Latest revision as of 13:29, 1 November 2017

Intermediate of Toehold activated alpha-neomycin (SynORI framework)

The intermediate of part:BBa_K2259034 - it has no promoter upstream the of the construct.

A composite part of the riboregulatory sequence Toehold 1 part:BBa_K2259014 coupled with the alpha subunit of the split antibiotic resistance gene Part:BBa_K2259018. It has a double terminator at the end.

This part (with up stream promoter) is used together with part:BBa_K2259038 to build a 3 or 4 plasmid SynORI selection gene circuit. Additionally, it can be used with signal activated trigger part:BBa_K2259042 to build a 5 plasmid SynORI selection system

Toehold 1 part is activated by the Trigger 1 part:BBa_K2259016

Sequence and Features

Introduction

Figure 1. The schematic representation of 4 plasmid SynORI selection system. First 2 plasmids constantly express Toehold 1 and 2 which are coupled with the split antibiotic alpha and beta subunits but has its translation locked. Trigger 1 and 2 are constantly expressed by other 2 to plasmids to unlock the resistance gene translation. If any of the plasmid is lost, the cell dies.

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