Part:BBa_K2213000

LacUV5_EutS

The ethanolamine utilisation bacterial microcompartment (BMC) protein, EutS, under control of the LacUV5 inducible promoter. Also contains a bidirectional terminator, RBS and all inducible components of the Lac operon. Thus, this part can be used to synthesize EutS at varying concentrations, depending on the task at hand. EutS is tagged with His6 (see Figure 1).

Fig 1: Schematic of the LacUV5_EutS part (BBa_K2213000)

This part is used in the composite part: https://parts.igem.org/Part:BBa_K2213012

Lac UV5 Promoter

The Lac expression system is one of the most commonly used systems for expressing recombinant proteins. The Lac UV5 promoter is very similar to the standard E.coli Lac promoter, with only two base mutations in the -10 hexamer region, compared to the lac promoter. The expression system is primarily composed of the Lac UV5 promoter, the Lac repressor (LacI) and an operator region. In our part, lactose (lac) can bind LacI, reducing its affinity for DNA. Thus upon lac addition, LacI dissociates from the operator , permitting transcription of any gene under control of the LacUV5 promoter.

EutS

EutS is one of the shell proteins that make up the Ethanolamine utilisation bacterial microcompartment (Eut BMC) in E. coli and other enterobacteria species. It is a hexameric protein, and seem to function as the outer edges of the BMC shell (Held et.al, 2013).

A study conducted by Held et.al (2016) and Choudhary et.al (2012) has shown that Eut S is necessary and sufficient for the successful formation of the Eut BMC. This property was also observed by the CU-Boulder iGEM team in 2016 (http://2016.igem.org/Team:CU-Boulder). While the Manchester team did not observe the sufficiency of EutS to form microcompartment, our data suggests that EutMN becomes more stable when co-expressed with EutS (see BBa_K2213001 https://parts.igem.org/Part:BBa_K2213001). This seems to be in line with previous findings on the necessity of EutS for proper BMC formation and further substantiates them.

Usage and Biology

The CU-boulder team has shown that it is possible to make functional BMCs using only EutS in 2016. However, when forced to produce BMCs, E. coli are placed under a large amount of strain and begin to experience slowed and abnormal growth (see characterisation data below). Therefore, the Manchester team recommends the use of a low copy number plasmid eg. pSB4A5 (https://parts.igem.org/Part:pSB4A5) for the expression of EutS. By using a low copy number plasmid, cellular stress is minimised, but the experimenter still has the ability to induce BMC formation.

Characterisation

Understanding growth defects upon Eut protein expression
Following the succesful transformation of Eut constructs into E. coli Manchester iGEM 2017 noticed that cultures grew at a slower rate after Eut subunit protein expression had been induced. This lead to the investigation of how each of Eut construct https://parts.igem.org/Part:BBa_K2213000 , https://parts.igem.org/Part:BBa_K2213001 and https://parts.igem.org/Part:BBa_K2213002 affected growth rate after it had been induced.

Manchester iGEM 2017 recorded optical density measurements at 600nM for EutS, EutMN, EutSMN and EutLK. OD measurements were taken at 0 hours, 4 hours and at 20 hours (see figure 3). It was observed that between 4 and 20 hours, the OD of cultures containing the constructs EutMN, EutSMN and EutLK were reduced by 75.53%, 81.77% and 67.93% respectively. In contrast to this, the OD of the EutS culture continued to rise and had increased by 45.28% when the final reading was taken at 20 hours. This suggests that the production of microcompartment subunits EutM, EutN, EutL and EutK are toxic to the cell, however, the production of EutS may be less toxic. This may be due to less strain being put on the cell due to the expression of a single microcompartment subunit, rather than multiple subunits being expressed simultaneously. Overall this data indicates that the expression of complete microcompartments is likely to be toxic to the cell and should be highly regulated.


Figure 2. Average optical density at 600 nM of EutS, EutMN, EutSMN constructs induced and non-induced. Measurements were taken at 0 hours, 4 hours and 20 hours.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1260
  
• 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.

References
-Held, M., Kolb, A., Perdue, S., Hsu, S., Bloch, S., Quin, M. and Schmidt-Dannert, C. (2016). Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli. Scientific Reports, 6(1).
-Held, M., Quin, M. and Schmidt-Dannert, C. (2013). Eut Bacterial Microcompartments: Insights into Their Function, Structure, and Bioengineering Applications. Journal of Molecular Microbiology and Biotechnology, 23(4-5), pp.308-320.
-Choudhary, S., Quin, M., Sanders, M., Johnson, E. and Schmidt-Dannert, C. (2012). Engineered Protein Nano-Compartments for Targeted Enzyme Localization. PLoS ONE, 7(3), p.e33342.