Part:BBa_K346085

Ptet-RBS-T7polymerase

Ptet-RBS-T7polymerase To drive our mercury absorption device, the T7polymerase is constitutively expressed with the Ptet promoter. Ptet is from the 2009 parts.

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]

Contribution From NNU-China 2021

Group: iGEM Team NNU-China 2021

Author: Yan Xu

Summary: Charactering the leakage ability of the Ptet and the transcription level of T7 RNAP

Characterization from iGEM21-NNU-China

        The intensity of T7 RNA polymerase (T7 RNAP) expression is essential to the production of the target gene on pET plasmid. In BL21 (DE3), the gene encoding the T7 RNAP is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and strong. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. While our project was focused on the translational level of T7 RNAP, we were also paying attention to the transcription level of T7 RNAP.

      In the composite part of(BBa_K346085), T7 RNAP was expressed under the Ptet, which was registered in 2010. Here, the composite part of(BBa_K346085)was inserted into the genome of BL21 (DE3) (Fig. 2A), yielding the strain of BL21 (DE3)-tet. We used the pET-eGFP plasmid to characterize the leakage ability of the Ptet and the transcription level of T7 RNAP. When addition of the inducer of anhydrotetracycline (aTc), the BL21 (DE3)-tet strain exhibited brighter fluorescent (Fig. 2A), indicating that the composite part of (BBa_K346085) can work in the BL21 (DE3). As shown in Fig. 2B, under the condition of no inducer, it can be seen that the Ptet exhibited the lowest leakage ability compared to that of the PlacUV5.

        Furthermore, the capacity of(BBa_K346085)for the production of recombinant proteins was investigated. As the proof of concept, the two membrane proteins of Ecb and Yidc were produced in BL21 (DE3)-tet, and the different concentration of aTc (0.6 μM, 2.4 μM, 7.0 μM) were performed, respectively (Fig. 2C). The production of Ecb was maximum when aTc concentration was 2.4 μM, which is 371.0 % and 44.6% higher than that of 0 uM and 7.0 μM, respectively. The production of Yidc in strain BL21 (DE3::tet) was optimized when aTc concentration was 7.0 μM, which is 17.0% higher than that of 2.4 uM. This results indicated that the production of the membrane proteins can be improved by(BBa_K346085). These results provide effective guide for future iGEM team to improve the production of hard-to-express proteins.

Fig.1 A. The fluorescence diagram of BL21 (DE3)-tet with or without inducer. B. Fluorescence intensity of different strains containing pET-eGFP plasmid. C. The expression of Ecb and Yidc under different inducer concentrations at 36 h.

Reference

1. Sun XM, Zhang ZX, Wang LR, Wang JG, Liang Y, Yang HF. Tao RS, Jiang Y, Yang JJ, Yang S. Downregulation of T7 RNA polymerase transcription enhances pET-based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis. Biotechnology and bioengineering. 2021; 118: 153–163.

Contribution From NNU-China 2022

Group: [ https://2022.igem.wiki/nnu-china/]

Author: Yaru Jiang

Ptet-RBS-T7polymerase

        T7 RNA polymerase (RNAP) from λ prophage can specifically recognize the T7 promoter (PT7) and achieve high gene expression at a transcriptional rate 8-fold higher than that of Escherichia coli RNAP. This expression system has been successfully introduced into a variety of yeast hosts and has driven the CRISPR-Cas9 system to enable gene editing. However, the above systems are expressed using constitutive promoters and do not allow for precise regulation[1]. Based on this, we introduced the component Ptet-RBS-T7polymerase into Y. lipolytica and tested its regulatory function. To ensure that this element is functional, we added nuclear localization sequences (BBa_K4343135) to both terminals of the T7 RNAP and integrated a green fluorescent protein expression cassette regulated by the PT7 in the genome (polf-T7RNAP-T7GFP or polf-T7GFP). The strain polf-T7RNAP-GFP exhibited brighter fluorescence when the anhydrotetracycline (aTC) inducer was added, with a more than 5-fold increase in unit fluorescence intensity after induction (Fig 1A). At the same time, the system has no significant growth burden on the host (Fig 1B). This shows that the optimised BBA_K346085 (BBa_K4343135) can work in lipolytic yeast and provides a reference for future iGEM teams.

Fig. 1. A. The GFP expression in the polf-T7GFP and polf-T7RNAP-T7GFP. B. The growth of Y. lipolytica and polf-T7RNAP-GFP.

Reference

[1] Morse, N. J., Wagner, J. M., Reed, K. B., Gopal, M. R., Lauffer, L. H., & Alper, H. S. (2018). T7 polymerase expression of guide RNAs in vivo allows exportable CRISPR-Cas9 editing in multiple yeast hosts. ACS synthetic biology, 7(4), 1075-1084.