Part:BBa_K5048058
LAP-6x His
Codes for the membrane protein of L.innocua, which can bind to HSP60 on the membrane of small intestinal epithelial cells, and another type of E.coli that we have engineered. Codon optimized for expression in E. coli, and a 6xHis tag was added for convenient detection.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1429
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1429
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2472
Illegal BglII site found at 2570 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1429
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1429
Illegal NgoMIV site found at 678
Illegal NgoMIV site found at 1944
Illegal AgeI site found at 310
Illegal AgeI site found at 1966 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 2292
Design
LAP(Listeria Adhesion Protein) from a kind of non-pathogenic Listeria that can interact with HSP60 and help our engineered bacteria adhere to the small intestine(Fig 1).
Fig 1 The expected effect for the Adhesion module, which adheres to the engineering bacteria and small intestine
Source
Listeria innocua strain CFSAN044836 chromosome, complete genomeSequence, ID: CP045743.1,Range 1: 55424 to 58024
Experiments
This part acts as an adhesin that can adhere to HSP60. We also cloned the improved LAP onto the LAP-GST plasmid(K5048066) and Neae-LAP plasmid(BBa_K5048068).
The overall experiments we conducted on BBa_K5048058 include:
1. Verification of transformation of plasmid LAP-6xHis
2. Verification of protein LAP
3. Detection of protein LAP locations
4. Clone LAP onto improved parts
Result
Verification of transformation
To evaluate the functions of the target proteins, we transform the pET-28a-[LAP-His] 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.
LAP 1 is a successful transcription sample
Fig 3 The sequencing result for transformation in ''E. coli'' BL21(DE3).
B: The sequencing result for pET-28a-[LAP-His].
Verification of protein LAP
We utilize protein purification resin(Fig 4) and Western Blot (WB)(Fig 5) to detect the Neae protein.
Fig 4 Coomassie Brilliant Blue stained gel image for LAP protein after protein purification resin treatment. LAP protein is expressed in E.coli BL21(DE3), under the induction of 0.1mM IPTG, 25 ℃, and 200rpm for 6 hours. LAP protein can be seen in samples of elution buffer2-5, which are marked by dark arrows, with sizes between 100kDa and 130kDa. Samples of non-denaturing wash buffer 1-4 are collected corresponding to the first through fourth pre-treatments of the purification resin. Sample supernatant is collected after the lysate of the non-denaturing wash buffer. Flow-through is collected after the supernatant flows through the purification resin. Samples of elution buffer 1-6 are collected corresponding to the first through sixth washes of elution buffer through the purification resin.
Fig 5 Western blot analysis for Neae protein.
B: WB analysis of LAP protein in ''E. coli'' BL21(DE3) under induction of 0.5mM IPTG, 25℃, and 250rpm for 1 hour. The exposure time is 30 seconds.
Detection of protein LAP locations
We examine the expression level of LAP in cell supernatant and outer membrane(Fig 6). Although the transcription level of LAP remains low, both of the contents include the LAP.
Fig 6 WB analysis of LAP 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 100 seconds.
Clone Neae onto improvement parts
See more in LAP-GST plasmid(K5048066) and Neae-LAP plasmid(BBa_K5048068).
Applications of BBa_K5048058
Cooperating with protein Neae and HSP60, we make two kinds of engineered bacteria adhere to each other. (Fig 7)
Fig 7 The expected effect for Adhesion construct
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