Toehold activated beta-neomycin (SynORI framework)

This is a full composite part of the constantly expressed riboregulatory sequence Toehold 2 part:BBa_K2259015 coupled with the beta subunit of the split antibiotic resistance gene Part:BBa_K2259019. It has an Anderson promoter part:BBa_J23101 and a double terminator at the end. Toehold 2 switch inhibits the translation of the downstream split antibiotic beta-subunit gene until the activator RNA - trigger 2 - is introduced into the system.

This part is used together with part:BBa_K2259040 to build a 3 or 4 plasmid SynORI selection gene circuit. Additionally, it can be used with signal activated trigger part:BBa_K2259043 to build a 5 plasmid SynORI selection system

Toehold 2 part is activated by the Trigger 2 part:BBa_K2259017

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
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 584
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 433
Illegal SapI.rc site found at 643

Introduction

The overview of 4 plasmid system

Figure 1. The principle scheme of SynORI 4 plasmid selection system

The overview of 5 plasmid system

Figure 1. The principle scheme of SynORI 4 plasmid selection system

Results

Figure 1. The results of 5 plasmid co-transformation using SynORI 5 plasmid selection system

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

Part:BBa_K2259035

Contents