Part:BBa_K5292127
DMtac57
DMtac is a series of mutated tac promoters designed using predictive models, with mutation sites concentrated in the 16 bp sequence between the -35 and -10 regions of the promoter. These mutants are intended to enhance the expression of the PET-degrading enzyme ICCG in Escherichia coli. Using a dataset built from the Mtac promoters previously obtained by random mutagenesis (BBa_K5292001 - BBa_K5292088), the DMtac series of mutants were predicted by models and exhibit different promoter strengths. Users can select the most suitable mutant based on their specific needs. We characterized the expression strength of the DMtac promoters by linking them to the target gene ICCG-GFP fusion protein. Fluorescence intensity measurements can be used for quantitative analysis of promoter activity, allowing for the selection of the optimal promoter.
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]
Abstract
The tac promoter is a hybrid of the trp and lac promoters, combining the strong promoter characteristics of both. It is widely used in Escherichia coli as a potent promoter for driving high-efficiency gene transcription. Frequently employed in expression systems, the tac promoter allows flexible regulation of downstream gene expression through lactose or IPTG induction. DMtac is a series of mutated tac promoters designed using predictive models, with mutations located in the 16 bp sequence between the -35 and -10 regions. These mutants are aimed at optimizing the expression of the PET-degrading enzyme ICCG in E. coli. The DMtac series was developed based on the Mtac promoter library (BBa_K5292001 - BBa_K5292088) generated through random mutagenesis, providing diverse options for efficient gene expression in synthetic biology projects.
To characterize the strength of DMtac promoters, we constructed ICCG-GFP fusion proteins to measure fluorescence intensity, allowing quantitative analysis of promoter activity. By using fluorescence intensity/OD600 data, we were able to compare the expression levels of different mutants and assess the regulatory effects of mutations on ICCG expression. These experiments also provide data for further optimization efforts.
Profile
Name：DMtac57
Base Pairs: 29
Origin: tac promoter (https://parts.igem.org/Part:BBa_J435360)
Properties: Tac promoter mutants designed using a model. The promoter activity was characterized by fluorescence intensity, and this mutant is capable of regulating ICCG expression.
Composite part used to characterize this basic part: BBa_K5292127; Vector: pET-26b(+)
Usage and Biology
The tac promoter is a hybrid of the trp and lac promoters, combining their strong promoter characteristics. It is widely used in Escherichia coli as a strong promoter to drive efficient transcription of genes. It is frequently used in expression systems, allowing flexible regulation of downstream gene expression via lactose or IPTG induction. DMtac is a series of tac promoter mutants designed using predictive models, with mutation sites located in the 16 bp sequence between the -35 and -10 regions. These mutants are designed to optimize the expression of PET-degrading enzyme ICCG in E. coli. The DMtac series is based on the Mtac promoter library (BBa_K5292001 - BBa_K5292088) generated through random mutagenesis, providing diverse gene expression control tools suitable for various synthetic biology projects.
To characterize the strength of DMtac promoters, we constructed an ICCG-GFP fusion protein to measure fluorescence intensity, allowing quantitative analysis of promoter activity. By comparing fluorescence intensity with OD600 data, we evaluated the expression levels of different mutants and confirmed the regulatory effects of mutations on ICCG expression, providing valuable data for further optimization.
DMtac promoters are a series of mutants of the tac promoter, designed using predictive models. The mutations are concentrated in the 16 bp sequence between the -35 and -10 regions, which are critical binding sites for RNA polymerase. The literature indicates that the spacer sequence in this 16 bp region is essential for promoter function, as it determines RNA polymerase binding efficiency and transcription initiation frequency (TIF). Mutating this region allows for fine-tuning of gene expression levels without altering the promoter's specificity for sigma factor recognition.
By optimizing this sequence region, DMtac provides stronger promoter activity than the wild-type tac promoter, particularly in driving ICCG expression with superior expression levels. These mutants offer a controllable tool for fine regulation of gene expression, suitable for applications that require flexible adjustment of expression levels.
