Difference between revisions of "Part:BBa K921002"

Revision as of 18:15, 3 October 2012

T7 RNAP + IPTG->PoPs (Mutant III)
This is a mutant T7 promoter that includes a lacO operator site. The promoter is inhibited by the LacI protein and can be induced by IPTG. The promoter sits in the 5' region of a gene and initiates transcription when cells express T7 RNA polymerase. Please use cell strains that are compatible with T7 expression vectors.Our promoters were characterized in a high copy plasmid (pIVEX); as a result, there was a measurable amount of background fluorescence values from uninduced cells. This measurement of leaky expression offers insight into the negative regulatory behavior of our hybrid promoters.

Mutant I
Mutant II

The following parameters were calculated and compared against the "wild-type" promoter.

Fluorogen-activating proteins were used as protein reporters in our characterization experiment (see details in here). 10uM malachite green was added to each well of a 96-well plate and fluorescence intensities were recorded over time. Our model was used to simulate the time lapse data using differential equations. We note that multiple dips were recorded in the readings of all three mutated promoters. This could be due to a strong metabolic burden and multiphasic growth of bacteria.


The Spinach aptamer was used as a RNA reporter in our characterization experiment (see details in here). 200uM DFHBI was added to each well of a 96- well plate and fluorescence intensities were recorded over time. Our model was used to simulate the time lapse data using differential equations.


The figures show the leaky expression of our T7Lac promoters in BL21(DE3) cells. Uninduced cells (without IPTG) were added to wells in a 96 well plate supplemented with either 200µM of DFHBI or 10µM of malachite green. Fluorescence intensities at the 3rd hour time point is shown for comparison between promoters.

Note: To learn more about how we characterized this set of promoters, click here.

Discussion:
The design of this mutant T7Lac promoter (BBa_K921002) was based on a different class of T7 promoters, which are weaker than the wildtype T7Lac promoter (BBa_K613007). Therefore, this promoter is expected to produce less protein than the wildtype promoter. However, this mutant promoter produces more protein than the wildtype promoter in our experiments. We hypothesize that this promoter does not burden cells as much as the wildtype T7 promoters, hence giving rise to higher translation rate of mRNA.

This promoter is ~2.5x more leaky than the wildtype promoter. The mutations in the LacO operator of this promoter could lower its affinity to the LacI repressor. As a result, transcription is more likely to occur even when IPTG is not present.

Functional Parameters