Regulatory

Part:BBa_K354000:Experience

Designed by: Gregory Meyer   Group: iGEM10_WITS-South_Africa   (2010-10-19)

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_K354000

IPTG activates the Lac/AraC promoter

A culture of 2ml of E. coli containing a construct with the Lac/AraC promoter in front of YFP variant Venus was grown overnight at 37°C and then transferred to 25 ml of ampicillin LB broth. This culture was then grown at 37°C in a shaking incubator for 2.5 hours so as to ensure that the bacteria were in their exponential growth phase. The culture was then imaged using fluorescent microscopy with the excitation and emission maximums set for yellow/green fluorescence to detect YFP.

A baseline reading (Fig 1) was taken at this point (Tile A), to determine the presence of non-specific promoter activation. 10% 1mM IPTG was then added and the culture returned to the shaking incubator. An aliquot was then imaged after 30min (Tile B), and again after 1 hour (Tile C). All aliquots used in imaging were 100 ul.


As is seen in the baseline image (Tile A), the degree of fluorescent activation is minimal before the addition of IPTG. Tile B shows the fluorescent activation after 30min incubation with IPTG - there is very little visible fluorescent activation. After an hour of incubation (Tile C) there is a marked increase in the degree of fluorescence, thus providing an indication that IPTG has a positive effect on the Lac/Ara-1 promoter, activating gene expression.

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Figure 1. Time course fluorescent microscopy of Lacto-detect before and after induction with IPTG



User Reviews

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we are team BGU-Israel from the 2013 competition and we used this part to control the expression of the Lamda cI protein. in order to characterize the behaviour of this promoter under different induction conditions we built a construct comprised of this promoter, strong RBS and the cI CDS. the promoter's performance under the presence of IPTG and Arabinose was analyzed via comassie staining. The cI protein is 29 KDa. the cI expession levels were dependent on the combination of inducers used to express the cI. this way we generated a 4-way switch to control the amount of protein the cell produces.

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