Team:Dundee/Biobricks

New Biobricks:

1. BBa_K895001 Salmonella Hcp protein. This biobrick encodes the Hcp protein (STM0279 or tssD). This is a key component of Type VI Secretion Systems and is often found in the cell supernatant so is commonly regarded as a secreted protein.

2. BBa_K895002 Salmonella VgrG protein. This biobrick is a gene encoding an important large protein thought to form the tip of the Type VI Secretion/Cell Puncturing device. VgrG (STM0289 or TssI) must have an extracytoplsmic localisation. This would be the initial point of contact between the Type VI Secretion System and any target cell.

3. BBa_K895003 Salmonella TtrRS two-component system. This biobrick encodes a bacterial two-component system. The TtrS protein is a membrane-bound histidine kinase. The kinase senses extracellular tetrathionate levels (a compound produced in the gut when the inflammation process is induced during infection), which activates its autophosphorylation activity. The phosphate is passed on to TtrR, the DNA-binding response regulator, which can then activate transcription of at least one operon its native organism (Salmonella. The divergent ttrB promoter/operator site is included in the biobrick. In the native organism, ttrB is regulated by oxygen levels (an FNR binding site is present in the promoter) and, predominantly, by extracellular tetrathionate levels via TtrRS.

4. BBa_K895004 Salmonella Hcp fused to φCD27 endolysin. This biobrick encodes Hcp (from BBa_K895001) fused via an HA epitope tag to a synthetic endolysin based on that from a C. difficile bacteriophage (φCD27).

5. BBa_K895005 Salmonella VgrG fused to φCD27 endolysin. This biobrick encodes VgrG (from BBa_K895002) fused via an HA epitope tag to a synthetic endolysin based on that from a C. difficile bacteriophage (φCD27).

6. BBa_K895007 Salmonella TtrRS two-components system linked to a GFP reporter. This biobrick is a working 'inflammation bisensor' comprising BBa_K895003 linked to GFP from BBa_J54103. Transcription, and subsequent translation, of GFP is linked to extracellular tetrathionate levels in the E. coli chassis organism.

Improving and de-bugging old biobricks:

1. BBa_K895000 Escherichia coli tat constitutive promoter. This small biobrick was originally designed by the Dundee iGEM 2011 Team. Though this was cloned as an EcoRI / PstI fragment into pSB1C3, this rookie team mistakenly omitted to include the correct prefix and suffix sequences to meet RFC[10]. In this work we have corrected this error and correctly assembled the tat promoter into pSB1C3.

2. BBa_K895006 Classic GFP-encoding gene cloned to RFC[10] standard in pSB1C3. For our project we decided to use GFP as a reporter protein. We wished to use a GFP-producing biobrick from the 2012 Distribution Kit and chose that from BBa_J54103. We noticed that this was in a ampicillin-resistant vector rather than pSC1C3. We thought it would be helpful to the synthetic biology community to move the open reading frame of the GFP encoded by BBa_J54103 onto pSB1C3 following RFC[10] standard rules.

3. BBa_K895007 Salmonella TtrRS two-components system linked to a GFP reporter. This is a working 'inflammation biosensor' comprising BBa_K895003 linked to GFP from BBa_J54103.

Favourite Biobricks:

1. BBa_K895007 Salmonella TtrRS two-components system linked to a GFP reporter. The 'inflammation biosensor' has great potential for health & medicine applications, particularly for devices designed to respond intelligently to the infection process.

2. BBa_K895001 Salmonella Hcp protein. The predicted extracellular localisation of this protein makes it potentially useful for future projects. If secretion of the protein can be controlled and characterised then it could be fused to any number of reporters or other enzymes. The BBa_K895001 polypeptide can be produced stably in E. coli. Fusion proteins are also stable.

3. BBa_K895002 Salmonella VgrG protein. The predicted extracellular localisation of this protein makes it potentially useful for future projects. It too could be fused to a broad spectrum of reporter proteins or other enzymes. The BBa_K895002 polypeptide can be produced stably in E. coli.