Difference between revisions of "Part:BBa K2718006"

(→‎Protein activity)
(→‎Protein activity)
 
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The Plac promoter <html><a href="https://parts.igem.org/Part:BBa_R0011">(BBa_R0011)</a></html> 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:  
 
The Plac promoter <html><a href="https://parts.igem.org/Part:BBa_R0011">(BBa_R0011)</a></html> 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).  
 
*repressed by LacI, the Lac inhibitor (i.e. repressor).  
−
*induced by [http://openwetware.org/wiki/IPTG IPTG] in E.Coli DH5-alpha.  
+
*induced by IPTG in E.Coli DH5-alpha.  
  
 
This promoter <html><a href="https://parts.igem.org/Part:BBa_R0011">(BBa_R0011)</a></html> is assembled with Methionine-y-lyase-Histag <html><a href="https://parts.igem.org/Part:BBa_K2718005">(BBa_K2718005)</a></html> to produce the methionine-y-lyase-histag.
 
This promoter <html><a href="https://parts.igem.org/Part:BBa_R0011">(BBa_R0011)</a></html> is assembled with Methionine-y-lyase-Histag <html><a href="https://parts.igem.org/Part:BBa_K2718005">(BBa_K2718005)</a></html> to produce the methionine-y-lyase-histag.
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<html><figure>
 
<html><figure>
−
<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
+
<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
 
</figcaption></figure></html>
 
</figcaption></figure></html>
  
 
<html><figure>
 
<html><figure>
−
<img src="https://static.igem.org/mediawiki/2018/2/2b/T--Aix-Marseille--WBDH5a.png" width="800" height="500"/><figcaption>Fig 2. Western Blot DH5a
+
<img src="https://static.igem.org/mediawiki/2018/2/2b/T--Aix-Marseille--WBDH5a.png" width="800" height="500"/><figcaption>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)
 
</figcaption></figure></html>
 
</figcaption></figure></html>
  
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<html><figure>
 
<html><figure>
−
<img src="https://static.igem.org/mediawiki/2018/d/d2/T--Aix-Marseille--WBBL21.png--Aix-Marseille--WBBL21.png" width="800" height="500"/><figcaption>Fig 3. Western Blot BL21
+
<img src="https://static.igem.org/mediawiki/2018/d/d2/T--Aix-Marseille--WBBL21.png--Aix-Marseille--WBBL21.png" width="800" height="500"/><figcaption>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)
 
</figcaption></figure></html>
 
</figcaption></figure></html>
  
 
===Purification of the protein===
 
===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).  
+
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).  
  
 
<html><figure>
 
<html><figure>
−
<img src="https://static.igem.org/mediawiki/2018/d/dd/T--Aix-Marseille--PurificationMGLDH5a1L.png" width="800" height="500"/><figcaption>Fig 4. Chromatogram of the purification of methionine-y-lyase-histag
+
<img src="https://static.igem.org/mediawiki/2018/b/b8/T--Aix-Marseille--Shcema_MGL_registre_elution.png" width="800" height="500"/><figcaption>Fig 4. Chromatogram of the purification of methionine-y-lyase-histag
 
</figcaption></figure></html>
 
</figcaption></figure></html>
  
−
[[File:Blue_after_purif.png|700px|thumb|left|Fig 5. SDS PAGE after purification, NR = non-restraint, 1, 8 and 10-15 correspond to the eluted fractions]]
+
[[File:Blue_after_purif.png|700px|center|thumb|Fig 5. SDS PAGE after purification, NR = non-restraint, 1, 8 and 10-15 correspond to the eluted fractions]]
−
 
+
  
 
===Protein activity===
 
===Protein activity===
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To characterize methionine-γ-lyase, activity tests were done :
 
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
 
* 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.
+
* 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.
−
 
+
−
[[File:Activity_test_mgl.png|700px|thumb|left|Figure 6 : Scheme of the reaction of the methionin-y-lyase activity test]]
+
−
 
+
−
 
+
−
[[File:Activity_test_withoutmgl_and_mgl_with_subtrate.png|700px|thumb|left|Figure 7 : Result of activity test of methione-y-lyase using DTNB. Both conditions are : DTNB only and protein + DTNB]]
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
  
 +
[[File:Activity_test_mgl.png|700px|center|thumb|Figure 6 : Scheme of the reaction of the methionin-y-lyase activity test]]
  
 +
<html><figure>
 +
<img src="https://static.igem.org/mediawiki/2018/3/39/T--Aix-Marseille--Shcema_MGL_activity_test_1.png" width="800"/>
 +
<figcaption>Fig 7.Results of activity test of methione-y-lyase using DTNB. Both conditions are : DTNB only and protein + DTNB
 +
</figcaption></figure></html>
  
 +
<html><figure>
 +
<img src="https://static.igem.org/mediawiki/2018/9/95/T--Aix-Marseille--Shcema_MGL_activity_test_2.png" width="800"/><figcaption>Fig 7. Results of activity test of methione-y-lyase using DTNB. Both conditions are : Protein only and protein + DTNB
 +
</figcaption></figure></html>
  
 +
The activity test for methionine-y-lyase is on our wiki[http://2018.igem.org/Team:Aix-Marseille/Experiments#Methionine_gamma_lyase_activity]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 01:16, 18 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 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