Part:BBa_K1675011
Bxb1 recombinase device
The Bxb1 integrase is a DNA recombinase, more precisely a member of serine recombinase family. Bxb1 could be divided to two parts, gp35 and gp47. The gp35 part could recognize specific sequences, called attB and attP, and then integrate, invert, or excise dsDNA depending on the orientation of recognition sequences. When it inverts DNA sequence, the attB and attP sequences are changed into attL and attR, as other DNA recombinases do. Another part called Bxb1 gp47 binds to integrase-DNA complex and this complex flips inverts DNA back into original sequence by regenerating attB and attP sequences. So the efficiency of inversion mediated by Bxb1 is half and half.
We constructed our verification circuits through OE-PCR, and linked them into vector pSB1C3 (Fig.1,2). And we divided it into recombinase part and fluorescence part.
Fig.1 The constructed result of FimE, P-atp2 and P-atp2+B0034+FimE(channel 1-3 are P-atp2, Bxb1 and P-atp2+B0034+Bxb1 respectively)
Fig.2 The construction result of FimE’s verification device K137058(channel 1~6 the construction result of K137058, channel 1-3 and 6 are positive.)
Meanwhile, we constructed the verification devices, fluorescence part, respectively, then co-transformed them into BMTOP10. We observed their fluorescent condition by fluorescence microscope and floe cytometry.
Fig.3 The fluorescence result of FimE’s verification device
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 825
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1099
Illegal XhoI site found at 1186 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1738
Illegal NgoMIV site found at 1825
Illegal AgeI site found at 875 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1933
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