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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+ | <center><img src="https://static.igem.org/mediawiki/parts/5/53/T--HZAU-China--SeF1.jpg" style="width:567px;height:420px"></center> | ||
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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).
![](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;