Difference between revisions of "Part:BBa I751101:Experience"

Tokyo-Tech iGEM 2011

Fluorescence intensity of BBa_I751101 was increased by both 3O-C6-HSL and IPTG induction.

Effect of 3O-C6-HSL induction and IPTG induction on fluorescence intensity
This work is done by Takuya Tsubaki.

To characterize Plux-lac hybrid promoter which has two LacI-operator parts and a LuxR-operator part on one regulator region, we used BBa_I751101. We introduced BBa_I751101 into a LacI-expressing E.coli strain, pTrc99A (JM2.300). For this reason, this E. coli expresses LuxR and LacI constitutively. Because IPTG controls the binding of LacI to two LacI-operator parts and 3O-C6-HSL controls the binding of LuxR to a LuxR-operator part, the transcription of gfp of BBa_I751101 is dually regulated by both IPTG induction and 3O-C6-HSL induction.

In the absence of the both inducers, the culture with Plux-lac hybrid promoter-GFP showed the background–fluorescence intensity generated by promoterless pSB3K3-GFP. The presence of either IPTG or AHL alone did not have any effect on increasing the fluorescence intensity. In the presence of both inducers, the culture showed 110-fold higher fluorescence intensity than that in the absence of both inducers. This result confirmed that the assembly of the two LacI operators and the LuxR operator integrated the inputs of IPTG and AHL into the output of GFP transcription. In other words, combination of the two LacI operators and the LuxR operator functioned as a genetic AND gate.

[Sample]

I751101 on pSB3K3 / pTrc99A

[Method]

①Overnight culture of BBa_I751101 grown at 37 in LB medium containing carbenicillin and kanamycin were diluted 1:100 in the medium, and then they were incubated at 37 °C as fresh cultures.

②After their OD600 reached 0.2, we added inducers into the fresh culture: 1 mM IPTG and/or 10 nM 3O-C6-HSL.

③After 3-hour incubation at 37 °C, 0.25 mL of each culture was harvested by centrifugalization and suspended by adding 1 mL of PBS (phosphate-buffered saline).

④We dispensed 500 µL of each suspension into a disposable tube through a cell strainer, and its fluorescence intensity was measured with a flow cytometer of Becton, Dickinson and Company.