Difference between revisions of "Part:BBa K1934000"
Line 24: Line 24:
 
<ul style="list-style-type:circle">
 
<ul style="list-style-type:circle">
 
   <li>Wash microcrystaline Cellulose five time in water. Then equilibrate in wash buffer (Amonium Sulfate 1M). Pack the cellulose (10x10 mm) in small chromatography columns (we used syringes barrels).  </li>
 
   <li>Wash microcrystaline Cellulose five time in water. Then equilibrate in wash buffer (Amonium Sulfate 1M). Pack the cellulose (10x10 mm) in small chromatography columns (we used syringes barrels).  </li>
−
   <li> Gently pour the lysate supernatant on the column. Once the liquid starts to flow out regularly measure the OD280 of the different fractions.  Continue pouring wash buffer until the OD<sub>280</sub> stabilizes around zero.  </li>
+
   <li> Gently pour the lysate supernatant on the column. Once the liquid starts to flow out regularly measure the OD<sub>280</sub>  of the different fractions.  Continue pouring wash buffer until the OD<sub>280</sub> stabilizes around zero.  </li>
−
   <li> Change the washing buffer to water. OD280 shortly rises. Keep the fractions with highest OD<sub>280</sub> . They should contain the protein. </li>
+
   <li> Change the washing buffer to water. OD<sub>280</sub>  shortly rises. Keep the fractions with highest OD<sub>280</sub> . They should contain the protein. </li>
 
<li>  Analyse collected fractions on an SDS PAGE.  
 
<li>  Analyse collected fractions on an SDS PAGE.  
 
  Optionally, proteins may be concentrated using ultrafiltration.</li>
 
  Optionally, proteins may be concentrated using ultrafiltration.</li>

Revision as of 23:28, 19 October 2016

RFP_CBDs

This part contains the sequence coding for the RFP protein linked to two cellulose binding domains, one located in N-terminal (CBD1) and one located in C-terminal (CBD2). It is a RFP-CBDs generator, following the model of our streptavidin-CBDs generator (BBa_K1934020).

The part BBa_K1934000 was designed for two reasons:

  • RFP properties and especially its red coloration enables the follow visually the purification process.
  • This colorimetric signal allows to determine if the covalent linkage of the CBDs on the protein disrupts the RFP folding.


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 380
    Illegal AgeI site found at 955
    Illegal AgeI site found at 1067
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

Purification Using Cellulose Affinity

The BBa_K1934000 part conceived by the 2016 INSA-Lyon team and synthesized by IDT was cloned into pSB1C3 and transformed into the E. coli NM522 strain. One recombinant clone was grown overnight in LB at 24°C, with IPTG 1 mM and glucose 5 mM. Cells were harvested and resuspended in 1 mL lysis buffer (50 mM Tris, 300 mM NaCl, 10% glycerol). Then the mix was sonicated 5 times 30 seconds on ice at moderate power. The lysate was centrifuged at 14.000 g for 10 min. The supernatant was treated as follow:

  • Wash microcrystaline Cellulose five time in water. Then equilibrate in wash buffer (Amonium Sulfate 1M). Pack the cellulose (10x10 mm) in small chromatography columns (we used syringes barrels).
  • Gently pour the lysate supernatant on the column. Once the liquid starts to flow out regularly measure the OD280 of the different fractions. Continue pouring wash buffer until the OD280 stabilizes around zero.
  • Change the washing buffer to water. OD280 shortly rises. Keep the fractions with highest OD280 . They should contain the protein.
  • Analyse collected fractions on an SDS PAGE. Optionally, proteins may be concentrated using ultrafiltration.

Figure 1. Purification of the chimeric RFP-CBDs protein on a cellulose column This elution graph shows a first peak, present for both the control and our expression culture. This first peak corresponds to unbound proteins. In presence of water, only one peak was observed: it’s the elution peak of our protein.

Verification of protein folding

Figure 2. Fluorescence measurement of the final supernatant of RFP-CBDs purification process. This figure was obtained with the Chemidoc image system. Tube A was filled with the purified protein fraction. Tube B is filled up with wash buffer. Tube D contains fraction 3 proteins. Only tube A shows hight levels of fluorescence. The RFP-CBDs was efficiently purified thanks to a cellulose column. A fluorescence signal is measured proving that the RFP is still functional even with the CBDs covalent ligation.

Conclusion

This experiment shows that the presence of CBDs on RFP-CBDs do not impaired neither the cellulose binding nor the protein folding.