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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2 Trancription Units
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*AckA <bbpart>BBa_K3866006</bbpart> and Pta<bbpart>BBa_BBa_K3866006</bbpart>regulated by arabinose inducible promoter araBAD for the prduction of Acetate
  
 
===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<bbpart>BBa_K3505010</bbpart> 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--grandACgel.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>]]
  
  
 
===Experimental Use and Experience===
 
===Experimental Use and Experience===
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Before we start producing SCFAs, we needed to optimize our protein expression system.
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[[File:T--Thessaly--SDSAC.png|700px|thumb|none|<i><b>Fig.3:</b>:SDS page (L=Lysate S=Souble I=Insoluble)(0%, 0,1%, 1% L-arabinose inducer)
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Expected bands:Pta 77,2 kDa and AckA 43,7 kDa</i>]]
  
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The enzymes are expressed in high levels. Even at induction with really low concentration 0,1% L-arabinose we observe that the enzyme is present in every collumn. There is a lot enzyme at the insoluble where it shouldn't be. So we must lower the protein production. This can be acheived by lowering the induction from L-arabinose, use a less powerfull promoter and less strong RBS.
  
 
===Sequence and Features===
 
===Sequence and Features===

Latest revision as of 11:22, 27 September 2021


Acetate production construct

Usage and Biology

2 Trancription Units

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 vectorBBa_K3505010 and has overhangs compatible for GoldenBraid cloning.

Figure 1. The final level omega module of the Actetate Production

Verification of Cloning

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

Before we start producing SCFAs, we needed to optimize our protein expression system.

Fig.3::SDS page (L=Lysate S=Souble I=Insoluble)(0%, 0,1%, 1% L-arabinose inducer) Expected bands:Pta 77,2 kDa and AckA 43,7 kDa

The enzymes are expressed in high levels. Even at induction with really low concentration 0,1% L-arabinose we observe that the enzyme is present in every collumn. There is a lot enzyme at the insoluble where it shouldn't be. So we must lower the protein production. This can be acheived by lowering the induction from L-arabinose, use a less powerfull promoter and less strong RBS.

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