Coding

Part:BBa_K3071002

Designed by: Leung Hei Man   Group: iGEM19_Hong_Kong-CUHK   (2019-10-07)
Revision as of 06:29, 21 October 2019 by Heimanleung (Talk | contribs) (→‎Description)


Two-component system response regulator protein RpfG from Xanthomononas campestris pv. campestris

Description

This the protein-encoding gene for the Two-component system response regulator protein RpfG from Xanthomononas campestris pv. campestris, which is codon-optimized to express in Escherichia coli Bl21(DE3) strain. This part is developed and improved based on the previous record part (BBa_K1315003). The SDS-PAGE gel and western blot data of our composite part (BBa_K3071019) shows it could be correctly expressed in E. coli.

Biology

RpfG consists of 2 domains, the REC domain, and the HD-GYP domain, which is a phosphodiesterase involved in the degradation of the second messenger cyclic -di-GMP. When it is phosphorylated by the RpfC, it is activated as phosphodiesterase and alter cyclic-di-GMP level. As a result, it promotes the synthesis of virulence factors, e.g. extracellular enzymes, dispersal of biofilm and motility of the bacterial cell.

Usage

T--Hong Kong-CUHK RpfG activation pathway.png
Figure 1 Activation pathway of Xcc bacteria by DSF signal (Ryan and Dow, 2011)


RpfG is the kinase involved in DSF sensing. When it is phosphorylated by the RpfC, it is activated as phosphodiesterase and alter cyclic-di-GMP level. As a result, it promotes the synthesis of virulence factors through activation of Clp, e.g. extracellular enzymes, dispersal of biofilm and motility of the bacterial cell.

Characterization

T--Hong Kong-CUHK RpfG optimization result.png
Figure 2 SDS-PAGE gel showing the RpfG expression of Old:BBa_K1315003 and New:BBa_K307100


T--Hong Kong-CUHK--RpfG western blot 1.png
Figure 3 Western Blot analysis of RpfG protein expression using Sigma F7425 ANTI-FLAG® antibody (1:5000)


By comparing the protein expression level of RpfG (Old:BBa_K1315003 and New:BBa_K3071002) in Escherichia coli K12 strain, it confirms that the codon optimization of our biocrick is successful. RpfG overexpression can be clearly seen in clones after codon optimization. The amount of RpfG proteins significantly increased compared to that in the old clones. The amount also rose with a longer incubation time of IPTG.

Comparing Figure 2 and Figure 3, we can deduce that RpfG expression reached saturation at 12hr and started degradation afterward.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 370
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None