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From 2012.igem.org

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  Data Page
• Project
  • Synthetic ecology
  • Overview
  • Motivation
  • Design
  • Possible applications

  • Schemes
  • Crossfeeding
  • Independent Population Control
  • Cross-population control
  • Stochastic State Transitions

  • Modeling
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  Data Page
• Results and Devices
  • Strains characterization
  • Strains description
  • Fluorescence screening
  • Screening of strain proportion
  • Auxotrophy confirmation
  • Coculture in liquid medium
  • Revertant control
  • Trp basal production
  • Growth dependence

  • BBs design
  • Trp/His export devices
  • Backup devices
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  • Testing
  • BioBricks testing
  • SynEco testing

  Data Page
• Human practices
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  • Spreading the word
  • Solving local problems

  • EMBO
  • Seeding SynBio in Latin America

  Data Page

BioBricks Design for Synthetic Ecology

In order for the cross-feeding scheme to work, we need the strains to export the amino acid they can produce and the other strains can’t. We are going to use yeast expression plasmids with inducible or constitutive promoters. The structure of the synthetic gene is represented in Figure 1.

Figure 1: Schematic representation of the synthetic gene for the cross-feeding design. RS: Restriction Sites, Prefix, Suffix: BioBrick standard, Signal: Secretion signal peptide. The ORF is highlighted in violet.

The synthetic gene has the following elements • Prefix of the BioBrick standard • Kozak consensus sequence for initiation of translation • Signal peptide that targets the product of the gene for secretion •Bold text Trojan peptide, to increase internalization in target cell • Payload: this is the exported amino acid rich domain of the protein • Suffix of the BioBrick standard

Beside, the gene will have convenient restriction sites for directional cloning (RS1 and RS3). RS2 will allow to easily remove the sequence coding for the trojan peptide, by restriction and re-ligation.

BioBrick Prefix and Suffix Because the entire ORF is contained within the prefix and suffix, no care for in-frame assembly has to be taken. We can use the original RFC10 BioBrick standard.

Figure2: RFC10 BioBrick standard

Kozak Sequence The Kozak sequence is analogous to the bacterial RBS, it is required for proficient initiation of translation. There is only one yeast Kozak sequence in the registry (part BBa_J63003, distributed in the 2012 kit). Note that this sequence codes for a glutamic acid (E) after the start codon. Alternatively we could use the sequence of the 5’UTR of the MF1 gene of yeast (see Signal Peptide).

Table1: DNA sequences for Kozak consensus