Difference between revisions of "Part:BBa K2718006"

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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.
 
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.
  
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[[File:T--Aix-Marseille--BluegelDH5a7D.png|900px|center|thumb|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 (1D)]]
 
  
 
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<img src="https://static.igem.org/mediawiki/2018/6/6b/T--Aix-Marseille--BluegelDH5a7D.png" width="800" height="500"/><figcaption>Fig 1. SDS-PAGE DH5a
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<img src="https://static.igem.org/mediawiki/2018/6/6b/T--Aix-Marseille--BluegelDH5a7D.png" width="800" height="500"/><figcaption>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
 
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Revision as of 20:42, 17 October 2018


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 [http://openwetware.org/wiki/IPTG 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


Fig 3. Western Blot BL21

Purification of the protein

The histidine tag present in c-terminal of the protein allows us to purify the protein on a nickel NTA 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
  • MTBH (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


Figure 7 : Results of activity test of methione-y-lyase using DTNB. Both conditions are : DTNB only and protein + DTNB
Figure 8 : Results of activity test of methione-y-lyase using DTNB. Both conditions are : Protein only and protein + DTNB



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