Design Notes
When designing the mutants, we used the sequences from the Mtac promoter library (BBa_K5292001 - BBa_K5292088) to train a predictive model. The predictive model then generated a series of mutants that could potentially enhance or reduce promoter activity. To construct the DMtac mutants, we designed single-point mutation primers and performed PCR amplification on the template to obtain a linearized vector. Seamless cloning technology was used to obtain recombinant plasmids carrying the DMtac mutants. The recombinant plasmids were then transformed into DH5α for plasmid amplification, followed by plasmid sequencing to confirm the correctly constructed recombinant plasmids.
Characterization
Here are the experimental data we used to characterize the activity of the Mtac promoter via the ICCG-GFP fusion protein. The table below lists the composite part numbers for our mutated Mtac promoters and the ICCG-GFP fusion protein. In the graph below, we present the fluorescence/OD600 data, which characterize the strength of the Mtac promoter mutants in E. coli BL21 (DE3). We developed a high-throughput cultivation screening system to test whether the Mtac promoter mutants can drive the expression of the ICCG-GFP fusion gene and to assess the effect of Mtac on fusion protein expression. These data validate the functionality of the Mtac promoter in various strains, providing experimental evidence for further optimization of ICCG expression.To characterize the activity of the DMtac promoters, we constructed an ICCG-GFP fusion protein, using fluorescence intensity as a quantitative measure of expression. The following steps were taken to characterize the mutants:
- The constructed DMtac promoter mutant plasmids were transformed into Escherichia coli BL21 (DE3) expression strains.
- The transformed strains were cultured in a high-throughput screening system, and the expression levels of different mutants were compared by measuring fluorescence/OD600.
The high-throughput screening system is as follows:
Induce Temperature (25℃), Induce Time (12 h), Concentration of IPTG (1.0 mM)
1. All the signal peptides and promoters mutants which we had designed were constructed on pET26-ICCG-GFP and were transformed into BL21 (DE3), and were selected for screening.
2. The above BL21 (DE3) strains containing recombinant protein-encoding plasmids were selected and grown in 96-deep-well plates with 600 μL of LB liquid medium with corresponding resistance per well for 6 h at 37 °C.
3. Then, 8 μL of the bacterial culture were transferred into 96-deep-well plates with 800 μL of LB liquid medium per well and grown for 6 h at 37 °C.
4. 100 μL samples were transferred into transparent 96-well microplates for OD600 measurements before the inducer was added.
5. Then, 7 μL inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) for each wells was added to a final concentration of 1.0 mM, and the cultivation was continued at 25℃ for 12 h.
6. Take 100 μL from each well add to test the OD600 before add the inducer.
7. 100 μL samples were transferred into transparent 96-well microplates for OD600 measurements and 100 μL samples were transferred into 96-well black microplates for GFP fluorescence measurement.
8. The fluorescence intensity was measured using an excitation wavelength of 485 nm and an emission wavelength of 520 nm by using EnVision Multilabel Reader.
The results of the fluorescence intensity characterization indicate that the DMtac promoter mutants can enhance ICCG expression to varying degrees, with some mutants showing higher expression levels compared to the wild-type tac promoter. These experimental data provide a strong foundation for further optimization, and the DMtac series offers more options for gene expression regulation in synthetic biology projects.
The design of the DMtac mutants combined predictive modeling with experimental validation, aiming to enhance the expression efficiency of target genes by regulating key regions of the tac promoter. The successful construction and characterization of this series of mutants provides new insights for optimizing gene expression systems in the future and demonstrates the potential for flexible regulation of gene expression under different conditions.
Reference
1.Zhang, S., Liu, D., Mao, Z., Mao, Y., Ma, H., Chen, T., Zhao, X., & Wang, Z. (2018). Model-based reconstruction of synthetic promoter libraryin Corynebacterium glutamicum. Biotechnology Letters, 40(5), 819–827.
2.Van Brempt, M., Clauwaert, J., Mey, F., Stock, M., Maertens, J., Waegeman, W., & De Mey, M. (2020). Predictive design of sigma factor-specific promoters. Nature Communications, 11, 5822.
3.Li, Z.-J., Zhang, Z.-X., Xu, Y., Shi, T.-Q., Ye, C., Sun, X.-M., & Huang, H. (2022). CRISPR-based construction of a BL21 (DE3)-derived variant strain library to rapidly improve recombinant protein production. ACS Synt, 11(1), 343-352.
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