Difference between revisions of "Part:BBa K921001"

Latest revision as of 01:09, 4 October 2012

T7 RNAP + IPTG->PoPs (Mutant II)
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 (pBR322 replication origin); 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 III

The following parameters were calculated and compared against the "wild-type" promoter. To learn more about how we characterized this set of promoters, click here.

Table 1: Relative transcription and translation efficiencies of three T7Lac promoters as compared to the wildtype promoter

Figure 1: FAP fluorescence (reporter for protein levels) over time.Fluorogen-activating proteins were used as protein reporters in our characterization experiment (see details in here). 10µM 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. Cells were grown in M9 media without casamino acids. Fluorescence intensities are normalized to optical density (absorbance @600nm). Lines are drawn as guide of eyes.


Figure 2: Spinach fluorescence (reporter for RNA levels) over time.The Spinach aptamer was used as a RNA reporter in our characterization experiment (see details in here). 200µM 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. Lines are drawn as guide of eyes.


Figure 3: Leaky RNA (top panel) and protein (bottom panel) expression levels 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.

Figure 4: Calculated values of transcription strength and translational efficiency. These values were calculated from the raw fluorescence values that were normalized by OD600 and fit into the differential equations that were developed to model our system. The units are in RFU/min due to time constraints and insufficient published information about our biosensor system. Further experiments are required to determine a conversion factor.

Discussion
The design of Mutant I (BBa_K921000) was based on random mutations throughout the promoter region including the recognition site, initiation site, and the lac operator. This promoter was expected to have a lower affinity to the T7 RNAP and therefore have a lower amount of protein expression. However, mutant I (BBa_K921000) of this set of T7/lac promoters produces more protein than the wild type promoter (BBa_K613007). We hypothesize that the difference between prediction and experimental results is due to the lower affinity between T7 promoter and T7 RNAP, which allows the polymerase to initiate transcription more frequently.

The leaky expression of this promoter is noticeably higher than the wild type promoter. Furthermore, this mutant promoter does not produce much protein after induction by IPTG. Therefore, a large percentage of the overall protein production after IPTG induction is likely due to the leaky expression. The leaky expression is likely due to a decrease in the affinity of the LacI repressor to the LacO operator.

Usage and Biology

Sequence and Features

Functional Parameters