Part Validation

NisQ Part Validation

Successful Cloning and Protein Production

Figure 1. PCR amplification of pSB1A3 plasmid containing GB1 (BBa_K4437001), using T7 promoter and terminator specific primers, indicating successful cloning. Lane 2 contains our expected band size at 331 bp

Figure 2. SDS-PAGE analysis of GST-NusA-NisQ (BBa_K4437003) samples from BL21 (DE3) E. coli strain autoinduced, using a Coomassie-blue stain. Large bands in lanes 2, 4, 5, 6, and 9 at 91kDa correspond to our expected protein size.

Figure 3. His-tag purified SDS-PAGE of GST-NusA-NisQ (BBa_K4437003) samples samples from BL21 (DE3) E. coli strain autoinduced, using a Coomassie-blue stain. Bands in lanes 3, 4, and 5 at 91kDa correspond to our expected protein size. Samples labelled "W-1" indicate wash 1, samples labelled "E-1" indicate elution 1.

Figure 4. Western blot of the whole cell lysate of GST+NusA+NisQ auto-induced in overnight express. A his-tagged positive control was also included. The protein ladder used was the Novex sharp pre-stained protein standard. The antibodies used were Mouse Anti-HIS-tag mAb (Abcam) for the primary antibody and Goat Anti-Mouse:HRP (Abcam) for the secondary antibody.

PHB Part Validation

Successful Cloning and Protein Production

Figure 5. Figure 5: The expression of phasin-hlyA with varying RBS strengths from the PHB construct (BBa_K934001) from BL21 (DE3) E.coli strain autoinduced for 48 hours on an SDS page using Anti-FLAG antibodies and the complementary capture antibody. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. The gel was loaded as follows: (1) Ladder, (2) Tokyo 2012 PHB construct only (BBa_K934001) (3) BBa_K4437500-phasin-hlyA (BBa_K4437501), (4) BBa_B0034-phasin-hlyA (BBa_K4437503), (5) BBa_B0035-phasin-hlyA (BBa_K4437504).