Difference between revisions of "Part:BBa K3904320"

 
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=Introduction=
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[[File:T--Vilnius-Lithuania--amebyeLogo dark.png|right|100px|AmeBye]]
  
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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.
===Usage and Biology===
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__TOC__
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3904320 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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===Usage and Biology===
<partinfo>BBa_K3904320 parameters</partinfo>
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Regular pET-28a(+) has not been suitable for our experiments as it contained additional nucleotides coding random amino acids between His-tag and protein-encoding sequence. Protein without any additional amino acids is needed because it later has been used in SELEX experiments where ssDNA sequences specifically bind to protein because of specific protein structure features.
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Keeping this in mind, we restricted pET-28a(+) with XbaI and NdeI and ligated with RBS, a spacer with no N-His tag. This way, we get plasmid with the ony C-His tag.
 
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===Sequence and Features===
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<partinfo>BBa_K3904320 SequenceAndFeatures</partinfo>

Latest revision as of 11:25, 20 October 2021


pET-28a(+) with C-His tag and TEV recoginition sequence

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

Regular pET-28a(+) has not been suitable for our experiments as it contained additional nucleotides coding random amino acids between His-tag and protein-encoding sequence. Protein without any additional amino acids is needed because it later has been used in SELEX experiments where ssDNA sequences specifically bind to protein because of specific protein structure features. Keeping this in mind, we restricted pET-28a(+) with XbaI and NdeI and ligated with RBS, a spacer with no N-His tag. This way, we get plasmid with the ony C-His tag.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 5115
    Illegal XbaI site found at 5030
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 5115
    Illegal NheI site found at 5076
    Illegal NotI site found at 5140
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 5115
    Illegal BglII site found at 4964
    Illegal BamHI site found at 5109
    Illegal XhoI site found at 5149
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 5115
    Illegal XbaI site found at 5030
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 5115
    Illegal XbaI site found at 5030
    Illegal NgoMIV site found at 137
    Illegal NgoMIV site found at 3184
    Illegal NgoMIV site found at 3344
    Illegal NgoMIV site found at 4932
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2263