Difference between revisions of "Part:BBa K2042004"
RATOVOMANANA (Talk | contribs) (→Characterization) |
RATOVOMANANA (Talk | contribs) (→Characterization) |
||
| Line 20: | Line 20: | ||
==Characterization== | ==Characterization== | ||
| − | This promoter was characterized by Fluorescent measurement. GFP was inserted under the control of the IPTG inducible promoter and transformed in Pseudomonas putida. Different | + | This promoter was characterized by Fluorescent measurement. GFP was inserted under the control of the IPTG inducible promoter and transformed in Pseudomonas putida. Different experiments were performed. |
For this experiment the cell grew in the 30ºC chamber with shaking until they reached OD of around 0.4 (spechtophotometre indicated that the OD was 0.4, the TECAN in which the fluorescence was read says 0.2 in the beginning, but this OD is irrelevant since the values only matter for normalization). | For this experiment the cell grew in the 30ºC chamber with shaking until they reached OD of around 0.4 (spechtophotometre indicated that the OD was 0.4, the TECAN in which the fluorescence was read says 0.2 in the beginning, but this OD is irrelevant since the values only matter for normalization). | ||
Revision as of 22:26, 20 October 2016
IPTG inducible promoter
This is the sequence of an leaky IPTG inductible promoter working in Pseudomonas putida.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
This promoter was characterized by Fluorescent measurement. GFP was inserted under the control of the IPTG inducible promoter and transformed in Pseudomonas putida. Different experiments were performed.
For this experiment the cell grew in the 30ºC chamber with shaking until they reached OD of around 0.4 (spechtophotometre indicated that the OD was 0.4, the TECAN in which the fluorescence was read says 0.2 in the beginning, but this OD is irrelevant since the values only matter for normalization). When the OD was reached, induction with the indicated concentrations of IPTG was made, the culture was put into a 96-wells plate, and measured on the TECAN for 1hour. (Note, the measure was made without shaking) These are the results that the TECAN automatically provides.
For this experiment the cell grew in the 30ºC chamber with shaking until they reached OD of around 0.4 (spechtophotometre indicated that the OD was 0.4, the TECAN in which the fluorescence was read says it is 0.2 in the beginning, but this OD is irrelevant since the values only matter for normalization). When the OD was reached, induction with the indicated concentrations of IPTG was made, the culture was putted back in the 30'C chamber with shaking. Timepoints was taken and measured directly in the TECAN. These are the results. Values for graph without correction of OD600
Values for graph normalizing with OD600
5h Long washed with water The experiment was repeated because LB emits fluorescence at the same range as GFP. This time it was done like this: For this experiment the cell grows in the 30ºC chamber with shaking until they reached OD of around 0.4 (spechtophotometre indicated that the OD was 0.4, the TECAN in which the fluorescence was read says 0.2 in the beginning, but this OD is irrelevant since the values only matter for normalisation). When the OD was reached, induction with the indicated concentrations of IPTG was made and the culture was putted back in the 30'C chamber with shaking. Timepoints was taken, washed them with water and measured them directly in the TECAN IN WATER These are the results.
Values for graph normalizing:
