Part:BBa_K2259043

Signal activated trigger 2 (SynORI framework)

This is a full composite part of the regulated expression activating RNA Trigger 2 part:BBa_K2259017 used to unlock the translation of gene inhibited by Toehold 2 switch part:BBa_K2259015. It is under a modified lambda Prm promoter part:BBa_I12006 and a double terminator at the end. RNA Trigger 2 is followed by the constantly expressed Prm promoter repressor - cI 434 part:BBa_K2259047 to minimize the expression when the activator - cI lambda part:BBa_K2259044 is absent.

This part is used together with part:BBa_K2259035 and part:BBa_K2259044 to build a 5 plasmid SynORI selection gene circuit.

Trigger 2 expression is upregulated when cI lambda part:BBa_K2259044 is present and activates the translation of Toehold 2 part:BBa_K2259015 locked gene.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 273
  Illegal NheI site found at 296
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1015
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

The overview of 5 plasmid system

Figure 1. The schematic representation of 5 plasmid SynORI selection system. First plasmid constantly expresses lambda, the activator of the modified phage promoter, which controls the expression of the Trigger 1 and 2. The Triggers unlock the translation of split resistance gene controlled by Toehold 1 and 2. Additionally, 434 repressor is constantly expressed to regulate the modified phage promoter. If any of the plasmid is lost, the cell dies.

Results

Figure 1. SynORI 5 plasmid co-transformation results. 1 - No trigger 1 (control). 2 - No trigger 2 (control). 3 - No lambda activator plasmid (control). 4 - Full System: lambda activator plasmid; toehold 1 alpha-neo; toehold 2 beta-neo; trigger 1; trigger 2

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]!

Toehold riboregulators in SynORI

Toehold switches together with their corresponding RNA triggers and split antibiotic genes 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).

Two groups of Toeholds

SynORI collection introduces two Toehold sequences termed Toehold 1 and Toehold 2 which only interact with its corresponding Trigger RNA, termed Trigger 1 and Trigger 2 and display no cross interaction.

References

Toehold Switches: De-Novo-Designed Regulators of Gene Expression

Green, Alexander A. et al. Cell, Volume 159, Issue 4, 925 - 939