Difference between revisions of "Part:BBa K3407019"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K3407019 short</partinfo> | <partinfo>BBa_K3407019 short</partinfo> | ||
+ | |||
+ | <span class='h3bb'>Sequence and Features</span> | ||
+ | <partinfo>BBa_K3407019 SequenceAndFeatures</partinfo> | ||
tetA/tetR - RBD - YmdB - T7t | tetA/tetR - RBD - YmdB - T7t | ||
− | |||
===Usage and Biology=== | ===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>. 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>. | ||
− | < | + | 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. |
− | < | + | |
− | < | + | |
+ | ==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). | ||
+ | |||
+ | <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. | ||
+ | |||
+ | |||
+ | ==References== | ||
+ | <html> | ||
+ | |||
+ | <head> | ||
+ | |||
+ | <title>Ordered List</title> | ||
+ | |||
+ | </head> | ||
+ | |||
+ | <style> | ||
+ | |||
+ | ol li { | ||
+ | |||
+ | counter-increment: OrderedList; | ||
+ | |||
+ | list-style: none; | ||
+ | |||
+ | } | ||
+ | |||
+ | ol li :before { | ||
+ | |||
+ | content: "[" counter(OrderedList) "]"; | ||
+ | |||
+ | } | ||
+ | |||
+ | </style> | ||
+ | <html> | ||
+ | <ol> | ||
+ | |||
+ | <li> | ||
+ | <a id="1" href="https://doi.org/10.1016/j.jsb.2015.10.010" target="_blank"> | ||
+ | Zhang, W., Wang, C., Song, Y., Shao, C., Zhang, X., & Zang, J. (2015). Structural insights into the mechanism of Escherichia coli YmdB: A 2’-O-acetyl-ADP-ribose deacetylase. Journal of Structural Biology.</a> | ||
+ | </li> | ||
+ | |||
+ | |||
+ | <li> | ||
+ | <a id="2" href="https://doi.org/10.1042/BJ20071047" target="_blank"> | ||
+ | Meng, W., & Nicholson, A. W. (2008). Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro. Biochemical Journal. </a> | ||
+ | </li> | ||
+ | |||
+ | |||
+ | <li> | ||
+ | <a id="3" href="https://doi.org/10.1101/gad.1729508" target="_blank"> | ||
+ | Kim, K. S., Manasherob, R., & Cohen, S. N. (2008). YmdB: A stress-responsive ribonuclease-binding regulator of E. coli RNase III activity. Genes and Development. </a> | ||
+ | </li> | ||
+ | |||
+ | |||
+ | <li> | ||
+ | <a id="4" href="https://doi.org/10.1002/wrna.1195" target="_blank"> | ||
+ | Nicholson, A. W. (2014). Ribonuclease III mechanisms of double-stranded RNA cleavage. Wiley Interdisciplinary Reviews: RNA. </a> | ||
+ | </li> | ||
+ | |||
+ | |||
+ | <li> | ||
+ | <a id="5" href="https://doi.org/10.1371/journal.pgen.1004977" target="_blank"> | ||
+ | Martin, J. E., Waters, L. S., Storz, G., & Imlay, J. A. (2015). The Escherichia coli Small Protein MntS and Exporter MntP Optimize the Intracellular Concentration of Manganese. PLoS Genetics. </a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a id="6" href="https://doi.org/10.1016/j.bbrc.2016.12.157" target="_blank"> | ||
+ | Kim, M., Kim, M., & Kim, K. sun. (2017). YmdB-mediated down-regulation of sucA inhibits biofilm formation and induces apramycin susceptibility in Escherichia coli. Biochemical and Biophysical Research Communications. </a> | ||
+ | </li> | ||
− | + | </ol> | |
− | + | </html> | |
− | + | ||
− | < | + |
Revision as of 11:24, 27 October 2020
YmdB regulator of RNAseIII in E. coli with tetA/tetR promoters - RBD - T1 terminator
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 59
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 59
- 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 59
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 59
- 1000COMPATIBLE WITH RFC[1000]
tetA/tetR - RBD - YmdB - T7t
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 [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).
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
- Zhang, W., Wang, C., Song, Y., Shao, C., Zhang, X., & Zang, J. (2015). Structural insights into the mechanism of Escherichia coli YmdB: A 2’-O-acetyl-ADP-ribose deacetylase. Journal of Structural Biology.
- Meng, W., & Nicholson, A. W. (2008). Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro. Biochemical Journal.
- Kim, K. S., Manasherob, R., & Cohen, S. N. (2008). YmdB: A stress-responsive ribonuclease-binding regulator of E. coli RNase III activity. Genes and Development.
- Nicholson, A. W. (2014). Ribonuclease III mechanisms of double-stranded RNA cleavage. Wiley Interdisciplinary Reviews: RNA.
- Martin, J. E., Waters, L. S., Storz, G., & Imlay, J. A. (2015). The Escherichia coli Small Protein MntS and Exporter MntP Optimize the Intracellular Concentration of Manganese. PLoS Genetics.
- Kim, M., Kim, M., & Kim, K. sun. (2017). YmdB-mediated down-regulation of sucA inhibits biofilm formation and induces apramycin susceptibility in Escherichia coli. Biochemical and Biophysical Research Communications.