Difference between revisions of "Part:BBa K3866009"
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===Usage and Biology===
 
===Usage and Biology===
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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.
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===Design Notes===
 
===Design Notes===
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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.
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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 omega1R vector and has overhangs compatible for GoldenBraid cloning.
  
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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>]]
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[[Image:T--Thessaly--grandAC.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--grandACgelgel.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 2.
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Expected bands: 3153 bp, 1769 bp, 1757 bp, 1447 bp, 1160 bp
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</i>]]
  
  

Revision as of 15:38, 25 September 2021


Acetate production construct

Usage and Biology

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 omega1R vector and has overhangs compatible for GoldenBraid cloning.

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 2. Expected bands: 3153 bp, 1769 bp, 1757 bp, 1447 bp, 1160 bp


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