Difference between revisions of "Part:BBa K3904229"

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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.  
 
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].
+
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 (Table 1). The same evaluation strategy was applied to measure the effectiveness of mRNA cyclization system [1].
 +
 
 +
<b>Table 1:</b> the evaluation results of the promoters without mRNA cyclization system.
 +
 
 +
{| class="wikitable sortable"
 +
|-
 +
! name
 +
! ratio
 +
! absolute value
 +
|-
 +
| BBa1033225 || 1.014962837 || 383.9908901
 +
|-
 +
| BBa1033222 || 0.602083582 || 227.7862817
 +
|-
 +
| BBa1033220 || 0.780394955 || 295.2468234
 +
|-
 +
| P-slpA || 1.343329840 || 508.2219782
 +
|-
 +
| J23118 || 0.466347714 || 176.4333307
 +
|-
 +
| J23117 || 0.411647170 || 155.7384740
 +
|-
 +
| J23115 || 0.358044033 || 135.4587989
 +
|-
 +
| J23114 || 0.462713712 || 175.0584787
 +
|-
 +
| J23113 || 0.477268368 || 180.5649417
 +
|-
 +
| J23107 || 0.669148942 || 253.1591191
 +
|-
 +
| J23106 || 0.627985537 || 237.5857682
 +
|-
 +
| J23103 || 0.472128151 || 178.6202433
 +
|-
 +
| J23102 || 0.496449817 || 187.8218593
 +
|-
 +
| J23101 || 0.597720501 || 226.1355971
 +
|}
  
 
=Introduction=
 
=Introduction=

Revision as of 06:43, 20 October 2021


mRNA cyclization system + slpA + sfGFP

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 (Table 1). The same evaluation strategy was applied to measure the effectiveness of mRNA cyclization system [1].

Table 1: the evaluation results of the promoters without mRNA cyclization system.

name ratio absolute value
BBa1033225 1.014962837 383.9908901
BBa1033222 0.602083582 227.7862817
BBa1033220 0.780394955 295.2468234
P-slpA 1.343329840 508.2219782
J23118 0.466347714 176.4333307
J23117 0.411647170 155.7384740
J23115 0.358044033 135.4587989
J23114 0.462713712 175.0584787
J23113 0.477268368 180.5649417
J23107 0.669148942 253.1591191
J23106 0.627985537 237.5857682
J23103 0.472128151 178.6202433
J23102 0.496449817 187.8218593
J23101 0.597720501 226.1355971

Introduction

AmeBye

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

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 [1]. A synthetic mRNA cyclization system was coupled with Lactobacillus acidophilus ATCC 4356 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 183

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