Revision as of 12:13, 8 October 2021

mRNA cyclization system + slpA + sfGFP

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.

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.

Part information

A synthetic mRNA cyclization system enables translation only when both ends of mRNAs are present and followed by circularization based on sequence-specific RNA–RNA hybridization. This system should improve the fraction of full-length proteins among all synthesized polypeptides by selectively translating intact mRNA and reducing abortive translation. A synthetic mRNA cyclization system was coupled with p-slpA promoter and gene encoding for green fluorescent protein (GFP) to evaluate how mRNA cyclization system improves production GFP under the aforementioned promoter.

Sequence and Features