Difference between revisions of "Part:BBa K3866031"
(→‎Experimental Use and Experience)
Line 20: Line 20:
 
===Experimental Use and Experience===
 
===Experimental Use and Experience===
 
Before we start producing SCFAs, we needed to optimize our protein expression system.
 
Before we start producing SCFAs, we needed to optimize our protein expression system.
−
[[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)
+
[[File:T--Thessaly--SDSPR.png|700px|thumb|none|<i><b>Fig.3:</b>: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</i>]]
+
Expected bands: sbm 85.6 kDa, ygfH 53.8 kDa & ygfG 29.17 kDa</i>]]
  
 
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.
 
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.

Revision as of 15:56, 6 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.

Fig.3::SDS page (L=Lysate S=Souble I=Insoluble)(0%, 0,1%, 1% L-arabinose inducer) Expected bands: sbm 85.6 kDa, ygfH 53.8 kDa & ygfG 29.17 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
    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