Team:Buenos Aires/Results/Bb1

From 2012.igem.org

(Difference between revisions)
(→Bb1)
(→BioBricks Design for Synthetic Ecology)
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Figure2: RFC10 BioBrick standard
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'''Figure2: RFC10 BioBrick standard'''
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== Kozak Sequence ==
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'''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).
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).
 +
 +
{| class="wikitable"
 +
|Name
 +
|DNA Sequence
 +
|-
 +
|BBa_J63003
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|cccgccgccaccatggag
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|-
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|MF1[-12,6]
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|acgattaaaagaatgaga
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|}
 +
 +
 +
'''Table1: DNA sequences for Kozak consensus'''
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 +
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== Signal Peptide ==
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 +
 +
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
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 +
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
 +
 +
{| class="wikitable"
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|Name
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|DNA Sequence
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|-
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|1: MF1 signal
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|'''atg'''agatttccttcaatttttactgcagttttattcgcagcatcctccgcattagct
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|-
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|2: BBa_K416003
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|'''atg'''aaagttttgattgttttgttggctattttcgctgctttgccattggctttggctcaaccagttatttctactactgttggttctgctgctgaaggttcactagataaaaga
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|}
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'''
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Table 2: DNA sequences for the signal peptides, atg codon in bold
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'''
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== Trojan peptide ==
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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].
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 +
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{| class="wikitable"
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|Penetratin
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|Residue sequence
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|-
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|TAT
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|YGRKKRRQRRR
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|-
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|polyarginine
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|RRRRRRRRRRR
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|}
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'''Table 3: Primary structure for penetratins'''
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 +
 +
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.
 +
 +
 +
{| class="wikitable"
 +
|R
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|Purine (A or G)
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|-
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|Y
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|Pyrimidine (C or T)
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|-
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|N
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|Any nucleotide
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|-
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|W
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|Weak (A or T)
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|-
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|S
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|Strong (G or C)
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|-
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|M
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|Amino (A or C)
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|-
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|K
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|Keto (G or T)
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|-
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|B
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|Not A (G or C or T)
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|-
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|H
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|Not G (A or C or T)
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|-
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|D
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|Not C (A or G or T)
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|-
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|V
 +
|Not T (A or G or C)
 +
|}
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 +
Table 4: Nucleotide convention for retro-translated sequences
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Table1: DNA sequences for Kozak consensus
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Penetratin DNA Sequence
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TAT TAYGGNMGNAARAARMGNMGNCARMGNMGN
 +
polyarginine MGNMGNMGNMGNMGNMGNMGNMGNMGNMGN

Revision as of 19:56, 17 September 2012


Contents

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

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