Coding

Part:BBa_K3071000

Designed by: Leung Hei Man   Group: iGEM19_Hong_Kong-CUHK   (2019-10-07)
Revision as of 16:51, 20 October 2019 by Heimanleung (Talk | contribs) (→‎Usage)


Sensory/regulatory protein RpfC from Xanthomononas campestris pv. campestris

Description

This the protein-encoding gene for the Sensory/regulatory protein RpfC, which is codon-optimized to express in Escherica coli K12 strain. This part is developed and improved based on the previous recorded part (BBa K1315002). The western blot data of our composite part (BBa_K3071018) shows it could be correctly expressed in E. coli.

Biology

RpfC is a kinase located in the cell membrane of bacteria acting as a sensor. It consists of three domains, including the input domain, histidine kinase (HisKA) domain, CheY-like two-component receiver domain (REC) and the C-terminal histidine phosphotransfer (HPT) domain. The sensory domain is composed of 5 transmembrane helices with periplasmic and cytoplasmic loops less than 20 amino acids long.

Usage

T--Hong Kong-CUHK Protein domain of RpfC.png
Figure 1 The protein domains in RpfC protein and its signal transduction upon activation by DSF


RpfC is mainly used as a receptor for diffusible signal factor (DSF) in cell-cell communication. When the DSF signal is received, active autophosphorylation starts at the His198 of HisKA domain. It will then be followed by a phosphorelay through the Asp512 and His657 in the REC domain and the HPT domain respectively. The phosphotransfer lastly activates the RpfG by phosphorylation at the REC domain of RpfG (figure 1).

Besides activating the RpfG signal protein, the second role of RpfC is to interact with RpfF, a DSF synthase, by its REC domain, in order to suppress the DSF production in that bacterial cell.

Characterization

Research showed that point mutations at His 198, Asp512 or His657 resulted in the reduction of virulence factors, extracellular cellulases, and proteases, demonstrating the importance of these three amino acids in the process of RpfC-RpfG signaling and the role of RpfC in virulence factor production.

T--Hong Kong-CUHK--RpfC western blot.png
Figure 2 Western Blot analysis of RpfC protein expression using Sigma Aldrich Monoclonal Anti-HA antibody(H9658) (1:20000)


Clone was induced by 1mM IPTG at 32℃ and collected at different time points, which are 12hr, 16hr, 20hr, 24hr, and 28hr. After blotting with corresponding antibodies, RpfC was confirmed with successful expression at all time points. Results of blots probing RpfC showed that clones collected at 12hr contained the highest amount of target proteins and the protein quantity decreased from 16hr to 28hr. This may due to degradation inside the cells.


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 303
    Illegal AgeI site found at 1135
    Illegal AgeI site found at 1614
    Illegal AgeI site found at 1698
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 352
    Illegal BsaI.rc site found at 216


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