"

From 2012.igem.org

• iGEM BsAs
  • The team
  • Official roster
  • Members
  • Where we come from

  • Safety
  • Key questions

  • Attributions
  • Attributions

  • Sponsors
  • Institutional

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

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

  • Modeling
  • Modeling synEcology
  • Advanced modeling

  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
  • Preparing for sending

  • Testing
  • BioBricks testing
  • SynEco testing

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

  • EMBO
  • Seeding SynBio in Latin America

  Data Page

Contents 1 BioBricks Design for Synthetic Ecology 2 Kozak Sequence 3 Signal Peptide 4 Trojan peptide

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).

Name	DNA Sequence
BBa_J63003	cccgccgccaccatggag
MF1[-12,6]	acgattaaaagaatgaga

Table1: DNA sequences for Kozak consensus

Signal Peptide

The signal peptide directs the secretion of the produced protein, and therefore allows for the exportation of the payload. This peptides are cleaved once the protein is in the lumen of the ER.

We have several options for yeast secretion signal peptides

1) MRFPSIFTAVLFAASSALA

2) MKVLIVLLAIFAALPLALAQPVISTTVGSAAEGSLDKR

Number 1 is the signal peptide for the yeast -mating factor [Water et al 1987]. Because its a part of a yeast gene, we don’t need to optimize it for yeast. Number 2 is part BBa_K416003 from the registry (not included in the kit), and was designed for yeast [Clements 1991].

The DNA sequence are shown in Table 2

Name	DNA Sequence
1: MF1 signal	atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagct
2: BBa_K416003	atgaaagttttgattgttttgttggctattttcgctgctttgccattggctttggctcaaccagttatttctactactgttggttctgctgctgaaggttcactagataaaaga

Table 2: DNA sequences for the signal peptides, atg codon in bold

Trojan peptide

Trojan peptides are short (15aa) sequences that penetrate through the plasma membrane inside the cell without the need of any receptor or endocitosis process [Derossi 1998]. We want to use them to increase the efficiency with witch the payload enters the target cell. Ideally they should not contain Trp or His, as those are the relevant amino acids for exportation. Two good candidates are the penetratin from the HIV TAT protein, and polyarginine [Jones et al 2005].

Penetratin	Residue sequence
TAT	YGRKKRRQRRR
polyarginine	RRRRRRRRRRR

Table 3: Primary structure for penetratins

This proteins are not from yeast, so we need to retro-translate them. Using the conventions in table 4, we get the DNA sequence in Table 5.

R	Purine (A or G)
Y	Pyrimidine (C or T)
N	Any nucleotide
W	Weak (A or T)
S	Strong (G or C)
M	Amino (A or C)
K	Keto (G or T)
B	Not A (G or C or T)
H	Not G (A or C or T)
D	Not C (A or G or T)
V	Not T (A or G or C)

Table 4: Nucleotide convention for retro-translated sequences

Penetratin DNA Sequence TAT TAYGGNMGNAARAARMGNMGNCARMGNMGN polyarginine MGNMGNMGNMGNMGNMGNMGNMGNMGNMGN