Difference between revisions of "Part:BBa K2718011"

Latest revision as of 23:45, 17 October 2018

IPTG inductible promotor RBS (strong) HmaS

Usage and Biology

HmaS is the first enzyme in the metabolic pathway shown in figure 1 used to produce benzaldehyde and 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 weight. Nevertheless, our inducible promotor is not perfect, and there is basal production of HmaS (line 1). However, negative control is as expected, 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 chromatography using an anion exchange column. For protocol, see our [http://2018.igem.org/Team:Aix-Marseille/Protocols#Protein_purification wiki]

Figure 3 Elution profile of HmaS during purification by ion exchange column performed by Akta

We can see 3 peaks in the elution profile because ion exchange chromatography is not a specific affinity technique to isolate protein. So we decided to check our protein purification using SDS-PAGE (figures 4a and 4b)

Figure 4a SDS-PAGE of the eluate after purifiction L. Ladder ; 1'Bacterial lysate ; 2'pellet of bacteria lysate ; 3' non purified fraction ; 4' flow through during protein injection ; 5' flow through 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 major protein at approxomatively 37kDa, this probably corresponds to HmaS. Nevertheless, the 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 mesured (S)Mandelate production by HPLC. For the protocol see [ here]. We tested 3 conditions, DH5α with empty an plasmid, DH5α pSB1A2_BBa K2718011, DH5α pSB1C3_BBa K2718011.

Figure 5 Measurements of (S)Mandelate production by HPLC

The first thing that can be noticed is the global increase in (S) mandelate concentrations. There is some mandelate production in the control strain possibly due to non-enzymatic degradation of the phenyl-pyruvate. We can conclude that there seems to be a (S) mandelate synthase activity, as the (S) mandelate rate are highter in our transformed strain than the E.coli strain with empty plasmid (in particular with the psb1c3 plamsid).In addition, the HPLC peak separation is not perfect, so a modification of the gradient parameters between solution A and B, as well as elution time, might improve the results. For more detailled results, visit our [http://2018.igem.org/Team:Aix-Marseille/Experiments#Mandelate_synthase_activity wiki]

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

Sources

Pugh, S., McKenna, R., Halloum, I., and Nielsen, D. R. (2015). EngineeringEscherichia coli for renewable benzyl alcohol production. Met. Eng. Commun.2, 39–45. doi: 10.1016/j.meteno.2015.06.002