Revision as of 16:12, 20 October 2021

Surface-layer protein A promoter (p-slpA)

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 (Fig. 1). 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.

Figure 1: first graph - comparison of promoter strength by indicating the curves of the two strongest promoters and the generalized curve of the remaining weaker promoters; second graph - comparison of acceleration vs velocity by indicating the curves of the two strongest promoters and the generalized curve of the remaining weaker promoters.

Introduction

Vilnius-Lithuania iGEM 2021 project AmeBye looks 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

The Lactobacillus acidophilus ATCC 4356 surface-layer protein A (slpA) promoter demonstrates the highest levels of transcription in all Lactobacillus strains examined in the study conducted by A. McCracken and colleagues. Combined with data from other researchers indicating that the slpA promoter is highly efficient in the natural host and in L. casei ATCC 393, this suggests that the slpA promoter sequence harbours motifs that interact effectively with RNA polymerase of several lactobacilli. This has striking implications for the development of heterologous gene-expression cassettes in lactobacilli by offering a powerful tool for achieving high levels of foreign protein production [1].

Sequence and Features

References

1. McCracken, A., Turner, M. S., Giffard, P., Hafner, L. M., & Timms, P. (2000). Analysis of promoter sequences from Lactobacillus and Lactococcus and their activity in several Lactobacillus species. Archives of microbiology, 173(5), 383-389.