Part:BBa_K5343004

code for phosphoribosyltransferase

Usage and Biology

Application value of shuttle promoters:

The application of shuttle promoters (e.g., from Corynebacterium glutamicum and applied to Escherichia coli) has important scientific and adaptive value. Firstly, Corynebacterium glutamicum is a common industrial microorganism commonly used in the production of amino acids, while E. coli is the most commonly used model organism in genetic research and industrial fermentation. By applying promoters from Corynebacterium glutamicum to E. coli, gene expression regulation between different hosts can be achieved. The primary significance of this cross-species promoter application is to achieve efficient expression of target genes, thereby facilitating genetic engineering applications in different microbial systems.

Specifically, the application of the promoter of Corynebacterium glutamicum in Escherichia coli enables the use of the properties of both organisms to optimise industrial production processes. For example, the promoter of Corynebacterium glutamicum may have the property of being sensitive to specific metabolic conditions, which, in combination with the rapid proliferation and ease of manipulation of E. coli, can improve the production of certain products during fermentation. In addition, in scientific research, the use of such shuttle promoters can help researchers to better understand the differences in gene expression in different host systems, reveal the regulatory mechanisms, and even provide opportunities for the development of novel metabolic engineering route lines

Possibilities for future applications:

When designing metabolic pathways for downstream products, our team (SDU-CHINA) wanted to expand the control of the expression strength of specific genes by introducing shuttle promoters. Therefore, we compared the transcriptome sequencing results of different Corynebacterium glutamicum strains, identified promoters and RBSs with different expression strengths, and applied them to E. coli chassis cells for expression, which will be beneficial in subsequent metabolic regulation targeting specific reaction pathways to increase the breadth and finesse

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]

Characteriation

We characterised this element by the intensity of expression of the reporter gene rfp：

We isolated their promoter sequences from the genome and subsequently cloned them upstream of the reporter gene rfp. This setup was used to assess the expression levels of various constitutive promoters under standardized conditions. The reporter genes rfp and P002/P009/P012/P022/P026/P038/P039/P040/P043/P048 were fused and assembled into plasmid PACYAC by seamless DNA cloning to obtain the characterisation plasmid PACYAC-002RFP、PACYAC-009RFP、PACYAC-012RFP、PACYAC-022RFP、PACYAC-026RFP、PACYAC-038RFP、PACYAC-039RFP、PACYAC-040RFP、PACYAC-043RFP、PACYAC-0048RFP.

Protocol

Our experimental conditions for characterizing this part were as follows:

• Plasmid Backbone: PACYC
• Chassis cell ：E. coli DH5α
• Medium: 1.5 mL of liquid LB medium
• Condition 37°C, 24 h, under vigorous shaking
• Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.)

Fluorescence intensity was detected using an enzyme marker at an excitation wavelength of 590 nm and an emission wavelength of 645 nm, and fluorescence intensity values (a.u.) were obtained by calculating the fluorescence intensity to OD600 ratio.

Result

Access to synthetic biology components through the transcriptome：

Based on differential analysis of transcriptome data, we identified previously unrecognized promoters associated with various genes. We isolated their promoter sequences from the genome and subsequently cloned them upstream of the reporter gene rfp. This setup was used to assess the expression levels of various constitutive promoters under standardized conditions.

Fig. 1 . Access to synthetic biology components through the transcriptome

Expression intensity results：

Analysis of the characterisation results showed that P038 is a moderate shuttle promoter for E. coli - Corynebacterium glutamicum.

Fig. 2 . Characterization results of PACYAC-038RFP in DH5α

Reference

[1] Hwang S, Choi KH, Yoon N, Cha J. Improvement of a Sulfolobus-E. coli shuttle vector for heterologous gene expression in Sulfolobus acidocaldarius. J Microbiol Biotechnol. 2015 Feb;25(2):196-205. doi: 10.4014/jmb.1407.07043. PMID: 25293629.

[2] Li K, Zhao Z, Zhang YZ, Wang Y, Ding JY. [Construction of Corynebacterium glutamicum/E. coli shuttle promoter-probe vector]. Wei Sheng Wu Xue Bao. 2007 Apr;47(2):191-6. Chinese. PMID: 17552218