Difference between revisions of "Part:BBa K3378001"
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Revision as of 12:20, 26 October 2020
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 826
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000COMPATIBLE 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).
![](https://static.igem.org/mediawiki/parts/5/53/T--HZAU-China--SeF1.jpg)
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;
Reference
[1] Yang, Hang, et al. "A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method." Scientific reports 5 (2015): 17257.[2] Yang, Hang, et al. "Antibiofilm activities of a novel chimeolysin against Streptococcus mutans under physiological and cariogenic conditions." Antimicrobial agents and chemotherapy 60.12 (2016): 7436-7443.
[3] Xu, Jingjing, et al. "Activity of the chimeric lysin ClyR against common Gram-positive oral microbes and its anticaries efficacy in rat models." Viruses 10.7 (2018): 380.