Difference between revisions of "Part:BBa K2718011"

Revision as of 17:00, 17 October 2018

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 a protein of approximatively 37kDa, which is consistent with HmaS's molecular weigth. Nonetheless, our inducible promotor is not perfect, and there is basal production of HmaS (line 1). Moreover negative control is correct, there is no prodution of HmaS with empty plasmid (line 5)

Purification

We tried to purify Hmas with Akta pure (FPLC) (GE healthcare) by ion exchange column. For protocol, see our [http://2018.igem.org/Team:Aix-Marseille/Protocols wiki]

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 check our protein purifiction using SDS-PAGE (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 purification 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 and contain others proteins in adition to HmaS. HmaS is on fractions 8 to 17, that's correspond to second spike on figure 3. This data show that we managed to produce and purify HmaS,while the purification step could be improved, for example using size exclusion chromatography.

Activity test

We mesure (S)Mandelate production by HPLC. For protocol see. 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