Difference between revisions of "Part:BBa K2812005:Design"

 
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===Design Notes===
 
===Design Notes===
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This biobrick contains the coding domain for truncated lysostaphin. Lysostaphin targets the cell wall peptidoglycan found in certain Staphylococci by cleaving its cross-linking pentaglycine bridges. Among others, it is effective for degrading S. aureus biofilm. The HlyA tag is a secretion signal peptide which is used for secretion via the Type 1 secretion pathway of gram-negative bacteria. In order to facilitate protein purification, a N-terminal HIS-tag is present. . In this biobrick, lysostaphin production is regulated by the T7 promoter. Expression can be induced by the presence of Isopropyl β-D-1-thiogalactopyranoside (IPTG).
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The sequence for the T7 promotor was has been taken from <partinfo>BBa_k525998</partinfo>. The initial starting sequence for truncated lysostaphin was taken from <partinfo>BBa_K748002</partinfo>. The amino acid sequence for HlyA was taken from uniprot (UniProtKB - Q1R2T5 (Q1R2T5_ECOUT)), residue 807-1024.<sup>1</sup> This is the C-terminal signal sequence, the N-terminal catalytic domain responsible for lysis of red blood cells is not included. The thrombin substrate sequence was obtained from Waugh, 2011<sup>2</sup> and all three components were linked via two flexible GGGGS linkers in between. This amino acid sequence was used to generate a codon optimized sequence for expression in ''E. coli''.
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===Source===
 
===Source===

Revision as of 22:50, 16 October 2018


Coding sequence for trunctated Lysostaphin with HlyA and His6-tag regulated by T7-promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1389
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 164
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The sequence for the T7 promotor was has been taken from BBa_K525998. The initial starting sequence for truncated lysostaphin was taken from BBa_K748002. The amino acid sequence for HlyA was taken from uniprot (UniProtKB - Q1R2T5 (Q1R2T5_ECOUT)), residue 807-1024.1 This is the C-terminal signal sequence, the N-terminal catalytic domain responsible for lysis of red blood cells is not included. The thrombin substrate sequence was obtained from Waugh, 20112 and all three components were linked via two flexible GGGGS linkers in between. This amino acid sequence was used to generate a codon optimized sequence for expression in E. coli.

Source

Uniprot

References