Measurement

Part:BBa_K1510820:Design

Designed by: Kao Jung Chang   Group: iGEM14_NYMU-Taipei   (2014-10-07)


RFP indicated INPNC performance evaluation


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 1036
    Illegal AgeI site found at 2220
    Illegal AgeI site found at 2332
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

To test whether or not our INPNC-C16 fusion protein can be successfully expressed on the surface of our E. coli, we constructed a testing circuit with RFP after C16.After constructing our testing circuit, we incubated the transformed bacteria together for 12 ~ 16 hours, before centrifugation. Theoretically, there are three possible results for our testing.

Possible Result 1: Fusion protein is on cell membrane

Possible Result 2: Fusion protein is inside cell membrane

Possible Result 3: Fusion protein is outside cell membrane


Here is the details of our method

Use 5ml LB culture and 5μl chloramphenicol antibiotic to incubate bacteria transformed by function test circuit (K523013 + CSP16 + E1010 and B0015) and control circuit (K523013 + CSP16 + and B0015) 12~16 hour. Centrifuge 5cc bacteria culture at 13000 rps for 2 minute.

Our result turn out to be a clear red color! This indicates that our fusion protein has been successfully displayed on the surface of the cell membrane.


Testing the display function and efficiency of INPNC We designed the experiment below.

Cultured our modified e-coli containing test circuit(promoter+INPNC+CSP16+RFP+terminator) in LB liquid culture for 12 hours. Used 96 well plate to culture above-mentioned e-coli. Used varioskan flash spectral scanning multimode reader to test fluorescence curve and OD curve every 2 hours. Also, compared culture in LB culture and PBS culture. Meanwhile, LB culture +competence cell and PBS culture+ competence cell as control.

Source

We truncated INPNC ourselves, derive C16 from Streptococcus Mutants and we ligate an igem BBa_E1010 RFP at the tail of this surface display gene. To round this gene up, we gave it a terminator B0015.

References