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Biobricks & Strategy

What are Biobricks?

The iGEM Standards

The iGEM competition is based on Biobrick standards; it is a standard form of plasmid for interchangeable parts, developed with a view to build biological systems in living cells. Parts are characterized by sequence of DNA that encodes for biological function. This standardization aims to facilitate shipping to the Registry (which gather all Biobricks) to maintain and test them; to measure them for evaluation and characterization; and then to assemble easily compatible parts without any issues.

Backbone, Prefix and Suffix

The backbone containing the part has two flanking parts: the prefix and suffix. The prefix and suffix contain restriction enzyme cut sites; that can be used to transfer and assemble parts. In prefix we find EcoRI and XbaI sites while in suffix we have SpeI and PstI sites. In order to standardize all parts, iGEM request for all parts to not have any of the restriction sites found in prefix or suffix. This justify why, in our strategy, we have tried to remove this illegal sites from our sequences. Restriction enzyme sites in prefix and suffix have been chosen in order to facilitate parts assembly.

iGEM basic parts

All Biobrick that have been created and used in iGEM are kept in the Registry. Each year all the team registered receive a kit. In 2015 the iGEM kit is composed of 5 plates with standards parts in dried DNA. In those plates we had the RBS in pSB1A2 plasmid and the double terminator T7 in pSB1C3 plasmid. For our construction we needed a promoter T7 and an endolysin/holin gene. So we have requested to the Registry a promoter T7 (BBa_I712074) and the endolysin/holin gene (BBa_K124003). However, as the demand with all iGEM team is important the time to receive those Biobrick was long.

Part of DNA

1 Backbone

Bacteria

1 Biobrick

Bio-Console parts

Fluoresence: VVD-split-YFP construction

In order to make our bacteria express fluorescence in response to a light stimulus, we aimed to construct a BioBrick that will link each VVD photoreceptor to one part of the split-YFP. In absence of blue-light, the conformation of the photoreceptor will prevent the formation of the complete fluorescent protein while in presence of the light signal the YFP protein will be reconstituted leading to the fast expression of a yellow fluorescence in our bacteria. Despite the possible dimerization of two similar VVD-Split-YFP, there is a probability of 50% for the YFP to be functional after dimerization. The fluorescence should be observable about 20 min after light illumination.

This BioBrick required the integration of some specific sequence between the split-YFP and the VVD receptor. Those ‘linker’ have been provided by Samuel Juillot, supervisor of our iGEM team. The linkers as well as the Split YFP (YC155-YN155) have been initially sent by Tom Kerppola, Ph. D, investigator at the Howard Hughes Medical Institute as well as Professor in the University of Michigan. The choice of linker is critical as their sequences must meet the flexibility and the length required for the two split part to join and to become effective. The linker chosen have been shown to work with the provided split-YFP. Hu, CD, Chinenov, Y and Kerppola, TK. Molecular Cell 9: 789-798, 2002.

VVS system strategy

The main point of our strategy is based on the Gibson Assembly. It is a method to assemble easily multiple DNA fragments independently of the fragments size or extremities compatibilities without any scars.

Kill switch biobricks: pDawn – Toxin & pDusk- Toxin

We aim to construct an efficient kill-switch triggering by light. In this way we will construct two different parts. The first one will be composed of pDawn and holin/endolysin. Toxins will be produced after light stimulation and induce lysis. The second part will be composed of pDusk and endolysin/holin complex, this will lead to the cell lysis when the bacteria won’t be exposed to light. This second part will be more difficult to characterized indeed all steps before characterization of the plasmid as plasmid amplification will have to be under light.

Strategy

As the VVD strategy; the construction of the BioBrick composed of pDawn and pDusk are based on the Gibson Assembly. The Gibson assembly allows to assemble together pDawn, endolysin/holin in pSB1C3 and also assemble pDusk, endolysin/holin in pSB1C3.