Difference between revisions of "Part:BBa K568002"

Revision as of 16:54, 21 September 2011

blue light promoter lacZ reporter part

blue light promotor + lacZ

This part is based on the blue light sensor (BBa_K238013) with an additional lacZ Gene. Part (BBa_B0034) was used as RBS.

Usage and Biology

The part provides the possibility to induce lacZ transcription via blue light irradiation.

Blue light leads to dimerisation of YcgF and binding of the repressor YcgE. The formation of the YcgE-YcgF complex leads to the unbinding of YcgE from the YcgZ promoter which activates the transcription of lacZ.

Sequence and Features

Functional Parameters

Induction ideally at 465 nm

The part works, however no complete on/off reaction was observed. Compared to a sample in the dark, cells under blue light (450 nm) expressed about 1.7 times as much beta-galactosidase. This was tested in a Miller Assay.

There is a temperature dependency, the blue light promotor works better at lower temperatures compared to 37 °C.

Results of the Miller Assay of BBa_K568002. Compared to the sample grown in room light and in the dark, blue light (450 nm) yielded a higher expression of beta-galactosidase. As the absolute values differed strongly, values were calculated relative to the sample "dark" which was set to 100 %.

Experimental Testing

two cultures in LB medium:

blue light - lacZ in DH5alpha with three incubation conditions: lighted in room light, dark and blue light (450 nm)

cpH8 in DH5alpha as a control under room light

conditions of bacterial growth: 23,5 °C, shaken, both cultures grown in the dark prior to the experiment, two hours before the experiments: cultures were subdivided and lit as described

volume of bacterial suspension used: 50 µl (or dilutions with LB medium)

incubation time for Miller Assay: 5 min (37 °C)

Start of Assay: - measurement of Abs(600nm)

- new eppi with 0,5 ml of Zbuffer + 20 μl of freshly prepared 0,1% SDS+ 40 μl of Chloroform (under fume hood) in 2 ml tube

- 50 μl (as a dilution 25 µl + 25 µl LB medium or 10 μl + 40 μl LB medium) of the samples are taken and transferred to the Zbuffer

- mix the solution by vortexing for 10 s (all samples with equal vortexing time)

- transferring of 100 μl supernatant to 96 well plate (for photometer)

- initiation of assay with 20 μl of ONPG (4mg/ml) = START

- incubation at 37 °C

- stop reaction with 50 μl of 1 M Na2CO3 = STOP after exactely 5 min

- measurement of Abs (420nm) and Abs (550nm)

for this diagram:

• calculation of Miller Units
• mean of triplicate measurement
• adjusted to 100 %
• corrected for outliers