Part:BBa_K921001
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.
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.
Note: To learn more about how we characterized this set of promoters, click here.
Discussion:
The design of this mutant T7Lac promoter (BBa_K921001) was based on random mutations throughout the promoter including the recognition site, melting box, initiation site, and the lac operator. This promoter is expected to have a significantly lower initiation frequency due to the T->C mutation in the melting box. RNA polymerase denatures DNA at the melting box to initiate transcription. The melting box TATA presents in all T7 promoters. Thymine and adenine have lower melting temperatures and are easily melted. Guanine and cytosine form an extra hydrogen bond and cause base stacking, which increases their melting temperature, making it more difficult for RNAP to initiate transcription. This mutation was rationally made to decrease an initiation frequency, resulting in a weaker T7Lac promoter. Indeed, mutant II (BBa_K921001) of this set of T7/lac promoters produces less protein than the wildtype T7Lac promoter (BBa_K613007).
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.
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.
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 | 90% |
| negative_regulators | LacI (Hypothesized LacIQ compatible) |
//function/regulation/transcriptional
//plasmid/expression/t7
//promoter
//regulation/negative
| direction | Forward |
| efficiency | 90% |
| negative_regulators | LacI (Hypothesized LacIQ compatible) |