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 .
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.
Usage and Biology
Construct containing mRNA cyclization system, CP44 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.
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).
Figure 1: comparison of different promoter strengths with and without mRNA cyclization system.
Sequence and Features
- 10Illegal SpeI site found at 1821
- 12Illegal SpeI site found at 1821
- 21Illegal BglII site found at 1630
- 23Illegal SpeI site found at 1821
- 25Illegal SpeI site found at 1821
Illegal AgeI site found at 186
Illegal AgeI site found at 429
Illegal AgeI site found at 786
- 1000Illegal SapI.rc site found at 1848
- 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