Team:BCCS-Bristol/Notebook/Biobricks

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<p>Finished biobricks to be submitted to the iGEM parstregistry at the competition are outlined in the tables below :</p>
<p>Finished biobricks to be submitted to the iGEM parstregistry at the competition are outlined in the tables below :</p>
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<td>AraC-RBS-FhuA-GFP-Terminator</td>
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Revision as of 21:25, 20 October 2009

BCCS-Bristol
iGEM 2009




Contents

Parts Submitted to The Registry

Finished biobricks to be submitted to the iGEM parstregistry at the competition are outlined in the tables below :

The table illustrates the finished biobricks that were created as a proof of principle to illustrate OMV directed delivery.
Finished Assemblies
AraC-RBS-GFP-Terminator
AraC-RBS-FhuA-GFP
The table illustrates biobricks that were created in the duration of the project and that will be of use to synthetic biologists.
Promoter-RBS Promoter-RBS-Carriers Reporters Bioscaffolds Proteins
AraC-RBS AraC-RBS-OsmE GFP-Terminator BpuEI-BseRI FhuA
- AraC-RBS-FhuA - BpuEI-CspCI(3 versions) OsmE


Why these biobricks?

We choose the AraC promoter because its activity can be controlled by the Arabinose levels (direct relationship) just like o rheostat. This means that we will be able to synthesize our fusion protein at will (i.e. when bacteria cell numbers are high) and at the required levels that won't cause cell lysis or non-specific aggregation in cytoplasm. The RBS J61100 is a ribosome binding site with strong affinity for RNA polymerase ensuring that once RNA polymerase binds to the RBS it will stay bound and won't dissociate giving us maximum transcription. The carrier proteins we selected for carrying our protein(s) of interest (cargo) had the following properties: 

           a.	small size
           b.	not involved in metabolic processes 
           c.	monomeric
           d.	non-toxic
           e.	commonly found in Bacterial OMVs
           f.	either outer membrane proteins or periplasmic

Based on a list of proteins that enriched OMVs (Eun-Young Lee et al, 2007) we selected OsmE, Fiu and FhuA.

The Bioscaffold BBa_K259002 is used for the efficient joining of the carrier and cargo partners in the protein fusion leaving a scarless region and a flexible Gly-Ser-Gly ([http://partsregistry.org/Part:BBa_K259002 more information]). Moreover GFP (Green Fluorescent protein) is used as our cargo just for proof of concept because it can be easily detected, enabling us to monitor the expression of the fusion protein and its introduction into OMVs. Also BBa_B0014 double terminator is used to stop transcription ensuring that no other DNA sequences are incorporated into our fusion protein that may interfere with the folding of the individual proteins.

Stucture of our Biobrick

General Structure of our Designed Biobrick Construct

Assembly Plan (timeline)

KEY: V=vector, I=insert. For the biobricks on the left hand side: the biobricks on the left were digested with SpeI and PstI whereas those on the right were digested with XbaI and PstI. For the biobricks on the right hand side: the biobricks on the left were cut with EcoRI and SpeI and those on the right with EcoRI and XbaI. For the final ligation the Promoter-RBS-carrier construct was cut with SpeI and PstI and the bioscaffold-cargo-terminator construct was cut with XbaI and PstI
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