Composite

Part:BBa_K3378001

Designed by: Shengkun Tong   Group: iGEM20_HZAU-China   (2020-10-17)
Revision as of 09:54, 26 October 2020 by TSK (Talk | contribs)


ClyR fused with PhoA signal peptide

ClyR is a chimeric lysine which can bind to S. mutans by a cell-wall binding domain and then lysis its cell wall from outside by a catalytic domain. ClyR has no activity for Gram-negative bacteria and can be expressed in Escherichia coli. PhoA fused ClyR can be secreted to the outside of the cells, killing S. mutans in the environment.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 826
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 190
    Illegal AgeI site found at 274
    Illegal AgeI site found at 589
    Illegal AgeI site found at 772
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional Parameters

To demonstrate the secretion of PhoA fused ClyR, plasmid pET-28a(+)-ClyR (with PhoA) was transferred to E. coli BL21(DE3), same plasmid just without PhoA signal peptide as control. E. coli strains was cultured to OD600~0.6, induced with 0.2 mM IPTG and 1 % glycine, and then overnight cultured at 16 ℃. On the second day, take the supernatant and bacterial liquid for SDS analysis, thus we can demonstrate that the PhoA fused ClyR can be secreted to the medium effectively (Figure 1).

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Figure 1. SDS-PAGE analysis of ClyR secretion. BL-PhoA-: Bacteria liquid of PhoA nagtive group; S-PhoA-: Supernatant of PhoA negative group; BL-PhoA+: Bacteria liquid of PhoA positive group; S-PhoA+: Supernatant of PhoA positive group;



[edit]
Categories
//cds/enzyme/lysis
Parameters
None