Part:BBa_K5048069
Neae-HSP60-FLAG
Codes for one of our team’s fusion proteins, presenting the HSP60 protein on the outer membrane of E.coli to enable the bacteria-bacteria adhesion(Fig 1). Codon optimized for expression in E.coli.
Fig 1 The expected effect for the Adhesion module, which adheres to the engineering bacteria and small intestine
Usage and Biology
Neae displays HSP60 on the outer membrane of our engineered E.coli to enable the bacteria-bacteria adhesion. Codon optimized for expression in E.coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 2006
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2347
Illegal AgeI site found at 2502
Illegal AgeI site found at 2980 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1244
Design
We constructed this part by restriction endonuclease cleavage and ligation. During the process of design, we had to consider the enzyme and the cleavage site we needed to use. When inserted into the pET-28a backbone, this part is included in Adhesion Construction(Fig 1).
Fig 1 The genetic circuit respectively for pET-28a-[Neae-LAP] (left)
Source
See our parts: BBa_K5048047, BBa_K5048060
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 2, A).
Fig 2 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 T4 ligation, we successfully constructed the target plasmid within E. coli DH5α and BL21(DE3)(Fig 2, B)(Fig 3)
Fig 3 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 4). 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 4 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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