Difference between revisions of "Part:BBa K3407003"
| Line 1: | Line 1: | ||
| − | |||
<partinfo>BBa_K3407003 short</partinfo> | <partinfo>BBa_K3407003 short</partinfo> | ||
| − | + | <span class='h3bb'>Sequence and Features</span> | |
| + | <partinfo>BBa_K3407003 SequenceAndFeatures</partinfo> | ||
| − | |||
===Usage and Biology=== | ===Usage and Biology=== | ||
| + | YmdB gene in E.coli codes for O-acetyl-ADP-ribose deacetylase, a small protein of 18.8 kDa (UniProt ID: P0A8D6) that catalyses the deacetylation of OAADPr signalling molecule to ADPr <html><a href="#1">[1]</a></html>[S]. Although the purpose of this protein in E.coli remains vastly unstudied, in some experiments it has shown its ability to inhibit E. coli RNAseIII, a member of the RNAseIII superfamily involved in RNA processing. E. coli RNAseIII has shown its catalytic activity upon dimerisation <html><a href="#2">[2]</a></html>[S], in which YmdB interferes by preventing it in vitro. These assays were performed in presence of the Mn2+ (0.4 mM) cation but not Mg2+ <html><a href="#3">[3]</a></html>[S], which was criticised because they do not reflect in vivo conditions <html><a href="#4">[4]</a></html>[S]. In vivo, E.coli keeps Mn2+ concentrations below 40 uM to prevent toxicity even when exposed in a media containing 0.5 mM of Mn2+ <html><a href="#5">[5]</a></html>[S]. | ||
| − | < | + | On the other hand, studies performing an overexpression of YmdB showed a reduction in biofilm formation and increased susceptibility to apramycin, two pathways known to be mediated by RNAseIII <html><a href="#6">[6]</a></html>[S], further suggesting YmdB is a modulator of its activity. More in-deep research should be performed to elucidate the exact mechanism of RNAseIII inhibition and confirm the reduction in activity is at a substrate level as a consequence of a direct interaction of both proteins in vivo. |
| − | < | + | |
| − | + | ||
| + | ==Experimental results== | ||
| + | <div><ul> | ||
| + | <center> | ||
| + | <li style="display: inline-block;"> [[File:T--TUDelft--YmdB.png|thumb|none|400px|<b>Figure 1:</b> SDS-PAGE gel showing the overexpression of YmdB. E. coli BL21 (DE3) is the negative control and E. coli BL21 (DE3) pBbA2k_YmdB contains the part <html><a href="https://parts.igem.org/Part:BBa_K3407019" target="_blank"><b>BBa_K3407019</b></a><html>. MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), 4h (4 hours after induction), ON (overnight). All the samples used corresponded to the same OD600.]] </li> | ||
| + | </center> | ||
| + | </ul></div> | ||
| − | + | As seen in the SDS-PAGE of the total protein content (Figure 1), a band corresponding to the molecular weight of the C-terminal His-tagged YmdB shows that it was successfully overexpressed after induction with 400 mM anhydrotetracycline. No additional bands can be observed in E. coli BL21 (DE3) pBbE8c_mini3 without induction, indicating that there is no leaky expression. | |
| − | + | ||
| − | + | ||
| − | + | ||
Revision as of 10:00, 27 October 2020
YmdB: a regulator of RNAseIII activity in E. coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
YmdB gene in E.coli codes for O-acetyl-ADP-ribose deacetylase, a small protein of 18.8 kDa (UniProt ID: P0A8D6) that catalyses the deacetylation of OAADPr signalling molecule to ADPr [1][S]. Although the purpose of this protein in E.coli remains vastly unstudied, in some experiments it has shown its ability to inhibit E. coli RNAseIII, a member of the RNAseIII superfamily involved in RNA processing. E. coli RNAseIII has shown its catalytic activity upon dimerisation [2][S], in which YmdB interferes by preventing it in vitro. These assays were performed in presence of the Mn2+ (0.4 mM) cation but not Mg2+ [3][S], which was criticised because they do not reflect in vivo conditions [4][S]. In vivo, E.coli keeps Mn2+ concentrations below 40 uM to prevent toxicity even when exposed in a media containing 0.5 mM of Mn2+ [5][S].
On the other hand, studies performing an overexpression of YmdB showed a reduction in biofilm formation and increased susceptibility to apramycin, two pathways known to be mediated by RNAseIII [6][S], further suggesting YmdB is a modulator of its activity. More in-deep research should be performed to elucidate the exact mechanism of RNAseIII inhibition and confirm the reduction in activity is at a substrate level as a consequence of a direct interaction of both proteins in vivo.
Experimental results
- [[File:T--TUDelft--YmdB.png|thumb|none|400px|Figure 1: SDS-PAGE gel showing the overexpression of YmdB. E. coli BL21 (DE3) is the negative control and E. coli BL21 (DE3) pBbA2k_YmdB contains the part BBa_K3407019. MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), 4h (4 hours after induction), ON (overnight). All the samples used corresponded to the same OD600.]]