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 ion exchange column.After HmaS production , we break ours cells, and with lysate we purify. We used like buffer 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 ion exchange column performed by Akta
We can see 3 spikes in the elution graph because ion exchange chromatography is not a perfect technique to isolate protein. So we decided to do SDS-PAGE to see if we have our protein (figures 4a and 4b)
Figure 4a SDS-PAGE of eluate after purifiction L. Ladder , 1'Bacterial lysate ; 2'pellet of bacteria lysate ; 3' non purified fraction ; 4' breakthrought during protein injection ; 5' breakthrought during wash before elution ; 3,8,9 and 10 elution samples
Figure 4b SDS-PAGE of eluate after purifiction L. Ladder 11 to 24 elution sample
We can see a big spot at approxomatively 37kDa, that's may correspond to HmaS. Nevertheless samples are not pure, others proteins are with HmaS. HmaS is on fractions 8 to 17, that's correspon to second spike on figure 3. So w have partly purifed HmaS. However, We can improve this purification, by using other methods like size eclusion chromatography.Bu
Activity test
We mesure (S)Mandelate production by HPLC. To prepare sample for HPLC, after ON production of HmaS (LB, glucose 20 g/L, indution OD 0.8 with 1mM IPTG, at 30°C.Centrifugation, and washing the pellet with PBS 1X pH7. Resupend te pellet with PBS 1X (pH7), glucose (5 g/L), phenylpyruvate (0,5 g/L) and begin the measurements with HPLC with C18 column with supernatent of aliquot (1mL) evry hours durinf 7 hours. We test 4 conditions, DH5α, DH5α with empty plasmid, DH5α pSB1A2_BBa K2718011, DH5α pSB1C3_BBa K2718011.
Figure 5 Measurements of (S)Mandelate production by HPLC
Design notes
It's a part whith BBa_B0030 like RBS and BBa_R0011 like inducible promotor And coding sequence comes from Amycolatopsis orientalis. And the sequence is optimized for E.coli thanks to IDT tool
This biobrick exists without promotor BBa_K2718010
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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