Part:BBa_K2107000:Experience

Validation and Proof of Concept of BBa_K2107000

1. Construct validation: After cloning and transformation we did growth curves using this part. Control groups consisted of the cell chassis alone and cell chassis with inducer. Test groups consisted of the transformed cell chassis with and without inducer. We saw that the cell transformed cell chassis exhibited a slight fitness cost indicating acceptance of BBa_K2107000. While the transformed cell chassis with inducer exhibited a large fitness cost indicating that the construct is being expressed.


Fig 1. Shows that induced origami cells cloned with PDGF-B Iso 1 exhibit the highest fitness cost on proliferation
rate when compared to all other groups. This indicates that the expression vector transformed into the origami
cells is successfully producing protein. n=3

2. Part validation: After confirmation via growth curve we induced and purified protein using periplasmic extraction and nickel column chromatography. To visualize the possible presence of BBa_K2107000 we performed SDS-PAGE of the purified fraction. The SDS showed bands of protein at the correct locations indicating that BBa_2107000 is being expressed and that the DsbA periplasmic translocation leader sequence works as expected.


Fig 2. SDS after osmotic shock

3. Part purification and correct folding via DsbA translocation proof of concept: Finally, to obtain more definitive conformation that BBa_k2107000 is being produced and correctly folded we purchased PDGF-B specific antibodies from Santa Cruz Biolabs and ran a western blot. To our delight we were able to visualized a band of protein at the correct location when compared to an industry standard western blot of PDGF-B using the same antibody. This indicates that BBa_K2107000 is being expressed and properly folded in the periplasm.

                        
Fig 3. Western Blot example of PDGF-B from Santa Cruz biotechnology.    Fig 4. Western blot of recombinant PDGF-B Iso 1 purified from E. coli

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