Part:BBa_K2718006

PLac Promoter-Methionine-y-Lyase-HisTag

The Plac promoter (BBa_R0011) is a hybrid regulatory region consisting of the promoter P(L) of phage lambda with the cI binding sites replaced with lacO1. The hybrid design allows for a strong promoter that can nevertheless be:

• repressed by LacI, the Lac inhibitor (i.e. repressor).
• induced by IPTG in E.Coli DH5-alpha.

This promoter (BBa_R0011) is assembled with Methionine-y-lyase-Histag (BBa_K2718005) to produce the methionine-y-lyase-histag.

Protein production

Different growing conditions were tested to determine the best growing conditions. The production was also tested two types E.coli bacteria :

• DH5a
• BL21 DE3

To verify the production of methionine-y-lyase, an SDS PAGE was performed and stained with Coomassie blue using cells containing this Biobrick and a Western Blot was performed with anti-his tag antibodies, revealed with alkaline phosphatase.

Fig 1. SDS-PAGE DH5a, Induced at 30°C with 1000mM IPTG(7D), Induced at 30°C with 500mM IPTG(6D), Induced at 30°C with 250mM IPTG(5D), Induced at 37°C with 1000mM IPTG(4D), Induced at 37°C with 500mM IPTG(3D), Induced at 30°C with 250mM IPTG(2D), No-induced

Fig 2. Western Blot DH5a, Empty plasmid (Ctrl), No-induced (1D), Induced at 30°C with 250mM IPTG(2D),Induced at 37°C with 500mM IPTG(3D), Induced at 37°C with 1000mM IPTG(4D),Induced at 30°C with 250mM IPTG(5D), Induced at 30°C with 500mM IPTG(6D), Induced at 30°C with 1000mM IPTG(7D), Induced at 16°C with 150mM IPTG(8D), Induced at 16°C with 500mM IPTG(9D), Induced at 16°C with 1000mM IPTG(10D)

Fig 3. Western Blot BL21, Empty plasmid (Ctrl), No-induced (1B), Induced at 30°C with 250mM IPTG(2B),Induced at 37°C with 500mM IPTG(3B), Induced at 37°C with 1000mM IPTG(4B),Induced at 30°C with 250mM IPTG(5B), Induced at 30°C with 500mM IPTG(6B), Induced at 30°C with 1000mM IPTG(7B), Induced at 16°C with 150mM IPTG(8B), Induced at 16°C with 500mM IPTG(9B), Induced at 16°C with 1000mM IPTG(10B)

Purification of the protein

The histidine tag present in c-terminal of the protein allows us to purify the protein on a NI sepharose 6 column. In our case, the protein was purified using an "Akta pure" on Histrap column (GE Healthcare).

Fig 4. Chromatogram of the purification of methionine-y-lyase-histag

Fig 5. SDS PAGE after purification, NR = non-restraint, 1, 8 and 10-15 correspond to the eluted fractions

Protein activity

To characterize methionine-γ-lyase, activity tests were done :

• DTNB (5,5′-Dithiobis 2-nitrobenzoic acid) test : DTNB reacts with thiol group so for methionine-y-lyase, DTNB reacts with thiol group of methanethiol
• MBTH (3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate) test : MBTH reacts with alpha-keto compounds so for methionine-y-lyase, MTBH reacts with 2 oxobutanoate.

Figure 6 : Scheme of the reaction of the methionin-y-lyase activity test

Fig 7.Results of activity test of methione-y-lyase using DTNB. Both conditions are : DTNB only and protein + DTNB

Fig 7. Results of activity test of methione-y-lyase using DTNB. Both conditions are : Protein only and protein + DTNB

The activity test for methionine-y-lyase is on our wiki[http://2018.igem.org/Team:Aix-Marseille/Experiments#Methionine_gamma_lyase_activity]

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 217
  Illegal NgoMIV site found at 730
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 523