Part:BBa_K5258005
pET28a-46#SBD
we are building and verifying the recognition ability of pET-28a+46#SBD. The general process is almost the same as the foregoing ones. Firstly, insert DNA, 46#SBD, and backbone, pET28a, are extracted from plasmids by restriction endonuclease, Xho1, and Nde1. Then, we use T4 DNA ligase to connect sticky ends on the vector and the 46#SBD. After constructing the recombinant plasmid, we transform the plasmid into the E.coli DH5α, a competent cell, by heat shock. The bacteria is cultivated, and a single colony is picked. The plasmid is extracted to verify the correctness of previous construction and has been inserted by gel electrophoresis and gene sequencing. Then, IPTG is used to induce the protein expression. SDS-PAGE is done to assess protein purity. Finally, the purified extracted target SBD proteins are tested for their binding ability with PT-DNA by carrying out EMSA.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4402
Illegal BglII site found at 4638 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2622
Illegal NgoMIV site found at 2782
Illegal NgoMIV site found at 4370 - 1000COMPATIBLE WITH RFC[1000]
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