Difference between revisions of "Part:BBa K921002"

Revision as of 03:35, 2 October 2012

T7 RNAP + IPTG->PoPs (Mutant III)
This is a mutant T7 promoter that includes a lacO sequence. This is repressible by the LacI protein and is induced in the presence of IPTG. The promoter sits in the 5' region of the gene of interest and initiates transcription when the cell expresses T7 RNA polymerase. Be sure you are using a cell strain that is compatible with T7 expression vectors.
The following parameters were calculated and compared against the "wild-type" [1] The following parameters were calculated:

Fluorogen activating proteins were used as protein reporters in our characterization experiment as described here. Here, a saturating amount of malachite green was added to each well of a 96 well plate and fluorescence values were recorded over time. Our model was used to describe the behavior of our time lapse data using differential equations and fitting algorithms.


The Spinach aptamer was used as an RNA reporter in our characterization experiment as described here. Here, a saturating amount of DFHBI was added to each well of a 96 well plate and fluorescence values were recorded over time. Our model was used to describe the behavior of our time lapse data using differential equations and fitting algorithms.

Note: Not included here are the properties of our characterization method. To learn more about how we characterized this set of promoters, click here.

Discussion:
Mutant III of this set of T7/lac promoters produces a lot of protein and seems to respond efficiently to the addition of IPTG. The design of this mutant was based on a different class of T7 promoters, which are weaker than the traditional variants that are popular in molecular biology. As a result, this promoter is expected to perform weaker than the wild type. We hypothesize that this promoter does not burden the cells as much as traditional T7 promoters that can overload cells and cause selective pressure, among other things.

It is important to note that across all three promoters, multiple dips were recorded in the readings even though our data is normalized against optical density to take into account viable cell number. This is due to the fact that strong promoters cause selective pressure and multiphasic growth curves. This can cause sinusoidal behavior in gene expression. The approximate period of protein production across all three promoters is 1 hour^-1.

Functional Parameters