Difference between revisions of "Part:BBa K2718006"

(→‎Purification of the protein)
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<partinfo>BBa_K2718006 short</partinfo>
 
<partinfo>BBa_K2718006 short</partinfo>
  
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The PLlac 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 strong promotion that can nevertheless be:  
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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:  
 
*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 [http://openwetware.org/wiki/IPTG IPTG] in E.Coli DH5-alpha.  
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* <i>DH5a</i>
 
* <i>DH5a</i>
 
*<i> BL21 DE3</i>
 
*<i> BL21 DE3</i>
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To verify the production of methionine-y-lyase, a SDS PAGE was performed and stained with coomassie blue using cells containing this biobrick and a Western Blot was performed with anti-histag antibodies, revealed with alkaline phosphatase.
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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.
  
  
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===Purification of the protein===
 
===Purification of the protein===
  
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The histidine tag present in c-ter of the protein allows to purify the protein on a nickel column. In our case the protein was purified using a "Akta pure" on Histrap column (GE Healthcare).  
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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).  
  
 
<html><figure>
 
<html><figure>

Revision as of 19:30, 13 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

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


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