Part:BBa_K921002

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 (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 II

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

Promoter	BBa K921000	BBa K921001	BBa K921002
Transcription Strength	97%	72%	127%
Translational Efficiency	169%	90%	160%

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 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 was expected to produce less protein than the wildtype promoter. However, this mutant promoter produces more RNA and protein than the wildtype promoter in our experiments.

This promoter is 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

direction	Forward
efficiency	160%
negative_regulators	LacI (Hypothesized LacIQ compatible)