Part:BBa_K5292069

Mtac726

To generate a dataset for training a predictive model of tac promoter strength, which can then be used to design promoters that enhance ICCG expression, we designed a pair of degenerate primers to randomly mutate the 16 bp sequence between the -35 and -10 regions of the tac promoter, creating the Mtac promoter. The tac promoter is a strong promoter capable of initiating robust gene transcription in E. coli.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE 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 powerful promoter for driving high-efficiency gene transcription. Commonly used in expression systems, it allows flexible regulation of downstream gene expression by controlling lactose or IPTG induction. To further enhance the potential of this promoter, we constructed a mutated version of the tac promoter (Mtac). Using a pair of degenerate primers, we randomly mutated the 16 bp sequence between the -35 and -10 regions of the tac promoter, generating a series of variants. To characterize the strength of these promoter variants, we constructed an ICCG-GFP fusion protein and quantified the promoter activity by measuring fluorescence intensity. This data will be used to train predictive models to optimize promoter design and improve the expression efficiency of the ICCG protein.

Profile

Name：Mtac726
Base Pairs: 29
Origin: tac promoter ( https://parts.igem.org/Part:BBa_J435360 )
Properties: A tac promoter mutant obtained through random mutagenesis. The promoter activity was characterized by fluorescence intensity, and this mutant is capable of downregulating ICCG expression compared to the wild-type tac promoter.
Composite part used to characterize this basic part: BBa_K5292069; Vector: pET-26b(+)

Usage and Biology

The Mtac mutant was generated through random mutagenesis to study and optimize promoter function, and to build a tac promoter design model tailored for ICCG expression. Mtac is effective in regulating downstream gene expression and is particularly useful for synthetic biology projects requiring precise or adjustable expression levels. The fluorescence intensity of the ICCG-GFP fusion protein can be used to characterize promoter strength, providing a quantitative reference for expression levels applicable to various scenarios.

The Mtac is a mutant of the tac promoter, with mutations located in the 16 bp region between the -35 and -10 sites. The -35 and -10 regions are critical for RNA polymerase binding to the promoter, and the spacer sequence between these regions is essential for regulating gene transcription. By altering the spacer sequence, the binding angle and efficiency of RNA polymerase can be modulated, optimizing the transcription initiation frequency (TIF) of the promoter and affecting the strength of gene transcription. This optimization ensures that the promoter retains its specific recognition by sigma factors while allowing precise regulation of transcription levels.

Design Notes

To construct the Mtac promoter mutants, we used a random mutagenesis approach targeting the 16 bp sequence between the -35 and -10 regions of the tac promoter, a critical area for RNA polymerase binding and transcription initiation. We designed a pair of degenerate primers, with the mutation region encoded by 16 consecutive N nucleotides, to introduce diversity into this sequence, generating a series of different promoter mutants.

We used PCR amplification to obtain a linearized vector and seamless cloning to obtain recombinant plasmids carrying the mutated tac promoters. The mixture of plasmids generated by random mutagenesis was then transformed into the expression strain BL21 (DE3). After expression screening through a high-throughput screening system, we selected samples for sequencing of the mutated regions, resulting in the identification of the Mtac sequence.

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.
  

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.

Figure 1. Normalized fluorescence intensity characterization of the mutants.

The fluorescence intensity characterization results show that the expression of the ICCG-GFP fusion protein initiated by the tac promoter mutant was either enhanced or reduced compared to the T7 promoter and the wild-type tac promoter. The data obtained were used to train the model, leading to the development of a promoter design and prediction model.

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.