Team:Freiburg/Biobricks

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Biobricks

1. Constructed BioBricks

Our project required the construction of fusion proteins that are displayed on the surface of the spores of B. subtilis. Anchoring an epitope-specific nanobody and glutathione S-transferase to coat proteins enables the spores to attach to surfaces and activate prodrugs on site. In addition to targeted drug delivery, site specific enzymatic activities play a crucial role in other scientific field and many further applications are imaginable ranging from the production of metabolites to bioremediation. Therefore, we would like to share our achievements with the iGEM community in the hope to support and facilitate future projects. Our supervisors Dr. Maximilian Ulbrich and Dr. Nicole Gensch provided us plasmids encoding the anti-GFP nanobody and the glutathione S-transferase that were inevitable for our project. The Integration vectors used to insert our constructs into the genome of B. subtilis were provided by Julia Bartels from the Mascher lab at the TU Dresden. Below you will find a table summarizing the submitted biobricks and a link guiding you to the entries in the registry.

	Biobrick	Part	pIG16_000
Surface Display	BBa_K2114001	aGFPnano_HA_aHelix_cotZ	pIG16_191
	BBa_K2114002	aGFPnano_HA_G4S_cotZ	pIG16_190
	BBa_K2114009	aGFPnano_HA_aHelix_CotG	pIG16_173
	BBa_K2114011	CotG_G4S_HA_GST	pIG16_066
	BBa_K2114017	aGFPnano_HA_aHelix_cgeA	pIG16_172
	BBa_K2114019	GST_HA_G4S_CgeA	pIG16_047
	BBa_K2114020	CgeA_G4S_HA_GST	pIG16_050
Improved Parts	BBa_K2114000	PCotYZ-RBS	pIG16_195
	BBa_K2114998	pelB anti-GFP nanobody	pIG16_162

We chose the parts BBa_K2114001, BBa_K2114002 and BBa_K2114011 as our favorite parts. They belong to the first fusion genes we could assemble. Also they were overall easy to handle regarding their construction compared to other fusion genes like the ones containg the spore coat gene cotB (which by the way proved to be quite a challenge).
We used them to generate functional Nanocillus displaying the anti-GFP nanobody that could bind its target or display an active glutathione-S‑transferase. Overall, those parts played a central role in our project ensuring that we pursue a promising approach for targeted drug delivery.

2. Improved Biobricks

pelB_anti-GFP nanobody

We submitted an improved anti-GFP nanobody containing the pelB leader sequence for the export of the protein in the periplasmatic space resulting in an increased yield of the over-expressed protein.

P_CotYZ-RBS

The B. subtilis promoter P_CotYZ regulates the expression of the late stage spore coat proteins CotY and CotZ. We included a ribosome binding site which simplifies the assembly of devices and the expression of subsequent genes.

Posted by: iGEM Freiburg

Nanocillus - 'cause spore is more!

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