Difference between revisions of "Part:BBa K3866031"

Revision as of 16:10, 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 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

@@ Line 23: / Line 23: @@
 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 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===