Composite

Part:BBa_K3904123:Design

Designed by: Rimvydė Čepaitė   Group: iGEM21_Vilnius-Lithuania   (2021-09-29)


CP44-TAL-sfGFP

Introduction

AmeBye

Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefore target E. histolytica from several angles: prevention and diagnostics. Our team's preventive solution consists of probiotics engineered to produce naringenin - an antiprotozoal compound. Two strains of genetically modified microorganisms were chosen as the main chassis - world-renowned Lactobacillus casei BL23 (Lactobacillus paracasei) and Escherichia coli Nissle 1917. Furthermore, the team made specific gene deletions to enhance naringenin production and adapted a novel toxin-antitoxin system to prevent GMO spreads into the environment. The diagnostic part includes a rapid, point of care, user-friendly diagnostic test identifying extraintestinal amebiasis. The main components of this test are aptamers specific to the E. histolytica secreted proteins. These single-stranded DNA sequences fold into tertiary structures for particular fit with target proteins.

To assure continuous and efficient naringenin production, we had to guarantee the proper expression of naringenin synthesis enzymes in our probiotic strains. To reach the maximum efficiency of the natural synthesis pathway, we decided to manipulate the expression rates of these proteins by finding the promoters of optimal strength for the expression of each of these enzymes using pTRKH2 vector. In addition to that, we also introduced an mRNA cyclization system to this project. The circularization of mRNA molecules is supposed to improve the fraction of full-length proteins among synthesized polypeptides by selectively translating intact mRNA and reducing abortive translation [1].

The effectiveness of the mRNA cyclization system was estimated by measuring how intensively the nissle transformants can produce sfGFP fused with tyrosine ammonia lyase (TAL) under the slpA promoter. Fluorescence intensity was evaluated by dividing the intensiveness of the signal by the OD600 during the course of 7 hours. Results in our particular case did not show the expected potential to improve the efficiency of our system (Figure 1).

AmeBye

Figure 1: comparison of different promoter strengths with and without mRNA cyclization system.

Usage and Biology

Construct containing mRNA cyclization system, slpA promoter and gene encoding for sfGFP fused with tyrosine TAL gene to evaluate the ability of mRNA system to enhance expression of fused proteins under the aforementioned promoter.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 1722
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 1722
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1531
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 1722
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 1722
    Illegal AgeI site found at 87
    Illegal AgeI site found at 330
    Illegal AgeI site found at 687
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1749

References

  1. Yang, J., Han, Y.H., Im, J. et al. Synthetic protein quality control to enhance full-length translation in bacteria. Nat Chem Biol 17, 421–427 (2021). https://doi.org/10.1038/s41589-021-00736-3