Difference between revisions of "Part:BBa K3904119"

 
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<partinfo>BBa_K3904219 short</partinfo>
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<partinfo>BBa_K3904119 short</partinfo>
  
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.
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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 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 [1].  
 
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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].
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=Introduction=
 
=Introduction=
 
[[File:T--Vilnius-Lithuania--amebyeLogo dark.png|right|100px|AmeBye]]
 
[[File:T--Vilnius-Lithuania--amebyeLogo dark.png|right|100px|AmeBye]]
  
Vilnius-Lithuania iGEM 2021 project [https://2021.igem.org/Team:Vilnius-Lithuania <b>AmeBye</b>]looks at amebiasis holistically and comprehensively, therefor target <i>E. histolytica</i> 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 <i>E. histolytica</i> secreted proteins. These single stranded DNA sequences fold into tertiary structures for particular fit with target proteins.
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Vilnius-Lithuania iGEM 2021 project [https://2021.igem.org/Team:Vilnius-Lithuania <b>AmeBye</b>]looks at amebiasis holistically and comprehensively, therefore target <i>E. histolytica</i> 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 <i>Lactobacillus casei</i> BL23 (<i>Lactobacillus paracasei</i>) and <i>Escherichia coli</i>  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 <i>E. histolytica</i> secreted proteins. These single-stranded DNA sequences fold into tertiary structures for particular fit with target proteins.
  
 
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=Usage and Biology=
 
=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.
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J23102 Anderson promoter and gene encoding for sfGFP fused with tyrosine ammonia lyase (TAL) gene to evaluate the expression of fused proteins under the aforementioned promoter.
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=Part characterization=
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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).
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[[File:MRNA_cyclization_system_evaluation_sfGFP+TAL.png|center|600px|AmeBye]]
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<b>Figure 1:</b> comparison of different promoter strengths with and without mRNA cyclization system.
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=Sequence and Features=
 
=Sequence and Features=
<partinfo>BBa_K3904219 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3904119 SequenceAndFeatures</partinfo>
  
 
=References=
 
=References=

Latest revision as of 21:32, 21 October 2021


J23102-TAL-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 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 [1].

Introduction

AmeBye

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

J23102 Anderson promoter and gene encoding for sfGFP fused with tyrosine ammonia lyase (TAL) gene to evaluate the expression of fused proteins under the aforementioned promoter.

Part characterization

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).

AmeBye

Figure 1: comparison of different promoter strengths with and without mRNA cyclization system.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 1698
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal SpeI site found at 1698
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1507
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 1698
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1725

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