Difference between revisions of "Part:BBa K2259018"
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<partinfo>BBa_K2259018 short</partinfo>
 
<partinfo>BBa_K2259018 short</partinfo>
  
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RNAII acts as a pre-primer and begins the synthesis of plasmid DNA leader strand. The transcript folds into a secondary structure which stabilises the interaction between the nascent RNA and the origin's DNA. This hybrid is attacked by RNase H, which cleaves the RNA strand, exposing a 3' hydroxyl group. This allows the extension of the leading strand by DNA Polymerase I. Lagging strand synthesis is later initiated by a primase encoded by the host cell.
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Split antibiotic resistance gene.
  
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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 as introduced by Calvin M. Schmidt et al.
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Alpha subunit, together with beta subunit [[Part:BBa_K2259019]] 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.
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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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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.
  
  
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=Introduction=
 
=Introduction=
 
==Biology==
 
==Biology==
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===ColE1 plasmid replication overview===
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===Split antibiotic resistance===
  
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[[Image:Cole1 horizontal cropped.png|center|500px|thumb|<b>Figure 1. </b> Main principles of ColE1 plasmid family replication. (Citation needed)]]
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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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<b>ColE1-type plasmid replication begins with synthesis of plasmid encoded RNA II</b> (also called primer transcript) by RNA polymerase which initiates transcription at a site 555bp upstream of origin of replication. The RNA transcript forms a RNA - DNA hybrid with template DNA near the origin of replication. Hybridized RNA is then cleaved at the replication origin by RNAse H and serves as a primer for DNA synthesis by DNA polymerase I (Figure 1. A).
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<b>Initiation of replication can be inhibited by plasmid encoded small RNA, called RNA I </b>. Synthesis of RNA I starts 445 bp upstream of the replication origin and proceeds in the direction opposite to that of RNA II synthesis, and terminates near the RNA II transcription initiation site. <b>RNA I binds to RNA II</b> and thereby prevents formation of a secondary structure of RNA II that is necessary for hybridization of RNA II to the template DNA (Figure 1. B).
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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
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For RNA I to inhibit primer formation, it must bind before the nascent RNA II transcript extends to the replication origin. Consequently, the concentration of RNA I and the rate of binding of RNA I to RNA II is critical for regulation of primer formation and thus for plasmid replication.
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Interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]].
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Rop dimer is a bundle of four tightly packed alpha helices that are held by hydrophobic interactions (Fig. 2).
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==Usage with SynORI (Framework for multi-plasmid systems)==
 
==Usage with SynORI (Framework for multi-plasmid systems)==
  
 
===About SynORI===
 
===About SynORI===
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[[Image:Aboutsynoritry1.png|600px|center|]]
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[[Image:sel.png|600px|center|]]
 
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]!
  
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===Regulative RNA II molecule in SynORI===
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===Split antibiotic resistance in SynORI===
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RNA II gene is foundational and central biobrick of SynORI system, and by far the only one that is mandatory for framework to run.  
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The two main functions of RNA II is as follows:
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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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# Initiating plasmid replication
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# Interacting with RNA I of specific plasmid group [[#Specific RNA II versions in multi-plasmid systems|(See below)]]
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SynORI selection gene circuits for multi-plasmid systems:
  
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===Specific RNA II versions in multi-plasmid systems===
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•  2 plasmids
  
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RNA II interacts with inhibitory RNA I with three secondary RNA stem loops. In order to create plasmid groups with independent copy number control, one group's RNA II molecule must interact only with the same group's RNA I molecule.
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Consisting of: Two split antibiotic genes ([[part:BBa_K2259018]] and [[part:BBa_K2259019]]).
  
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<b>For example</b> if there are two plasmid groups in a cell - A and B - RNA II of A group
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•  3 plasmids
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would only interact with RNA I A, and not RNA I B.
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[[Image:RnainteractionIII.png|center|500px|thumb|<b>Figure 1. </b> RNA I AND II group interaction example]]
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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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===Origin of RNA II biobrick===
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•  4 plasmids
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In order to flexibly control the synthesis of RNA I (Why RNA I ? <link to RNA I biobrick>), the RNA I gene first needed to be inactivated in ColE1 origin of replication. That, however, was not a trivial task, as ColE1 ORI is an antisense system, which means that by changing RNA I promoter sequence, one also changes the RNA II secondary structure, which is crucial for plasmid replication initiation (Find out more about how team Vilnius-Lithuania solved this problem by pressing this link! <LINK REQUIRED>). This is the main reason why, in SynORI framework, the wildtype ColE1 ORI is split into two different parts - <b> RNR I and RNA II </b>.
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<Picture of how RNA I promoter mutations might destroy RNA II secondary structure.>
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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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=Characterization of RNA II (Vilnius-Lithuania 2017)=
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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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==Constitutive Rop protein effect on plasmid copy number==
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To be updated!
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==References==
 
==References==
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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 19:42, 1 November 2017


Alpha-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 (this part!) and Beta between amino acid residues 59 and 60 as introduced by Calvin M. Schmidt et al.

Alpha subunit, together with beta subunit Part:BBa_K2259019 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.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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 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

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