Part:BBa_K3904119
No part name specified with partinfo tag.
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. In addition to that, we also introduced an mRNA cyclization system to this project. The circularization of mRNA molecules improves the fraction of full-length proteins among synthesized polypeptides by selectively translating intact mRNA and reducing abortive translation.
Promoter strength was estimated by measuring how intensively the nissle transformants can produce GFP under the promoter of interest. Fluorescence intensity was evaluated by dividing the intensiveness of the signal by the OD600 of the medium during the course of 6 hours. The greater ratio would indicate the stronger promoter. The data were compared between all the promoters of our interest and the sequences demonstrating required expression rates were selected for the construction of naringenin synthesis cassete. The same evaluation strategy was applied to measure the effectiveness of mRNA cyclization system [1].
Introduction
Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefor target E. histolytica from several angles: prevention and diagnostics. As a tool to prevent amebiasis, team created probiotics capable of naringenin biosynthesis. For the diagnostic part, project 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
J23102 Anderson promoter and gene encoding for green fluorescent protein (GFP) fused with tyrosine ammonia lyase (TAL) gene to evaluate the expression of fused proteins under the aforementioned promoter.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 1698
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal SpeI site found at 1698 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1507
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 1698
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 1698
Illegal AgeI site found at 63
Illegal AgeI site found at 306
Illegal AgeI site found at 663 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1725
References
- 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
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