Difference between revisions of "Part:BBa K875001:Experience"

Revision as of 23:17, 26 September 2012

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.

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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.

These two graphs are made with the same data. Both of them show the relation between p-cumate concentration and the fluorescence intensity of GFP at different times. Is interesting to note that the culture induced with 30 µM of p-cumate has much the same GFP fluorescence intensity than the culture induced with 60 µM or with 120 µM of p-cumate.

In the plate assay:

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

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+These two graphs are made with the same data. Both of them show the relation between p-cumate concentration and the fluorescence intensity of GFP at different times. Is interesting to note that the culture induced with 30 µM of p-cumate has much the same GFP fluorescence intensity than the culture induced with 60 µM or with 120 µM of p-cumate.
 ''In the plate assay:''