Part:BBa_K3633015
T7 promoter
T7 promoter (19 bp)
Description
This is a common promoter that can be induced by IPTG. The iGEM20_Shanghai_SFLS_SPBS use the promoter to accomplish experiments including production of betacyanins and laccase.
Experiments and Results
Successful production of betacyanins in E.coli BL21(DE3)(T7 promoter)
The team members constructed the plasmid T7-4,5-DODA using Gibson Assembly. They added 0.1 mM IPTG for induction. Again, after 20 h culture at 37℃, 220 rpm, we centrifuged the cells, discarded the LB medium, and resuspended the cell pellet in sterilized water. They then cultured the cells at 20℃, 120 rpm for 102 h and acquired expected results. They did not test the production in Vibrio natriegens because the bacterial strain we acquired (ATCC 14048) does not have the T7 promoter.
Successful production of laccase in E.coli BL21(DE3)(T7 promoter)
The plasmid was transformed into E. coli BL21(DE3). 0.1 mM IPTG induction was added when the culture reached an OD600 of 1.0. Then, the shake flask was cultured at 25℃ for 20 h (cultured at 25℃ instead of 18℃ because no more shakers were available in the laboratory). Then, the bacterial solution was centrifuged, the cell pellet was resuspended in PBS (pH=7.2-7.4), and the resuspension was sonicated.
Sequence & 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]
Contribution by CU-Egypt 2022
We measured the expression yield of the T7 promoter with different genes [BBa_K3396000, BBa_K4165003] to compare the expression yield and validate our Dry-Lab mathematical model with the Wet-Lab experimental results while keeping the promoter, RBS, lac operator, and terminator unchanged in the biobrick.
GST-DOC vs GST-COH
Figure 3. This figure shows the extraction of GST-DOC vs GST-COH using Chemical and sonication methods, this figure
proves that there is a significant difference between the expression of the Docs and Coh2 proteins. Although
modulatory parts for transcription and translation are the same.
His-DOC vs His-COH
Figure 4. This figure shows the extraction of His-DOC vs His-COH using Chemical and sonication methods, this figure
proves that there is a significant difference between the expression of the Docs and Coh2 proteins. Although
modulatory parts for transcription and translation are the same.
Mathematical Modeling of GST-DOC with T7 Promoter
Transcription rate and translation rate under T7 promoter
The mathematical modeling was based on our code for the calculation of transcription and translation according to thermodynamic laws and ODE (You can find it on our wiki). The results were validated and compared with the wet lab showing the same rates.
Figure 5. this figure shows the results from the transcription and translation code, showing the variation of mRNA and protein concentrations with time compared with the wet lab results (2mM IPTG concentration)
Mathematical modeling of His-Doc with T7 promoter
The mathematical modeling was based on our code for the calculation of transcription and translation according to thermodynamic laws and ODE (You can find it on our wiki). The results were validated and compared with the wet lab showing the same rates.
Transcription rate and translation rate under T7 promoter
the mathematical modeling was based on our code for the calculation of transcription and translation (you can find it in the code section) besides the estimated results from the wet lab.
Figure 6. this figure shows the results from the transcription and translation code, showing The mRNA and protein concentrations variation with time compared with the wet lab results (2mM IPTG concentration).
//direction/forward
//chassis/prokaryote/ecoli
//promoter
//regulation/constitutive
//chassis/bacteriophage/T7
| negative_regulators | |
| positive_regulators |