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.
The following parameters were calculated and compared against the "wild-type" promoter.
| Promoter | BBa K921000 | BBa K921001 | BBa K921002 |
|---|---|---|---|
| Transcription Strength | 97% | 72% | 127% |
| Translational Efficiency | 169% | 90% | 160% |
Cells were grown in M9 media without casamino acids. Fluorescence intensities were normalized to optical density (absorbance @600nm).
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.
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 leaky expression of our system after 3 hours in BL21(DE3) cells is plotted here across all of the promoters, including the wild-type. Uninduced cells (without IPTG) were added to wells in the 96 well plate and 200µM of DFHBI and 10µM of malachite green were added to the wells. Fluorescence intensities were recorded at regular intervals. Here, the last value (3:15 after plating the cells) is shown here 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 has a high level of leaky expression. In comparison, it is roughly 2.5x more leaky than the wildtype. The mutations in the lacO of this promoter lower its affinity to the LacI repressor. As a result, transcription is more likely to occur even when the inducer is not present.
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.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
| direction | Forward |
| efficiency | 160% |
| negative_regulators | LacI (Hypothesized LacIQ compatible) |
//function/regulation/transcriptional
//plasmid/expression/t7
//promoter
//regulation/negative
| direction | Forward |
| efficiency | 160% |
| negative_regulators | LacI (Hypothesized LacIQ compatible) |