Part:BBa_K4307028

pT7-AffiPmrB-PmrA-PmrC-EGFP

In order to enable the engineered bacteria to react to the proteins on the sperm surface, a protein detection system is constructed in engineered bacteria. We call it affipmrABC system. This system is modified from the two-component system PmrB/A in E.coli. AffiPmrB (BBa_K4307025）protein is the receptor of our system. The extracellular iron (III)-binding motif of PmrB receptor(Trp34 to Glu64) is replaced with another protein named affibody to build AffiPmrB. Affibody is a kind of engineered protein that can recognize Fc fragment of antibody(Stahl et al., 2017). Through our design, bacteria can use antibodies as an intermediate to respond to external protein molecules. PmrA (BBa_K4307026） is corresponding response regulator to mediate cellular responses. PmrC (BBa_K4307027） is the promoter regulated by PmrA. EGFP is the reporter gene of this system.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 47
  Illegal PstI site found at 388
  Illegal PstI site found at 676
  Illegal PstI site found at 1108
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1855
  Illegal PstI site found at 388
  Illegal PstI site found at 676
  Illegal PstI site found at 1108
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 939
  Illegal BamHI site found at 2283
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 47
  Illegal PstI site found at 388
  Illegal PstI site found at 676
  Illegal PstI site found at 1108
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 47
  Illegal PstI site found at 388
  Illegal PstI site found at 676
  Illegal PstI site found at 1108
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The following figure demonstrates our successful construction.

               Figure 1:  The construction results of pT7-AffiPmrB-PmrA-PmrC-EGFP.
               
          

Confocal fluorescent microscope assay was done to characterize the bio brick.

First, IPTG was used to induce the expression of affiPmrB receptor and PmrA protein, and then human IgG antibody was added to the experimental group. Then we detect the fluorescence of EGFP under confocal fluorescence microscope. The experiment shows that compared with the culture medium without antibody, more engineering bacteria produce fluorescence in the culture medium with antibody concentration of 100mM.

               Figure 2:  The confocal fluorescent microscope photos for validation experiment.Cells without antibody induction(A and C) and cells with antibody induction(B and D) were observed under confocal fluorescence microscope.
               
         

Conclusion

The EGFP fluorescence intensity of the E.coli which express this part increases after being induced, however the increment is indeed very low, and is rather unstable. Therefore, in order to use the part in diagnose system, we built a new composite part with AffiPmrBAC and amplifier system T7-T3 (BBa_K4307033).

References

[1] Chen, H. D., & Groisman, E. A. (2013). The biology of the PmrA/PmrB two-component system: the major regulator of lipopolysaccharide modifications. Annu Rev Microbiol, 67, 83-112.

[2] Stahl, S., Graslund, T., Eriksson Karlstrom, A., Frejd, F. Y., Nygren, P. A., & Lofblom, J. (2017). Affibody Molecules in Biotechnological and Medical Applications. Trends Biotechnol, 35(8), 691-712.