Difference between revisions of "Part:BBa K3866009"
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The coding sequence was domesticated . We removed BsmBI and BsaI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in seva a2 vector and has overhangs compatible for GoldenBraid cloning.
 
The coding sequence was domesticated . We removed BsmBI and BsaI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in seva a2 vector and has overhangs compatible for GoldenBraid cloning.
  
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[[Image:T--Thessaly--arac-pta-term.png|900px|thumb|none|<I><b>Figure 1.</b> The level a module of the Actetate Production : a2:ParaBAD:RBS-Pta-Double terminator </i>]]
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[[Image:T--Thessaly--grandaAC.png|900px|thumb|none|<I><b>Figure 1.</b> The final level omega module of the Actetate Production</i>]]
  
 
===Verification of Cloning===
 
===Verification of Cloning===
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[[File:T--Thessaly--arac-ptagel.png|700px|thumb|none|<i><b>Fig.2:</b>: (U=Uncut , C= Cut)Positive Clone 1  Expected Bands 6345, 3544</i>]]
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[[File:T--Thessaly--grandACgelgel.png|700px|thumb|none|<i><b>Fig.2:</b>: (U=Uncut , C= Cut)Positive Clone 1  Expected Bands 6345, 3544</i>]]
  
  
 
===Experimental Use and Experience===
 
===Experimental Use and Experience===
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This part showed functionality at this part <bbpart>BBa_K3866009</bbpart>
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===Sequence and Features===
 
===Sequence and Features===

Revision as of 15:35, 25 September 2021


Acetate production construct

Usage and Biology

This TU includes the Pta gene placed under the control of the arabinosed-induced promoter. The inducible promoter is used because cloning wasn't done. After the constitutive expression, the metabolism went out of balance so no colonies survied to be chosen for screening.

Design Notes

The coding sequence was domesticated . We removed BsmBI and BsaI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in seva a2 vector and has overhangs compatible for GoldenBraid cloning.

File:T--Thessaly--grandaAC.png
Figure 1. The final level omega module of the Actetate Production

Verification of Cloning

File:T--Thessaly--grandACgelgel.png
Fig.2:: (U=Uncut , C= Cut)Positive Clone 1 Expected Bands 6345, 3544


Experimental Use and Experience

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 2853
    Illegal PstI site found at 3399
    Illegal PstI site found at 3699
    Illegal PstI site found at 3864
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 2853
    Illegal PstI site found at 3399
    Illegal PstI site found at 3699
    Illegal PstI site found at 3864
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3503
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 2853
    Illegal PstI site found at 3399
    Illegal PstI site found at 3699
    Illegal PstI site found at 3864
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 2853
    Illegal PstI site found at 3399
    Illegal PstI site found at 3699
    Illegal PstI site found at 3864
    Illegal AgeI site found at 1959
    Illegal AgeI site found at 2357
    Illegal AgeI site found at 3603
    Illegal AgeI site found at 3801
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  • Dittrich, C. R., Bennett, G. N., & San, K. Y. (2005). Characterization of the acetate-producing pathways in Escherichia coli. Biotechnology progress, 21(4), 1062–1067. https://doi.org/10.1021/bp050073s