Part:BBa_K2718011
IPTG inductible promotor RBS (strong) HmaS
Usage and Biology
HmaS is used in the metabolic pathway shown in figure 1 to produce benzyl alcohol. HmaS catalyzes the oxidative decarboxylation of phenylpyruvate to produce (S)Mandelate.
Figure 1 Metabolic pathway to produce benzyl alcohol
Production
We producted HmaS in E.coli DH5alpha, with 1mm IPTG at OD 0.8, 3 hours
Figure 2 SDS-PAGE of HmaS production with induction (2, 3 and) 4, without induction ( 1,NI) and with empty plasmid (5)
We see overproduction of HmaS of approximatively 37kDa, that's correspond to molecular wheigt of HmaS. Nonetheless, our inducible promotor is not perfect, and there is basal production of HmaS. Moreover negative control is correct, there is noprodution of HmaS with empty plasmid
Purification
We tried to purify Hmas with Akta pure (GE healthcare) by exchange ion column. We used to eluate 60mL of Tampon B (1M NaCl and Tris 20mM)with gradient of 0 to 100%, figure 3.
Figure 3 Elution of HmaS after purification by exchange ion column performed by Akta
We can see
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 607
Illegal BamHI site found at 716 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 556
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