Difference between revisions of "Part:BBa K3398003"
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====Plasmid construction==== | ====Plasmid construction==== | ||
We designed the fusion protein sequence ordered gene synthesis service from GenScript Biotech Corporation and obtained the recombinant pGEX-2TK plasmid with a GST tag. To confirm the sequence of the fusion protein, we designed a couple of primers to amplify the sequence by PCR and sent to Zhejiang Sunya Biotechnology Co., Ltd for sequencing.The vector map and PCR results are shown in Figure 1 and Figure 2 below. | We designed the fusion protein sequence ordered gene synthesis service from GenScript Biotech Corporation and obtained the recombinant pGEX-2TK plasmid with a GST tag. To confirm the sequence of the fusion protein, we designed a couple of primers to amplify the sequence by PCR and sent to Zhejiang Sunya Biotechnology Co., Ltd for sequencing.The vector map and PCR results are shown in Figure 1 and Figure 2 below. | ||
| − | + | ====Expression of mamC_ZZ==== | |
| − | ===Expression of mamC_ZZ=== | + | |
After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pGEX-2TK construct was transformed into BL21 (DE3). | After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pGEX-2TK construct was transformed into BL21 (DE3). | ||
Revision as of 02:40, 22 October 2020
mamC_ZZ fusion protein
This part is encodes a fusion protein of mamC and ZZ. And for separation from other proteins at the purification step, we introduced a GST tag at the N-terminus and a thrombin site between the fusion protein and tag for cleavage after purification, which has been submitted as BBa_K3398004. To maximize the expression of a functional protein, we employed codon optimization to maximize the expression of a functional protein in Escherichia coli. Not only in E.coli, these parts can also be expressed by magnetotactic bacterium such as MSR-1 to produce magnetosomes modified with ZZ protein on the surface as a self-assembly platform for polymer varied antibodies.
Usage and Biology
MamC protein is a membrane protein which covers the most area of the magnetosome surface formed by magnetotactic bacteria MSR-1, and ZZ is the Fc-binding domain of staphylococcal protein A (SPA), an immunoglobulin-binding protein from the cell wall of Staphylococcus aureus. This fusion protein can be expressed and anchored onto the surface of magnetosomes by transferring the recombinant plasimd into MSR-1.The resulting recombinant magnetosomes will be capable of self-assembly with the Fc region of mammalian antibodies and will ve therefore useful for functionalization of magnetosomes such as being developed to a antibody vector or serving as specifically tumor-targeting contrast agents for MRI.
Characterization
Plasmid construction
We designed the fusion protein sequence ordered gene synthesis service from GenScript Biotech Corporation and obtained the recombinant pGEX-2TK plasmid with a GST tag. To confirm the sequence of the fusion protein, we designed a couple of primers to amplify the sequence by PCR and sent to Zhejiang Sunya Biotechnology Co., Ltd for sequencing.The vector map and PCR results are shown in Figure 1 and Figure 2 below.
Expression of mamC_ZZ
After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pGEX-2TK construct was transformed into BL21 (DE3).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 36