Part:BBa_K3722009
T7_Promoter-RBS-SttH-T7_Terminator
Flavin-dependent Trp halogenases use halide ions (X−) and FADH2 for halogenation，if you use it in cell-free systems, requiring supplementation of the high-cost cofactor FADH2 and additional cofactor regenerating enzymes.
Biology
SttH is a Tryptophan halogenases from Streptomyces toxytricini. It can substitute the 6-CH of Trp by a Bromine atom.
Usage
Here we use BBa_K3722009 to verify the expression of SttH. We transformed the composite part BBa_K3722009 into E.coli DH5α&E.coli BL21(DE3) to produce 6-Br-Trp. The positive clones were cultured.
Characterization
1.Identification
We received the synthesized DNA, then performed PCR with the primer of SttH. We used agarose gel electrophoresis to confirm that we got the right DNA fragment. The experimental results were shown below and ideally fit the length we want.
2.The proof of expression
We connected the composite part Bba_K3722009 with pET-28a(+), then transformed it into E. coli BL21(DE3). We added 0.1 mM IPTG and incubated the bacterial culture at 18℃ for 16 hours to get a high expression with low noise protein. We also incubated the other
E. coli BL21(DE3) bacterial culture which transformed only with pET-28a(+) as a control group,
the expressed proteins were collected by centrifugation. Our target bands were observed through SDS-PAGE. The experimental results were shown below. We can easily find the target band which only shown in the left lane.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1635
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 268
Illegal NgoMIV site found at 526
Illegal AgeI site found at 755 - 1000COMPATIBLE WITH RFC[1000]
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