Part:BBa_K4941095

PlacUV5MB8-EutS-T7t

The EutS expression frame was initiated by the promoter placUV5MB8 to express the T7t-terminated EutS expression frame, which was used to find the right intensity of EutS expression and to reduce the host burden.

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 by Team ZJFH-NANJING 2023

        For the Contribution, we supplemented the experimental characteristics of the part elements in Escherichia coli (BBa_I732021, BBa_K2213000). These studies involve two main aspects: construction of a T7RNAP library based on the PlacUV5 mutant and replacing the lacUV5 promoter with different expression strengths to alleviate the burden on the host organism. These data were added to the corresponding BioBricks.

1. Construction of T7 RNAP expression library

        lacUV5 promoter is mostly utilized for the efficient expression of T7RNAP, further combined with the T7 promoter to achieve high-level expression of recombinant proteins or target genes (Fig.1). In our investigation, we initially employed the pET system for expressing xylose reductases from different sources to produce xylitol. However, achieving high-level expression of T7RNAP does not necessarily enhance the yield of the target gene. On the contrary, it is essential to consider the physiological characteristics of the target gene, aiming to balance the expression relationship between the two. This approach allows for obtaining an optimal expression intensity ratio that not only enhances the expression of the target gene but also reduces the burden on the growth host of E.coli [1].

Fig.1: Schematic Diagram of pET Expression System

        Therefore, based on the obtained promoter library, we assembled the T7 RNAP onto plasmids with different strengths of promoters for testing. The resulting pET expression system can achieve the production of various intensities of combinations and can be applied to different types of E.coli (such as DH5α, MG1655, etc.). Here, we chose DH5α as a representative strain for application. Through qPCR analysis of transcriptional levels, we found that by optimizing in this manner, we obtained a T7RNAP library with different intensities, and the transcriptional levels showed a positive correlation with the previously measured fluorescence intensities (Fig.2).

Fig.2: Relative transcription levels of T7 RNAP

2. LacUV5_EutS

        EutS constitutes one of the shell proteins comprising the Ethanolamine Utilization Bacterial Microcompartment (BMC) in E. coli and various other enterobacteria species. In 2016, the CU-Boulder team demonstrated that it is possible to engineer a functional BMC solely using EutS. However, when compelled to produce BMC, E. coli experiences a heightened burden, consequently impacting the normal growth of the bacterial cells. Therefore, we attempted mutations in the -35 and -10 regions of the lacUV5 promoter and constructed a promoter expression library. The EutS gene was cloned downstream of the lacUV5 promoter mutants and tested in DH5α. We monitored changes in OD600 values over 24 hours. The results indicate that strategically reducing transcription levels effectively alleviates the host burden in E.coli. The mutant strain with a weaker lacUV5 promoter showed a 1.27-fold increase in OD600 compared to the control (Fig.3, BBa_K4941095).

Fig.3: The impact of different lacUV5 promoters driving mutS on host growth.

        All of these may be helpful to other teams and we hope it will make some contribution to the iGEM community.

Reference

        [1] Zhang ZX, Nong FT, Wang YZ, Yan CX, Gu Y, Song P, Sun XM. Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity. Microb Cell Fact. 2022 Sep 15;21(1):191.