Part:BBa_K875001:Experience

Trieste Team 2012

To test this promoter we performed two different assays. First of all we cloned this promoter in pSB1C3 and then we inserted downstream the GFP BBa_I13504. In the same plasmid we cloned J23100-CymR-B0015 in order to repress the promoter. To test this system we used different concentrations of cumate which binds CymR preventing its repression, thus allowing the GFP expression.

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.

In liquid assay:

We inoculated a 20ml culture. After overnight growth, we diluted the culture to OD600 = 0.2. Then we aliquoted 200 μl in 8 replicates in a microtiter plate at different concentrations of cumate. The reading was performed in a monochromator at 485-510nm.

Both graphs show the relation between p-cumate concentration and the GFP fluorescence intensity at different times. In the first one, the modulation range is explored and it has been relevated that the maximum yield is reached at 30uM. In the second one the fluorescence of a culture is followed in the time and it has been noticed an interesting difference between the induced and not induced cultures. A little increase tendency is due to the bacteria population growth. The data were normalized over the background given by the LB media.

In the plate assay:

We streaked the culture on LB Agar plates containing different p-cumate concentrations.

User Reviews

UNIQ785e1b25e9b532b2-partinfo-00000000-QINU UNIQ785e1b25e9b532b2-partinfo-00000001-QINU