Part:BBa_K921000
T7 RNAP + IPTG->PoPs (Mutant I)
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 II
Mutant III
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). 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.
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.
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 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 lower than the wild type promoter. However, this promoter also produces a large amount of protein once induced. Therefore, the LacI repressor could bind very tightly in order to inhibit the strong expression of mRNA. This promoter produces the highest protein level and has the lowest leaky expression of the three constructs.
Usage and Biology
Sequence and Features
- 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 | 169% |
| negative_regulators | LacI (Hypothesized LacIQ compatible) |
//function/regulation/transcriptional
//plasmid/expression/t7
//promoter
//regulation/negative
| direction | Forward |
| efficiency | 169% |
| negative_regulators | LacI (Hypothesized LacIQ compatible) |