|Make It: Diesel Production||Break It: Gluten Destruction|| iGEM Toolkits: Gibson Assembly|
|We designed and constructed a modular alkane production BioBrick, the PetroBrick, to generate alkanes, the main constituent of diesel. By expressing this BioBrick in E. coli, we were able to produce alkanes of yields over 100 mg/mL.||Gluten intolerance stems from an inappropriate immune response to PQLP, the most common motif in the immunogenic peptide. We reengineered a protease, active at low pH, for strongly enhanced activity against PQLP.||We built two iGEM toolkits. The first is a set of five BioBrick vectors optimized for Gibson assembly. The second is a set of genes essential for magnetosome formation characterized in E. coli.|
Data for Favorite New Parts
- 1, 2. BBa_K590025: The PetroBrick - A modular and open platform for the biological production of diesel fuel. The PetroBrick consists of AAR and ADC, each behind a standard Elowitz RBS. All of this is under regulation by a high constitutive promoter in pSB1C3.
- 3 The FabBrick - An add-on module to the PetroBRick that causes the production of odd chain length Fatty acids. This part consists of [ http://partsregistry.org/Part:BBa_K590034 FabH2] expressed on a low copy number IPTC inducible vector These are converted into even chain length alkanes by BBa_K590025 the PetroBrick, completing the spectrum of linear alkane compounds that can be produced using the
- 4. BBa_K590087: KumaMax- A modified version of the enzyme Kumamolisin, a protease ofthe sedolisin family native to Alicyclobacillus sendaiensis known to be active at low pH and elevated temperatures. To Kumamolisin, the mutations N291D, G319S, D358G, D368H increase activity to the PQLP peptide, an antigenic epitope in gliadin, 118-fold.
Gibson Assembly Toolkit
- 5. BBa_K590010: pGA1A3, BBa_K590011: pGA1C3, BBa_K590012: pGA4C5, BBa_K590013: pGA4A5, BBa_K590014: pGA3K3 - These are plasmid backbones based on the bglBrick standard (BBF RFC 21) and optimized for use in Gibson assembly. These vectors are suitable replacements for the equivalent pSB vectors for iGEM teams using Gibson cloning to assemble their constructs.
- 6. BBa_K590015: sfGFP_mamK_pGA1C3 - This part consists of the mamK gene from Magnetospirillum magneticum strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamK creates an actin-like filament that orients itself along the long-axis of the cell and acts as the scaffold for the alignment of magnetosome vesicles.
- 7. BBa_K590016 sfGFP_mamI_pGA1C3 - This part consists of mamI gene from Magnetospirillum magneticum strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamI is a membrane-localized protein required for magnetosome vesicle formation that also binds the poly-MamK filament.
Data for Existing Parts
- 8. K314100: High Constitutive Expression Cassette (Washington, iGEM 2010) - We used this part to express our Petrobrick, found that it works well for expression, and entered this information in the part experience page.
- 9. pSB1A3 - As part of the Gibson Vector Toolkit we characterized the cloning efficiency of this plasmid backbone for Gibson assembly, using the prefix and suffix regions as primers. We found that pSB1A3 had a proper insert efficiency of 11%, compared to 99% for the equivalent Gibson-optimized pGA1A3 vector.
- 10. BBa_K590059, BBa_K590060, BBa_K590061: LuxC, D, and E (Cambridge, iGEM 2010) - Formerly part of the LuxBrick the genes luxC, D, and E were not separated or codon-optimized. We codon-optimized these genes and put them under the control of standard Elowitz RBS's (B0034). This was accomplished by the Alternate Aldehyde branch of the Alkane Production team.