Difference between revisions of "Part:BBa K639003:Experience"
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<h2>Imperial iGEM 2013</h2>
 
<h2>Imperial iGEM 2013</h2>
  
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We intended on using [https://parts.igem.org/Part:BBa_K639003 BBa_K639003] to detect whether our cells were stressed when grown in mixed waste, waste conditioned media or in the presence of plastic toxic byproducts. However, as the data below shows, this mCherry biosensor is very leaky, with clear expression in an un-induced or non-stressed state. However based upon our characterisation data, IPTG does induce mCherry expression as expected and therefore we have confirmed that the sensor is likely to be functional. We recommend that future teams try to improve the part by reducing its leakyness.
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<h3>Stress biosensor characterisation (BBa_K639003)</h3>
  
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We did however find the sensor to be useful to our project as a marker of our chassis in extended waste toxicity assays. All of our data is included below.
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Originally we intended on using [https://parts.igem.org/Part:BBa_K639003 BBa_K639003] to detect whether our cells were stressed when placed in various toxic byproducts. However, as the data below shows, this biobrick is very leaky. As such, as an alternative we utilised the stress sensor as a marker for our chassis E. coli (MG1655) for an array of qualitative and quantitative waste growth and toxicity assays.
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mCherry production is induced by the stress pathway and detection of ppGpps. Induction with IPTG bypassess this mechanism through an inhibition of LacI, resulting in mCherry expression.  
  
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<h3>IPTG induction</h3>
 
  
 
{| class="wikitable" style="margin: 1em auto 1em auto;"
 
{| class="wikitable" style="margin: 1em auto 1em auto;"
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| [[File:OD_stress.png|thumbnail|right|400px|Cell growth over 6h with IPTG induction. mCherry production is induced by the stress pathway and detection of ppGpps. In order to bypass this, we induced with IPTG which inhibits LacI, resulting in mCherry expression.]]
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|[[File:OD_stress.png|thumbnail|right|400px|<b>Stress sensor growth assay</b>E. coli (MG1655) transformed with stress biosensor (BBa_K639003) and grown at 37oC, with shaking for 6 hours. Error bars represent S.E.M. n=4 ]]
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| [[File:Fluorescence_stress.png|thumbnail|right|400px|Fluorescence of the cells under IPTG induction over a 6h period.]]
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|[[File:Fluorescence_stress.png|thumbnail|right|400px|<b>Stress sensor IPTG induced fluorescence</b>E. coli (MG1655) transformed with stress biosensor (BBa_K639003) and grown at 37oC, with shaking for 6 hours. Error bars represent S.E.M. n=4]]
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[[File:StressResponse.jpg|400px|thumb|left|BBa_K639003 transformed into E coli. strain MG1655. Pink colonies are visible, which relate to 'leaky' RFP production]]
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|[[File:StressResponse.jpg|400px|thumb|left|<b>BBa_K639003 transformed into E coli. strain MG1655.</b> Pink colonies are visible, which relate to 'leaky' mCherry production]]
  
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[[File:K639003IPTG.jpeg|500px|thumb|left|BBa_K639003 transformed into E coli. strain MG1655. Cells were grown at 37oC in 4ml LB with 0, 1 or 2mM IPTG. At 6 hours post IPTG induction, cells were spun down.]]
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|[[File:K639003IPTG.jpeg|500px|thumb|left|<b>BBa_K639003 transformed into E coli. strain MG1655.</b> Cells were grown at 37oC in 4ml LB with 0, 1 or 2mM IPTG. At 6 hours post IPTG induction, cells were spun down and imaged.]]
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<h3>Waste growth assays</h3>
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Insert data
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Revision as of 23:17, 28 September 2013

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Applications of BBa_K639003

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Imperial iGEM 2013

Stress biosensor characterisation (BBa_K639003)

Originally we intended on using BBa_K639003 to detect whether our cells were stressed when placed in various toxic byproducts. However, as the data below shows, this biobrick is very leaky. As such, as an alternative we utilised the stress sensor as a marker for our chassis E. coli (MG1655) for an array of qualitative and quantitative waste growth and toxicity assays.

mCherry production is induced by the stress pathway and detection of ppGpps. Induction with IPTG bypassess this mechanism through an inhibition of LacI, resulting in mCherry expression.


Stress sensor growth assayE. coli (MG1655) transformed with stress biosensor (BBa_K639003) and grown at 37oC, with shaking for 6 hours. Error bars represent S.E.M. n=4
Stress sensor IPTG induced fluorescenceE. coli (MG1655) transformed with stress biosensor (BBa_K639003) and grown at 37oC, with shaking for 6 hours. Error bars represent S.E.M. n=4
BBa_K639003 transformed into E coli. strain MG1655. Pink colonies are visible, which relate to 'leaky' mCherry production
BBa_K639003 transformed into E coli. strain MG1655. Cells were grown at 37oC in 4ml LB with 0, 1 or 2mM IPTG. At 6 hours post IPTG induction, cells were spun down and imaged.