Part:BBa_K5048069:Experience

Experiments

This part acts as an adhesin construct that can display protein HSP60 on the outer membrane of E. coli and adhere to LAP.

The overall experiments we conducted on BBa_K5048069 include the following:

1. Verification of PCR products;
2. Verification of transformation of the plasmid;
3. Verification of target protein.

Results

Verification of PCR products

We employed PCR to introduce restriction enzyme cleavage sites for EcoRI and HindII and amplified target segments(Fig 1, A).

Fig 1 The construction and verification of pET-28a-[Neae-HSP60-FLAG]
A: The PCR product of Neae backbone and HSP60 segments. Both ends of the DNA fragment were introduced to restriction enzyme cleavage sites for EcoRI and HindIII. Neae Backbone 1-2 and HSP60 1-2 all have segments corresponding to the estimated DNA length. EcoRI-Neae backbone-HindIII: 7087bp; HindIII-HSP60-EcoRI: 1955bp.
B: The colony PCR for E. coli BL21(DE3) strains. Neae-HSP60 3-5 and 7 all have segments corresponding to the estimated DNA length. Neae-HSP60: 3983bp

Verification of transformation of the plasmid

By performing DNA purification, restriction enzyme digestion, and ligation, we successfully constructed the target plasmid within E. coli DH5α and BL21(DE3)(Fig 1, B)(Fig 2)

Fig 2 The sequencing results for Neae-HSP60, indicate that we successfully transfer the plasmid into E. coli BL21(DE3) without mutations

Verification of targeted protein

The expression level of Neae-HSP60 is also detected by WB (Fig 3). The translation characteristics of protein Neae-HSP60 are quite similar to protein Neae and HSP60. All of them exhibit a better yield at a higher temperature.

Fig 3 WB analysis of Neae-HSP60 protein in E. coli BL21(DE3) under 250rpm induction for 1.3 hours. The uninduced group shows slight protein leakage while the induced group demonstrates an abundant protein expression. Neae-HSP60 is estimated at around 130kDa. The exposure time is 10 seconds.

Applications of BBa_K5048069

A part of Adhesion Construct that can display protein HSP60 on the outer membrane of E. coli and adhere to LAP in engineered bacteria and small intestine.

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