Difference between revisions of "Part:BBa K3866031"

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The coding sequences were domesticated. We removed BsmBI and BsaI sites in order to be compatible with GoldenBraid and MoClo. The sequences are cloned in seva ω2 vector and have overhangs compatible for GoldenBraid cloning.
 
The coding sequences were domesticated. We removed BsmBI and BsaI sites in order to be compatible with GoldenBraid and MoClo. The sequences are cloned in seva ω2 vector and have overhangs compatible for GoldenBraid cloning.
  
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[[Image:T--Thessaly--GRAND-term.png|600px|thumb|none|<I><b>Figure 1.</b> The ω module of the Propionate Production: α1:ParaBAD:RBS-sbm-Double terminator:ParaBAD:RBS-ygfG-Double terminator:ParaBAD:RBS-ygfH-Double terminator </i>]]
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[[Image:T--Thessaly--GRAND-term.png|900px|thumb|none|<I><b>Figure 1.</b> The ω module of the Propionate Production: α1:ParaBAD:RBS-sbm-Double terminator:ParaBAD:RBS-ygfG-Double terminator:ParaBAD:RBS-ygfH-Double terminator </i>]]
  
 
===Verification of Cloning===
 
===Verification of Cloning===
 
[[File:T--Thessaly--grand1gel.png|800px|thumb|none|<i><b>Figure 2.</b>: (C=Cut, U=Uncut) Restriction digestion of α1-sbm-ygfG-ygfH (C5-C6-C7-C8-C9) with: EcoRV, Expected bands: 5727bp, 2167bp, 1509bp, 1434bp, 745bp, Positive result: C5-C6-C7-C8-C9</i>]]
 
[[File:T--Thessaly--grand1gel.png|800px|thumb|none|<i><b>Figure 2.</b>: (C=Cut, U=Uncut) Restriction digestion of α1-sbm-ygfG-ygfH (C5-C6-C7-C8-C9) with: EcoRV, Expected bands: 5727bp, 2167bp, 1509bp, 1434bp, 745bp, Positive result: C5-C6-C7-C8-C9</i>]]
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[[File:T--Thessaly--grand2gel.png|800px|thumb|none|<i><b>Figure 3.</b>: (C=Cut, U=Uncut) Restriction digestion of α1-sbm-ygfG-ygfH with: BamHI, Expected bands: 5727bp, 2612bp, 1903bp, 1076bp, 264bp, Positive result: C1-C2-C3-C4-C5</i>]]
  
 
===Experimental Use and Experience===
 
===Experimental Use and Experience===
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This part showed functionality at part <bbpart>BBa_K3866031</bbpart>
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Before we start producing SCFAs, we needed to optimize our protein expression system.
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[[File:T--Thessaly--SDSPR.png|850px|thumb|none|<i><b>Figure 3:</b> SDS page (L=Lysate S=Souble I=Insoluble)(0%, 0,1%, 1% L-arabinose inducer)
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Expected bands: sbm 77.8 kDa, ygfH 53.8 kDa & ygfG 29.17 kDa</i>]]
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The three enzymes are not being produced in a desired way. The two large enzymes, sbm and ygfH, are not being produced at all, even after a relatively "strong" induction with 1% arabinose. As for the third and smaller enzyme, ygfG, we see that we have an overproduction, not only at low levels of arabinose (at 0.1%), but there is also a big amount of enzyme at the insoluble where it shouldn't be. This means that we should opt to test a series of different combinations of promoters and RBSs, in order to achieve optimal production of all enzymes; a stronger expressing combination of promoter and RBS for sbm and ygfH and a less powerful promoter and RBS for the ygfG.
  
 
===Sequence and Features===
 
===Sequence and Features===

Latest revision as of 22:30, 19 October 2021


Propionate Production Construct

Usage and Biology

This composite part consists of the following two Composite Parts:

sbm:stuffer: BBa_K3866029 & ygfG:ygfH: BBa_K3866030

Design Notes

The coding sequences were domesticated. We removed BsmBI and BsaI sites in order to be compatible with GoldenBraid and MoClo. The sequences are cloned in seva ω2 vector and have overhangs compatible for GoldenBraid cloning.

Figure 1. The ω module of the Propionate Production: α1:ParaBAD:RBS-sbm-Double terminator:ParaBAD:RBS-ygfG-Double terminator:ParaBAD:RBS-ygfH-Double terminator

Verification of Cloning

Figure 2.: (C=Cut, U=Uncut) Restriction digestion of α1-sbm-ygfG-ygfH (C5-C6-C7-C8-C9) with: EcoRV, Expected bands: 5727bp, 2167bp, 1509bp, 1434bp, 745bp, Positive result: C5-C6-C7-C8-C9
Figure 3.: (C=Cut, U=Uncut) Restriction digestion of α1-sbm-ygfG-ygfH with: BamHI, Expected bands: 5727bp, 2612bp, 1903bp, 1076bp, 264bp, Positive result: C1-C2-C3-C4-C5

Experimental Use and Experience

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

Figure 3: SDS page (L=Lysate S=Souble I=Insoluble)(0%, 0,1%, 1% L-arabinose inducer) Expected bands: sbm 77.8 kDa, ygfH 53.8 kDa & ygfG 29.17 kDa

The three enzymes are not being produced in a desired way. The two large enzymes, sbm and ygfH, are not being produced at all, even after a relatively "strong" induction with 1% arabinose. As for the third and smaller enzyme, ygfG, we see that we have an overproduction, not only at low levels of arabinose (at 0.1%), but there is also a big amount of enzyme at the insoluble where it shouldn't be. This means that we should opt to test a series of different combinations of promoters and RBSs, in order to achieve optimal production of all enzymes; a stronger expressing combination of promoter and RBS for sbm and ygfH and a less powerful promoter and RBS for the ygfG.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4655
    Illegal BamHI site found at 1148
    Illegal BamHI site found at 1424
    Illegal BamHI site found at 3343
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 983
    Illegal AgeI site found at 3178
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 965
    Illegal SapI site found at 3160
    Illegal SapI.rc site found at 1883


References

Akawi L, Srirangan K, Liu X, Moo-Young M, Perry Chou C. Engineering Escherichia coli for high-level production of propionate. J Ind Microbiol Biotechnol. 2015 Jul;42(7):1057-72. https://doi.org/10.1007/s10295-015-1627-4