Part:BBa_K2718011
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 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 4 conditions, DH5α, DH5α with empty an plasmid, DH5α pSB1A2_BBa K2718011, DH5α pSB1C3_BBa K2718011. In the figure we show the difference between the test condtiions and DH5α.
http://2018.igem.org/File:T--Aix-Marseille--Hplc_s_mandelate_rate_evolution_new.png
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
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
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