Part:BBa_K4941076

PlacUV5-T7RNAP-T7t

T7 RNAP expression frames initiated by the promoter placUV5 and terminated by T7t were used to construct T7 RNAP expression libraries with different expression strengths.

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]

placUV5-MB7

        The provided sequence is an improved version of the wild-type lacUV5 promoter (PlacUV5, BBa_I732021), known as placUV5-MB. This is a naturally occurring strong inducible promoter in Escherichia coli. placUV5-MB is part of a series of derivatives obtained by mutating specific sites in the -35 and -10 regions of PlacUV5. As registries typically don't allow multiple sequences to be inputted, we are presenting the sequence of PlacUV5_MB7 as a representative (with the highest fluorescence intensity). For completeness and ease of use by other teams and researchers, the remaining sequences are also provided below.

Biology

        The T7 RNA polymerase from the λ phage is controlled by the strong inducible lacUV5 promoter and can be induced by IPTG. The conventional host for the T7 expression system utilizing T7RNAP and the T7 promoter is BL21(DE3), a host naturally susceptible to λ phage infection. However, this has two major limitations: (1) the T7 expression system can only be applied in this specific host, and (2) different types of proteins or metabolic pathway gene expressions may require different intensities, and higher intensity is not necessarily better.

        Based on this observation, we drew inspiration from the book "Basic Molecular Biology (4th Edition)" written by Zheng Yonglian, published by China Higher Education Press. We discovered that the -35 and -10 regions of promoters have standard sequences. If both regions of a promoter are close to the consensus sequence, the expression strength is enhanced; conversely, if they deviate, the strength is weakened. Therefore, we performed base mutations on the PlacUV5 region (Table 1) based on the standard consensus and existing sequences of strong and weak promoters (Plac, PlacUV5, ParaBAD, PrecA) as references. Simultaneously, we used a plasmid to express GFP under the control of placUV5 to serve as a reporter protein, indicating changes in transcriptional intensity.

Table 1: The -35 and -10 region sequences of different promoters

placUV5-MB sequences

        The table below displays ten newly designed lacUV5 promoters. All promoters underwent mutations targeting the -35 and -10 region sequences with reference to the standard sequence and the known sequences of strong and weak promoters (Fig.1). The specific sequences are detailed in Table 2.

Fig.1: Legend of the LacUV5 promoter

Table 2: Newly designed lacUV5 promoters with -35 and -10 region mutations

Design

        Experimental design of promoter strength

        To assess the strength of the mutated lacUV5 promoters, we employed the Golden Gate assembly protocol. Ten mutant promoter variants were cloned, along with a weak RBS (B0031), the GFP reporter gene (BBa_E0040), and the T7 terminator (BBa_B0012), onto a plasmid vector simultaneously. The use of a weak RBS was chosen to minimize transcriptional level differences that might arise due to strong translation levels. Simultaneously, these fragments were assembled onto a low-copy-number plasmid (RK2 Broad Host Range Vector with Kanamycin Resistance, BBa_K2491030), aiming to avoid excessive host burden and reduce noise, as illustrated in Fig.2.

Fig.2: Schematic representation of the mutant lacUV5 Promoter strength assay

        The mixture obtained through the Golden Gate assembly protocol was chemically transformed into DH5α and plated on agar plates containing kanamycin for selection. Transformed colonies containing different lacUV5 promoter variants were inoculated into tubes containing 5 ml of Terrific Broth (TB) medium and cultured at 37°C with shaking at 220 rpm. When the OD600 reached 0.6-0.8, induction was initiated by adding IPTG to a final concentration of 0.5 mM, and fermentation continued for 24 hours. Samples were taken at 12 hours and 24 hours, and the fluorescence intensity was measured using a microplate reader on a 96-well plate.

Result

        Through fluorescence intensity testing of different lacUV5 promoter mutants, we observed results consistent with our expectations. The lacUV5 variant (placUV5-MB7), identical to the standard sequence, exhibited the highest transcriptional expression intensity. Conversely, as deviations from the standard sequence increased, expression intensity exhibited a decreasing trend, particularly when the -35 region was replaced with a sequence from a weaker promoter (placUV5-MB8), resulting in a noticeable reduction in fluorescence intensity (Fig.3). These observations indicate that mutating the -35 and -10 regions of the promoter, especially when using sequences based on standard motifs and known promoter strengths, is feasible. This approach allows for the construction of variant libraries of the target promoter with different expression intensities.

fig.3: Fluorescence Intensity Changes in Different lacUV5 Mutants

Application

        In order to verify the effect of three catalytic properties of the pET expression system on PsXR by modulating the intensity of T7RNAP expression and thus affecting the catalytic properties of PsXR, we transformed T7 RNAP expression plasmids and pet28a-PsXR containing different lacUV5 promoters into Escherichia coli BL21 hosts at the same time. Here is an example of transforming the T7RNAP plasmid driven by PlacUV5MB and the pet28a-PsXR plasmid.         The transformants containing different lacUV5 promoters were screened out by transformation and colony PCR, and transfected into 96-well dishes containing LB+Kana+Cm liquid medium for culture and fermentation testing. The results showed that MB7-PsXR catalyzed the production of 6.8 g/L xylitol from the substrate 8 g/L xylose after 36h (Fig.4), which proved the feasibility of the strategy applied to optimize the pET expression system by constructing a promoter library expressing T7RNAP.

fig.4: Xylitol production under different promoter variants.

Conclusion

        In summary, by referencing standard sequences and the -35 and -10 regions of different strength promoters, we have successfully achieved precise modification of the placUV5 expression intensity. This optimization strategy has not only been successfully applied to the construction of the T7RNAP expression library but has also yielded significant results in optimizing the expression of the EutS protein. We are confident that this library will be of great assistance to other research teams in future studies.

Reference

        [1] Sun, X. M., Zhang, Z. X., Wang, L. R., Wang, J. G., Liang, Y., Yang, H. F., ... & Yang, S. (2021). Downregulation of T7 RNA polymerase transcription enhances pET‐based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis. Biotechnology and Bioengineering, 118(1), 153-163.         [2] Romano, E., Baumschlager, A., Akmeric, E. B., Palanisamy, N., Houmani, M., Schmidt, G., ... & Di Ventura, B. (2021). Engineering AraC to make it responsive to light instead of arabinose. Nature Chemical Biology, 17(7), 817-827.