Difference between revisions of "Team:TCU Taiwan/Project/Experimental"

 
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To increase efficiency in isolating our AMPs, we introduced a signal peptide upstream of the N-terminal of antimicrobial peptides to facilitate peptide production. This signal peptide is obtained from chitinase C of <I>S.lividans</I> (MGFRHKAAALAATLALPLAGLVGLASPAQA). After translation process, this signal peptide will lead AMPs to the secretion system of <I>E. coli</I>. When the premature peptides enter the periplasmic space, peptidase will identify the cleavage site Ala-Gln-Ala and cut at the double Ala at the signal and mature peptide linkage site. This separates the signal peptide from the AMP. To verify the <I>E. coli</I> secretion of AMPs to the LB culture medium, we attached an Ala at the N-terminal of AMPs.<a href="#tcu_sing_references_1">[1,2]</a>
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To increase efficiency in isolating our AMPs, we introduced a signal peptide upstream of the N-terminal of antimicrobial peptides to facilitate peptide production. This signal peptide is obtained from chitinase C of <I>S.lividans</I> (MGFRHKAAALAATLALPLAGLVGLASP</br>AQA). After translation process, this signal peptide will lead AMPs to the secretion system of <I>E. coli</I>. When the fusion peptides enter the periplasmic space, peptidase will identify the cleavage site Ala-Gln-Ala and cut at the double Ala at the signal and AMPs linkage site. This separates the signal peptide from the AMPs. To verify the <I>E. coli</I> secretion of AMPs to the LB culture medium, we attached an Ala at the N-terminal of AMPs.<a href="#tcu_sing_references_1">[1,2]</a>
 
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By modifying the amino acid sequence of Signiferin and Epinecidin-1 to facilitate the secretion process, we can apply structure prediction software to analyze the attached Ala and it’s effect on the peptide folding process, as described in our <a href="https://2015.igem.org/Team:TCU_Taiwan/Modeling/Protein_structure">modeling page</a>.
 
By modifying the amino acid sequence of Signiferin and Epinecidin-1 to facilitate the secretion process, we can apply structure prediction software to analyze the attached Ala and it’s effect on the peptide folding process, as described in our <a href="https://2015.igem.org/Team:TCU_Taiwan/Modeling/Protein_structure">modeling page</a>.
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<img src="https://static.igem.org/mediawiki/2015/e/e6/TCU_Taiwan_Signal_Peptide.jpeg">
 
<img src="https://static.igem.org/mediawiki/2015/e/e6/TCU_Taiwan_Signal_Peptide.jpeg">
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<font size="5">Fig.1 Signiferin fusion peptide design model</font>
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<img src="https://static.igem.org/mediawiki/2015/3/3c/TCU_Taiwan_Signal_Peptide_2.jpeg">
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<font size="5">Fig.2 Epinecidin-1 fusion peptide design model</font>
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Latest revision as of 18:52, 18 September 2015



Signal Peptide

To increase efficiency in isolating our AMPs, we introduced a signal peptide upstream of the N-terminal of antimicrobial peptides to facilitate peptide production. This signal peptide is obtained from chitinase C of S.lividans (MGFRHKAAALAATLALPLAGLVGLASP
AQA). After translation process, this signal peptide will lead AMPs to the secretion system of E. coli. When the fusion peptides enter the periplasmic space, peptidase will identify the cleavage site Ala-Gln-Ala and cut at the double Ala at the signal and AMPs linkage site. This separates the signal peptide from the AMPs. To verify the E. coli secretion of AMPs to the LB culture medium, we attached an Ala at the N-terminal of AMPs.[1,2]

By modifying the amino acid sequence of Signiferin and Epinecidin-1 to facilitate the secretion process, we can apply structure prediction software to analyze the attached Ala and it’s effect on the peptide folding process, as described in our modeling page.



Fig.1 Signiferin fusion peptide design model





Fig.2 Epinecidin-1 fusion peptide design model

References


[1] Fujii, T. and K. Miyashita, Multiple domain structure in a chitinase gene (chiC) of Streptomyces lividans. J Gen Microbiol, 1993. 139(4): p. 677-86.

[2] Tokuyasu, K., et al., Production of a recombinant chitin deacetylase in the culture medium of Escherichia coli cells. FEBS Lett, 1999. 458(1): p. 23-6.





             
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