Part:BBa_K1934000

RFP_CBDs

This part contains the sequence coding for the RFP protein linked to two cellulose binding domains, one located at the N-terminus (CBD1 - BBa_K1934080) and one located at the C-terminus (CBD2 - BBa_K1934090). It is a RFP-CBDs generator, based on 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 the purification process visually;
• 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 mmol.L^-1 and glucose 5 mmol.L^-1. Cells were harvested and resuspended in 1 mL lysis buffer (50 mmol.L^-1 Tris, 300 mmol.L^-1 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 microcrystalline cellulose five time in water. Then equilibrate in wash buffer (ammonium sulfate 1 mol.L^-1). 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 flowing out evenly, 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.
• Analyze collected fractions on a 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 was filled with wash buffer. Tube D contains Fraction nº3 proteins. Only tube A shows high 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 does not impair neither the cellulose binding nor the protein folding.