Part:BBa_K3407019

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 pBbA2k_YmdB. 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) pBbA2k_YmdB without induction, indicating that there is no leaky expression.

References

Ordered List