1. BioBrick Design
We registered 51 BioBricks, of which we sent in 41. Our cloning work was either performed by Standard Assembly according to RFC10 standards or by Golden Gate Assembly with subsequent cloning into pSB1C3. Our parts bring sustainability to synthetic biology. We designed parts which both detect and respond changes to the environment, and help maintaining E. coli cultures over extended periods of time. Additionally, we created Parts to lure and kill mosquitoes, and make sustainable, no-maintenance malaria prevention an emerging reality.
2. Growth Inhibition Module
To create a Sustainable H.I.E.L.D. we needed the bacteria not to grow and divide, but to maintain a stable cell density. Our growth inhibition module BBa_K2588021 was confirmed by characterization. It inhibits E. coli cell division and growth to a third of its natural growth within only 6 hours after induction. BBa_K2588021 induces expression of growth inhibiting proteins cbtA, cspD and mraZ in absence of diaminopimelic acid (DAP). This allows to grow bacteria wearing this construct by addition of DAP, but becomes active in natural environments without high amounts of DAP.
3. Lure Modules
To bait mosquitoes in the environment of our trap, we designed three Composite Parts:
Lactate dehydrogenase A (ldhA) from E. coli, which converts pyruvate to lactate. We amplified this gene from E. coli genomic DNA by PCR. ldhA was cloned downstream of pBAD promoter BBa_K206000 and BBa_0034 ribosome binding site.
We confirmed the overexpression of ldhA by SDS-PAGE with subsequently Coomassie Blue staining.
Furthermore, the reaction rate of ldhA in cell lysates was determined by an activity assay using a coupled test system. The reaction rate of lactate dehydrogenase was 2.1 nmol/(mL*min).
3.2 Myristic Acid and 3-Methyl-1-butanol
Myristic acid and 3-methyl-1-butanol are well-known odor baits to attract Anopheles gambiae and can be synthesized in E. coli. We designed and submitted all basic parts required to construct both synthetic metabolic pathways, but we were not able to assemble them to their Composites in time.
3.3. Thermogenesis Module
Mosquitoes are attracted by heat. Registry Part BBa_K410000 was described to generate heat at low temperatures. It is regulated by cold-shock promoter HybB (BBa_J45503). We analysed BBa_J45503 sequence and found room for major improvements: The original Part does not only contain the promoter sequence, but also the 5’ UTR and parts of the coding sequence of its natural downstream gene, interfering with expression of genes of interest cloned downstream of BBa_J45503. We improved it by reducing it to its essential sequence. Improved HybB promoter, which we sent in as BBa_K2588001, showed an over two-fold improved expression and a tighter activation pattern. To complete the thermogenesis module, we also sent in Nelumbo nucifera Alternative Oxidase 1a (NnAOX1a, BBa_K2588018), taken directly from BBa_K410000. Even after attempting to recreate results written on BBa_K410000 five times, we could not confirm that NnAOX1a increases temperature, contradicting characterizations by previous iGEM Teams.
5. Hydrogel Synthesis
We synthesized a poly ammonium salt hydrogel, providing a surface for mosquitoes to land on, by polymerisation of the monomers ODA (4,4-´oxydianiline) and PMDA (pyromellitic dianhydride) and subsequently treatment with triethylamine.
6. Alginate Beads Synthesis
Gel beads consisting of alginate were synthesized and their decomposition in lactic acid containing environment examined. The alginate beads are stable in different for E. coli and S. elongatus physiological concentrations of lactic acid present and can therefore be used for 3D-embedded culture of S. elongatus.
7. Human Practices
We talked with various experts including Dr. Mirko Himmel, Dr. David Hutchinson, Dr. Andrew Hammond and Dr. Thomas Jacobs, gaining valuable insights to integrate into our project. We significantly improving our project’s Biosafety and -security, and environmental feasibility based on their advice, and learned much about the fight against malaria.
We designed the S.H.I.E.L.D. to withstand harsh environmental conditions while still maintaining utility using simple yet multifunctional 3D-printed parts. We employ novel techniques like 3D gel-embedded culture and spatially separated co-culture to create an application that redefines standards of environmental synthetic biology.
We designed Part BBa_K2588003 as RNA-based transcriptional inverter to create the energy-efficient glucose-induced regulator BBa_K2588002. In BBa_K2588002, transcription induced by a constitutive promoter is terminated using short noncoding RNA RnaG120. We built a novel kinetic circuit model to select the constitutive promoter with the best predicted signal to noise ratio of the resulting construct. Despite we were unable to confirm BBa_K2588002 functionality in the lab, we are confident in our modelling, both for its results, and for its potential use for future applications.
For these years InterLab study we followed the standard protocol by transforming and measuring the GFP expressing plasmids.