Part:BBa_K2259019

Beta-neomycin resistance protein

Split antibiotic resistance gene.

Amino 3'-glycosyl phosphotransferase (APH(3')) – a protein granting the resistance to aminoglycoside family antibiotics was split into two subunits Alpha and Beta (This part!) between amino acid residues 59 and 60 as introduced by Calvin M. Schmidt et al and Part:BBa_K1442031.

Beta subunit, together with alpha subunit Part:BBa_K2259018 are improved versions of Part:BBa_K1442031 part. The antibiotic has been properly split to two basic parts and includes additionally added termination codon at the end of the alpha subunit to terminate the translation after the peptide is synthesized. We removed the leucine zipper domains, as the heterodimerization occurs naturally. We did not include the start codon at the 5' end of the gene as reports say it has an alternative start codon.

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.

This part is used in SynORI multi-plasmid framework selection system, in which up to 5 unique plasmid groups can be maintained in a single cell using only one antibiotic resistance protein.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 452
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 301
  Illegal SapI.rc site found at 511
  

Introduction

Biology

Split antibiotic resistance

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.

Aminoglycoside-3'-phosphotransferase gene was split by Calvin M. Schmidt et al to produce a protein that is enzymatically active only when a from alpha and beta subunits is formed

Usage with SynORI (Framework for multi-plasmid systems)

About SynORI

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