Coding

Part:BBa_K2978100

Designed by: Jose Pablo Delgado Navarro   Group: iGEM19_diffEASY   (2019-10-15)


Auto Inducer Protein for QS in Clostridioides difficile

This composite part was constructed to reproduce the Quorum Sensing signaling of C. difficile. It's mechanisms is based on the Lubkowicz, et al., (2018) assays who reproduces the QS signaling from S. aureus.
Naturally, in C. difficile AgrD protein is code in higher expression levels when conditions are favorable for the pathogen. This peptide is modified and transport to the medium by the transmembrane AgrB protein, such modification results in the auto inducer protein (AIP) that recreates an infection/colonization induction in the bacteria.
AIP is sensing by the AgrC membrane protein, which phosphorylate AgrA protein when AIP is detected in the outsider of the bacteria. Once two AgrA protein are phosphorylated creates a dimer protein, and this joining form a DNA binding domain, still unknows in C. difficile, but well reported in S. aureus.

Characterization

Genetic Construct

Both, AgrD and AgrB, have a pLac promoter with the objective to produce AIP when IPTG is applied. Also, a GFP reporter is code polycistronically after the AgrD peptide, as a way to indirectly quantify the AIP. Is important to note that nickel affinity chromatography isn't used for this protein considering the insoluble properties of the peptide reported in literature, and the possible interference of a his tag during the process of AgrD by AgrB.

Protein Extraction

After induction, proteins were extracted and analyzed on a SDS-PAGE. As you can see in the next figure, GFP (intracellular protein, 26 kDa) (Slade et al., 2009), AgrB (transmembrane protein, 21 kDa) (Zhang et al., 2002) and AIP protein (~5 kDa) are located in the intracellular fraction.

T--Costa Rica--team-R3.png

Figure 1: Polyacrylamide gel electrophoresis of intracellular proteins of a recombinant AIP bacterial culture. Protein with 26 kDa correspond to GFP, 21 kDa to AgrB and approx. 5 kDa to AIP.

It is worth to mention that, considering the size of the annotated bands in figure 1, we hypothesize that our desire proteins are present in this fraction. Nevertheless, sequencing is necessary to confirm this argument. Also, in other SDS PAGE, extracellular fractions didn’t show any intense bands related to our desired proteins. Therefore, we recommend modifications to our system and further characterization in order to transfer AIP to the extracellular space.


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 AgeI site found at 499
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1696


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