Difference between revisions of "Part:BBa K3378001"
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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. | 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. | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K3378001 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3378001 SequenceAndFeatures</partinfo> | ||
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===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K3378001 parameters</partinfo> | <partinfo>BBa_K3378001 parameters</partinfo> | ||
| + | <p>To demonstrate the secretion of PhoA fused ClyR, plasmid pET-28a(+)-ClyR (with PhoA) was transferred to <i>E. coli</i> BL21(DE3), same plasmid just without PhoA signal peptide as control. <i>E. coli</i> strains was cultured to OD<sub>600</sub>~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 (<b>Figure 1</b>).</p> | ||
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| + | <p align="center"><b>Figure 1.</b> SDS-PAGE analysis of ClyR secretion. <b>BL-PhoA-:</b> Bacteria liquid of PhoA nagtive group; <b>S-PhoA-:</b> Supernatant of PhoA negative group; <b>BL-PhoA+:</b> Bacteria liquid of PhoA positive group; <b>S-PhoA+:</b> Supernatant of PhoA positive group;</p> | ||
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Revision as of 09:54, 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).
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;