Difference between revisions of "Part:BBa K2259018"
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<partinfo>BBa_K2259018 short</partinfo> | <partinfo>BBa_K2259018 short</partinfo> | ||
− | Amino 3'-glycosyl phosphotransferase (APH(3')) – a protein granting the resistance to aminoglycoside family antibiotics was split into two subunits Alpha (this part!) and [https://parts.igem.org/Part:BBa_K2259019 Beta]. | + | Amino 3'-glycosyl phosphotransferase (APH(3')) – a protein granting the resistance to aminoglycoside family antibiotics was split into two subunits Alpha (this part!) and [https://parts.igem.org/Part:BBa_K2259019 Beta] between amino acid residues 59 and 60 and introduced by Calvin M. Schmidt et al and [[Part:BBa_K1442031]]. Additionally, we added the termination codon at the end of the alpha subunit. |
+ | |||
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. | 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 | + | This part is used in SynORI multiplasmid framework selection system, in which up to 5 unique plasmid groups can be maintained in a single cell using only one antibiotic resistance protein. |
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=Introduction= | =Introduction= | ||
==Biology== | ==Biology== | ||
− | === | + | ===Split antibiotic resistance=== |
[[Image:Cole1 horizontal cropped.png|center|500px|thumb|<b>Figure 1. </b> Main principles of ColE1 plasmid family replication. (Citation needed)]] | [[Image:Cole1 horizontal cropped.png|center|500px|thumb|<b>Figure 1. </b> Main principles of ColE1 plasmid family replication. (Citation needed)]] | ||
− | <b> | + | <b> 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 to produce proteins that are enzymatically active only as a dimer Calvin M. Schmidt et al. | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
==Usage with SynORI (Framework for multi-plasmid systems)== | ==Usage with SynORI (Framework for multi-plasmid systems)== | ||
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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]! | 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]]). | |
− | + | ||
− | |||
Revision as of 16:38, 31 October 2017
Alpha-neomycin resistance protein
Amino 3'-glycosyl phosphotransferase (APH(3')) – a protein granting the resistance to aminoglycoside family antibiotics was split into two subunits Alpha (this part!) and Beta between amino acid residues 59 and 60 and introduced by Calvin M. Schmidt et al and Part:BBa_K1442031. Additionally, we added the termination codon at the end of the alpha subunit.
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 multiplasmid 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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 to produce proteins that are enzymatically active only as a dimer Calvin M. Schmidt et al.
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).
Characterization of RNA II (Vilnius-Lithuania 2017)
Constitutive Rop protein effect on plasmid copy number
To be updated!
References