Part:BBa_K5048060

HSP60-FLAG

Codes for the membrane protein of small intestinal epithelial cells, which can adhere to LAP. Codon optimized for expression in E. coli, and a FLAG tag was added for convenient detection.

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 325
  Illegal AgeI site found at 480
  Illegal AgeI site found at 958
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design

HSP60 is a human protein expressed on the membrane of intestine epithelia. Codon optimization should be done to ensure the expression in E. coli, and a FLAG tag was added for easy detection. Cooperated with Neae and LAP, we expected to achieve cell-cell adhesion and cell-intestine adhesion in the small intestine(Fig 1).

Fig 1 The expected effect for the Adhesion module, which adheres to the engineering bacteria and intestine

Source

HSPD1 heat shock protein family D (Hsp60) member 1 [Homo sapiens (human)] - Gene - NCBI (nih.gov) CCD：CCDS Report for Consensus CDS (nih.gov)

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 improved 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 2). Moreover, The Sanger sequencing conducted by the cooperated company ensures the specific sequence (Fig 3).

Fig 2 The confirmation of transformation.
HSP60 1-3 are successful transcription samples

Fig 3 The sequencing result for transformation in E. col 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 4) to detect the HSP60 protein.

Fig 4 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 improved parts

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

Applications of BBa_K5048060

Cooperating with protein Neae and LAP, we make two kinds of engineered bacteria adhere to each other(Fig 5).

Fig 5 The expected effect for Adhesion construct