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Team:Aberdeen Scotland/WetLab/quorumsensing/results
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<br><br>Those results suggest that <partinfo>BBa_J37032</partinfo> is not responsive to Quorum Sensing as it does not need LuxI/LuxR to produce GFP. Or as an alternative the pLux promoter could be extremely leaky. Hence, the pIR construct was further tested with the pLux responsive promoter construct <partinfo>BBa_K182103</partinfo> this team created. <br><br> | <br><br>Those results suggest that <partinfo>BBa_J37032</partinfo> is not responsive to Quorum Sensing as it does not need LuxI/LuxR to produce GFP. Or as an alternative the pLux promoter could be extremely leaky. Hence, the pIR construct was further tested with the pLux responsive promoter construct <partinfo>BBa_K182103</partinfo> this team created. <br><br> | ||
| - | Apart from testing the pIR construct, the AND-gate, i.e. part <partinfo>BBa_K182103</partinfo> (further referred to as pLG) was also tested in this experimental set-up. <partinfo>BBa_K182103</partinfo> needs two inputs to initiate the transcription of GFP, firstly IPTG to release LacO repression and secondly Quorum Sensing for triggering the pLux promoter. <br><br> SCS1 cells were transformed with either 1) ptrc99A and pIR, 2) ptrc99A and pLG or 3) ptrc99A, pIR and pLG. The plasmid ptrc99A contains lacI<sup>q</ | + | Apart from testing the pIR construct, the AND-gate, i.e. part <partinfo>BBa_K182103</partinfo> (further referred to as pLG) was also tested in this experimental set-up. <partinfo>BBa_K182103</partinfo> needs two inputs to initiate the transcription of GFP, firstly IPTG to release LacO repression and secondly Quorum Sensing for triggering the pLux promoter. <br><br> SCS1 cells were transformed with either 1) ptrc99A and pIR, 2) ptrc99A and pLG or 3) ptrc99A, pIR and pLG. The plasmid ptrc99A contains lacI<sup>q</sup> which over-produces LacI. This is needed to sufficiently suppress the Lac operator in the pLG construct in the starting cultures. |
[[Image:PIR_figure.jpg|center|600 px]] | [[Image:PIR_figure.jpg|center|600 px]] | ||
Revision as of 21:21, 27 September 2009
University of Aberdeen - Pico Plumber
Results and Discussion
Aims and background for testing our Quorum Sensing construct
In our first experimental set-up, we wanted to test whether our Quorum Sensing construct (further referred to as pIR) works on its own, and induces a pLux promotor to enhance trancription. The testing construct , which was used, consists of a pLux promoter and the gene for the green fluorescence protein. Therefore, it is expected that without Quorum Sensing construct no fluorescence occur.
For this purpose SCS1 E.coli cells were transformed with 1) pIR alone, 2) alone, and 3) pIR and J37032 together. The first one is the negative control. Without any GFP gene inside the cells they should not fluorescence. The second set of cells should show no fluorescence as the promoter needs to be activated by Quorum Sensing or only to a small degree due to leakiness of the promoter. The third set should show low fluorescence at low cell density and high fluorescence at high cell density.
Pictures were taking from over night cultures, i.e. at high cell density, under a light microscope (left hand-side) and a fluorescence microscope (right hand-side) from the 3 different SCS1 E.coli cell transformances. No fluorescence were shown on the pIR only transformance. However, the J37032 transformance shows as high a fluorescent as the double transformance.
Those results suggest that is not responsive to Quorum Sensing as it does not need LuxI/LuxR to produce GFP. Or as an alternative the pLux promoter could be extremely leaky. Hence, the pIR construct was further tested with the pLux responsive promoter construct this team created.
Apart from testing the pIR construct, the AND-gate, i.e. part (further referred to as pLG) was also tested in this experimental set-up. needs two inputs to initiate the transcription of GFP, firstly IPTG to release LacO repression and secondly Quorum Sensing for triggering the pLux promoter.
SCS1 cells were transformed with either 1) ptrc99A and pIR, 2) ptrc99A and pLG or 3) ptrc99A, pIR and pLG. The plasmid ptrc99A contains lacIq which over-produces LacI. This is needed to sufficiently suppress the Lac operator in the pLG construct in the starting cultures.
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