponential growth of cells carrying different LasR autoinducing loops in presence of 100 nM HSL.




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