Part:BBa_K5048060:Experience

Experiments

This part is expressed on the outer membrane of E. coli BL21(DE3), and we conducted a range of experiments to confirm the presentation of DNA and protein. We also cloned the HSP60 segment onto the Neae-HSP60 plasmid(BBa_K5048069) and SUMO-HSP60-Flag plasmid(BBa_K5048065).

The overall experiments we conducted on BBa_K5048060 include:

1. Verification of transformation of plasmid HSP60-FLAG
2. Verification of protein HSP60
3. Clone HSP60 onto improvement parts

Result

Verification of transformation

To evaluate the functions of the target proteins, we transform the pET-28a-[HSP60-Flag] into strain E. coli BL21(DE3). We conducted colony PCR to verify the transformation(Fig 1). Moreover, The Sanger sequencing conducted by the cooperated company ensures the specific sequence (Fig 2).

Fig 1 The confirmation of transformation.
HSP60 1-3 are successful transcription samples

Fig 2 The sequencing result for transformation in E. coli BL21(DE3).
A: The sequencing result for pET-28a-[HSP60-Flag]. The base deletion at the end is due to the limitations of the Sanger method, rather than a base mutation.

Verification of protein HSP60

We utilize Western Blot (WB)(Fig 3) to detect the HSP60 protein.

Fig 3 Western blot analysis for HSP60 protein.
A: WB analysis of HSP60 protein in E. coli BL21(DE3) under induction of 0.5mM IPTG and 250rpm for 3 hours. The differences in induction temperatures are marked on the image. The exposure time is 50 seconds.

Clone HSP60 onto improvement parts

See more in Neae-HSP60 plasmid(BBa_K5048069) and SUMO-HSP60-Flag plasmid(BBa_K5048065).

Applications of BBa_K5048060

Cooperating with protein Nea and LAP, we make two kinds of engineered bacteria adhere to each other(Fig 4).

Fig 4 The expected effect for Adhesion construct

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