20.20(S10):Notebook/BEI/2010/05/11

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Entry title
All the research that was not previously uploaded belongs here, namely:

References:

1. "Synthetic cooperation in engineered yeast populations." Shou, et al. 2. "Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology." Huang & Casmund, et al. 3. "A synthetic mutlticellular system for programmed pattern formation." Basu, et al. 4. "Production and secretion of fatty acids in genetically engineered cyanobacteria." Liu, et al. 5. "Engineering synthesis and export of hydrophilic products from cyanobacteria." Niederholtmeyer, et al. 6. "Genome sequence of the cyanobacterium Prochlorococcus marinus SS120, a nearly minimaloxyphototrophic genome." Haselkorn, et al. 7. "Locality versus globality in bacterial signalling: can local communication stabilize bacterial communities?" Venturi, et al. 8. "Synthetic ecosystems based on airborne inter- and intrakingdom communication." Weber, et al. 9. "A synthetic Escherichia coli predator–prey ecosystem." Balagadde, et al. 10. "Photoautotrophic Nutrient Utilization and Limitation During Semi-Continuous Growth of Synechocystis sp. PCC6803." Kim, et al. 11. "Performance comparison of two photobioreactors configurations (open and closed to the atmosphere) treating anaerobically degraded swine slurry." Molinuevo-Salces, et al. 12. KEGG: Kyoto Encyclopedia of Genes and Genomes.

Also, some minor points and information that did not make it into our final documentation can be found below.

Overarching idea: Population control as a useful technique for sustainable and continuous production, which improves not only overall output as suggested by (Article One) and (Article Two), but guarantees a safeguard against organism escape and reduces the need for labor in an industrial or laboratory setting, freeing time for more pressing concerns. Note that nutrient recycling is not explicitly a part of this technique, but is a useful potential application of this system.

[Concerning choice of growth factor]

We selected a variant of thymidylate synthase as a regulated growth factor for both organisms in an attempt to impose upon each the same order of growth restriction. E. Coli have the traditional version of ThyA in their genomes, but the cyanobacteria instead express an alternative enzyme ThyX. Although it may be a bold assumption that growth restriction is comparable given that ThyA and ThyX differ, we believe that relatively minor variation in growth restriction will occur and have confidence that the shape of our population dynamics will be in keeping with our presumed system.

[Concerning Inactivation of Amino Acids / Auxotrophy; Note that specific proteins must still be isolated; consult CoSMO and PubMed]

We selected a Lysine/Leucine combination, wherein the E.Coli possess a Leucine deficiency and the Cyanobacteria a Lysine deficiency. The former was chosen because there is already in existence a strain of E.Coli possessing a LeuB mutation (and which subsequently requires leucine to grow) while the latter was chosen because (the CoSMO paper) has already identified target enzymes for induced Lysine deficiency. Our system is at variance with (the CoSMO paper) in that we selected two amino acids rather than amino acid and a purine.

[Concerning upregulation of amino acids]

Our system follows (the CoSMO paper) closely in this regard, although we must select a differing set of enzymes in our Cyanobacteria in order to account for genomic and proteomic differences.

[The way they upregulated Trp-or-whatever synthesis in the other species was by removing end-product inhibition. They don't state exactly what they changed, but I think we could probably get away with saying we'd do the same thing. -KAD]

[Concerning the choice of promoters; note that we must also add the new promoter selected from the list of promoters given by the cyanobacteria article.]

Courtesy of (the CoSMO paper), we have at our disposal a series of useful promoters. The two we selected for this project were the PrnpB and Ptrc20 promoters. The former is used for the production of LasI and LasR in order to enable quorum sensing and the second is used for the production of the ThyX growth factor, which is regulated by the previously noted enzymes or their products.

[Concerning the possiblity that an element of our system may not work because the RNA Polymerase of Cyanobacteria varies; there is admittedly one point at which activation of a promoter by a protein complex is required, and it is not clear that this would be completely viable given the present promoters that exist in Cyanobacteria. Specifically, the issue is the Ptrc10 promoter and the LuxRAHLComplex. Although it is apparently properly inhibited by LacI and activated by IPTG, it is not necessarily the case that it will be activated by a LuxRAHLComplex.


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