Difference between revisions of "Part:BBa K3407019"

Revision as of 22:12, 27 October 2020

YmdB regulator of RNAseIII in E. coli with tetA/tetR promoters - RBD - T1 terminator

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 59
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 59
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 59
Illegal BglII site found at 68
Illegal BamHI site found at 673
Illegal XhoI site found at 682
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 59
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 59
1000
COMPATIBLE 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) (BBa_K3407003) that catalyses the deacetylation of OAADPr signalling molecule to ADPr [1]. 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], 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], which was criticised because they do not reflect in vivo conditions [4]. 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].

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], 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

To overexpress Mini-3, we incubated E. coli BL21 (DE3) (negative control) and E. coli BL21 (DE3) transformed with the plasmids pBbE8c_mini3. When the cultures reached OD600 ≈ 0.6, they were induced with 20 mM L-arabinose for 4 hours at 37ºC and overnight at 20ºC. The total protein content of the cells was analysed by SDS-PAGE electrophoresis (Figure 3).

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.

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.

References

Ordered List

@@ Line 7: / Line 7: @@
 ===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) (<html><a href="https://parts.igem.org/Part:BBa_K3407003" target="_blank"><b>BBa_K3407003</b></a></html>) that catalyses the deacetylation of OAADPr signalling molecule to ADPr <html><a href="#1">[1]</a></html>. 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>, 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>, which was criticised because they do not reflect in vivo conditions <html><a href="#4">[4]</a></html>. 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>.
+YmdB gene in E.coli codes for O-acetyl-ADP-ribose deacetylase, a small protein of 18.8 kDa (UniProt ID: P0A8D6) (<html><a href="https://parts.igem.org/Part:BBa_K3407003" target="_blank"><b>BBa_K3407003</b></a></html>) that catalyses the deacetylation of OAADPr signalling molecule to ADPr <html><a href="#1">[1]</a></html>. Although the purpose of this protein in <i>E. coli</i> remains vastly unstudied, in some experiments it has shown its ability to inhibit <i>E. coli</i> RNAseIII, a member of the RNAseIII superfamily involved in RNA processing. <i>E. coli</i> RNAseIII has shown its catalytic activity upon dimerisation <html><a href="#2">[2]</a></html>, 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>, which was criticised because they do not reflect in vivo conditions <html><a href="#4">[4]</a></html>. 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>.
 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>, 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.
@@ Line 13: / Line 13: @@
 ==Experimental results==
-To overexpress Mini-3, we incubated E. coli BL21 (DE3) (negative control) and E. coli BL21 (DE3) transformed with the plasmids pBbE8c_mini3.  When the cultures reached OD600 ≈ 0.6, they were induced with 20 mM L-arabinose for 4 hours at 37ºC and overnight at 20ºC. The total protein content of the cells was analysed by SDS-PAGE electrophoresis (Figure 3).
+To overexpress Mini-3, we incubated <i>E. coli</i> BL21 (DE3) (negative control) and <i>E. coli</i> BL21 (DE3) transformed with the plasmids pBbE8c_mini3.  When the cultures reached OD600 ≈ 0.6, they were induced with 20 mM L-arabinose for 4 hours at 37ºC and overnight at 20ºC. The total protein content of the cells was analysed by SDS-PAGE electrophoresis (Figure 3).
 <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>
+    <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. <i>E. coli</i> BL21 (DE3) is the negative control and <i>E. coli</i> 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.
+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 <i>E. coli</i> BL21 (DE3) pBbE8c_mini3 without induction, indicating that there is no leaky expression.