Difference between revisions of "Part:BBa K2259049"

 
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<partinfo>BBa_K2259049 short</partinfo>
 
<partinfo>BBa_K2259049 short</partinfo>
  
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This construct defines a constitutive copy number of plasmid by using an anderson promoter to transcribe RNA I at a specific rate. RNA I controls the copy number by binding to RNA II (plasmid replication iniator) and inhibiting its ability to form primer for polymerase.
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This composite part has a constant expression of amino 3'-glycosyl phosphotransferase (APH(3')) – a protein granting the resistance to aminoglycoside family antibiotics - Beta subunit [[Part:BBa_K2259019]].
  
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<!-- Add more about the biology of this part here
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It is used in conjunction with with the alpha subunit [[Part:BBa_K2259018]] to produce 2 and 3 plasmid selection genetic circuit.
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2259049 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2259051 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2259049 parameters</partinfo>
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<partinfo>BBa_K2259051 parameters</partinfo>
 
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__TOC__
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=Introduction=
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==Biology==
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Aminoglycoside-3'-phosphotransferase (APH(3')), sometimes called aminoglycoside kinase, is an enzyme that catalyzes the addition of phosphoryl group from ATP to the 3'-hydroxyl group of a 4,6-disubstituted aminoglycoside, such as kanamycin, neomycin.
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If both alpha and beta DNA sequences are transcribed and translated in the cell, they can combine and form a fully functional antibiotic resistance protein.
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==Results==
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2 vectors with constant expression of alpha and beta subunits were co-transformed into TG1 competent cells and plated on agar with kanamycin antibiotic
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[[Image:alfabeta.jpg|600px|center|]]
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==Usage with SynORI (Framework for multi-plasmid systems)==
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===About SynORI===
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[[Image:Sel.png|600px|center|]]
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SynORI is a framework for multi-plasmid systems created by ''Vilnius-Lithuania 2017'' which enables quick and easy workflow with multiple plasmids, while also allowing to freely pick and modulate copy number for every unique plasmid group! Read more about [http://2017.igem.org/Team:Vilnius-Lithuania SynORI here]!
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===Split antibiotic resistance in SynORI===
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Split antibiotic coding gene, together with toehold switches and their corresponding RNA triggers completes the dynamic SynORI selection system. The switches lock the translation of downstream split antibiotic genes and form an AND type gate genetic circuit which functions to stably maintain multiple plasmids in the SynORI collection.
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SynORI selection gene circuits for multi-plasmid systems:
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•  2 plasmids
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Consisting of: : Two split antibiotic genes ([[part:BBa_K2259018]] and [[part:BBa_K2259019]])
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•  3 plasmids
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Consisting of:
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One Toehold ([[part:BBa_K2259014]] or [[part:BBa_K2259015]]),
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one Trigger RNA ([[part:BBa_K2259016]] or [[part:BBa_K2259017]]) and
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split neomycin antibiotic resistance genes ([[part:BBa_K2259018]] and [[part:BBa_K2259019]]).
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•  4 plasmids
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Consisting of: Two Toeholds ([[part:BBa_K2259014]] and [[part:BBa_K2259015]]), two Trigger RNAs ([[part:BBa_K2259016]] and [[part:BBa_K2259017]]) and split neomycin antibiotic resistance genes ([[part:BBa_K2259018]] and [[part:BBa_K2259019]]).
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•  5 plasmids
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Consisting of: Modified phage control system [[part:BBa_K2259044]], two Toeholds ([[part:BBa_K2259014]] and [[part:BBa_K2259015]]), two repressed Trigger RNAs ([[part:BBa_K2259042]] and [[part:BBa_K2259043]]) and split neomycin antibiotic resistance genes ([[part:BBa_K2259018]] and [[part:BBa_K2259019]]).
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==References==
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Stable Maintenance of Multiple Plasmids in E. coli Using a Single Selective Marker
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Calvin M. Schmidt, David L. Shis, Truong D. Nguyen-Huu, and Matthew R. Bennett
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ACS Synthetic Biology 2012 1 (10), 445-450
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DOI: 10.1021/sb3000589

Latest revision as of 13:27, 1 November 2017


Constitutive beta-neomycin device (SynORI framework)

This composite part has a constant expression of amino 3'-glycosyl phosphotransferase (APH(3')) – a protein granting the resistance to aminoglycoside family antibiotics - Beta subunit Part:BBa_K2259019.

It is used in conjunction with with the alpha subunit Part:BBa_K2259018 to produce 2 and 3 plasmid selection genetic circuit.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Introduction

Biology

Aminoglycoside-3'-phosphotransferase (APH(3')), sometimes called aminoglycoside kinase, is an enzyme that catalyzes the addition of phosphoryl group from ATP to the 3'-hydroxyl group of a 4,6-disubstituted aminoglycoside, such as kanamycin, neomycin.

If both alpha and beta DNA sequences are transcribed and translated in the cell, they can combine and form a fully functional antibiotic resistance protein.

Results

2 vectors with constant expression of alpha and beta subunits were co-transformed into TG1 competent cells and plated on agar with kanamycin antibiotic

Alfabeta.jpg

Usage with SynORI (Framework for multi-plasmid systems)

About SynORI

Sel.png

SynORI is a framework for multi-plasmid systems created by Vilnius-Lithuania 2017 which enables quick and easy workflow with multiple plasmids, while also allowing to freely pick and modulate copy number for every unique plasmid group! Read more about [http://2017.igem.org/Team:Vilnius-Lithuania SynORI here]!

Split antibiotic resistance in SynORI

Split antibiotic coding gene, together with toehold switches and their corresponding RNA triggers completes the dynamic SynORI selection system. The switches lock the translation of downstream split antibiotic genes and form an AND type gate genetic circuit which functions to stably maintain multiple plasmids in the SynORI collection.

SynORI selection gene circuits for multi-plasmid systems:

• 2 plasmids

Consisting of: : Two split antibiotic genes (part:BBa_K2259018 and part:BBa_K2259019)

• 3 plasmids

Consisting of: One Toehold (part:BBa_K2259014 or part:BBa_K2259015), one Trigger RNA (part:BBa_K2259016 or part:BBa_K2259017) and split neomycin antibiotic resistance genes (part:BBa_K2259018 and part:BBa_K2259019).

• 4 plasmids

Consisting of: Two Toeholds (part:BBa_K2259014 and part:BBa_K2259015), two Trigger RNAs (part:BBa_K2259016 and part:BBa_K2259017) and split neomycin antibiotic resistance genes (part:BBa_K2259018 and part:BBa_K2259019).

• 5 plasmids

Consisting of: Modified phage control system part:BBa_K2259044, two Toeholds (part:BBa_K2259014 and part:BBa_K2259015), two repressed Trigger RNAs (part:BBa_K2259042 and part:BBa_K2259043) and split neomycin antibiotic resistance genes (part:BBa_K2259018 and part:BBa_K2259019).


References

Stable Maintenance of Multiple Plasmids in E. coli Using a Single Selective Marker Calvin M. Schmidt, David L. Shis, Truong D. Nguyen-Huu, and Matthew R. Bennett ACS Synthetic Biology 2012 1 (10), 445-450 DOI: 10.1021/sb3000589