Part:BBa_K1655000
Propane 1 codes three of the ten enzymes necessary to produce propane in Escherichia coli.
Propane 1 includes 1)acyl-CoA thioester hydrolase (YciA) from Haemophilus influenzae, which catalyzes the reaction: acyl-CoA + H2O <-> CoA + a carboxylate, 2) Carboxylic acid reductase (CAR) from Mycobacterium marinum (strain ATCC BAA-535 / M), which catalyzes the reaction: a carboxylate + reduced acceptor <-> an aldehyde + acceptor + H2O, and 3) maturation factor phosphopantetheinyl transferase (sfp) from Bacillus subtilis, which activates CAR by catalyzing the transfer of a prosthetic phosphopanteine group to CAR.
All coding regions are assembled as an operon after a T7 promoter and RBS's from common cloning vectors (such as pCDF-Duet1 by Novagen). Terminator sequence after the last coding sequence, CAR, is also derived from common cloning vectors (e.g. pCDF-Duet1 by Novagen uses this terminator).
All coding sequences have been codon optimized twice for E. coli. First with Thermo Fisher's codon optimizer and then with IDT's codon optimization software.
Propane 1 can be used as a part to produce propane in E. coli. The propane production pathway is shown below in figure 1, and the enzymes included in Propane 1 are highlighted in blue.
The plasmid has been assembled from IDT's gBlocks with NEBuilder assembly, similar to Gibson Assembly.
Validation: We restricted our Propane 1 and ran the insert on an agarose gel. From the picture we can tell that the insert's size is correct. The result can be seen in Figure 2.
Additionally, we did a colony PCR with VR and our primer P001. The VR primer attaches to our plasmid's backbone while P001 anneals with the very beginning of our construct. With this colony PCR we were able to show that the insert is present and it is indeed in the pSB1C3 backbone. See figure 3 for results, where the product in wells 1, and 5-10 is of the right size.
The colony which produced the product seen in figure 3, well 10 was sent to the registry.
Click here to download the full sequence of Propane 1 in pSB1C3 backbone.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2902
Illegal BamHI site found at 573
Illegal BamHI site found at 1271
Illegal BamHI site found at 3791
Illegal BamHI site found at 3929
Illegal XhoI site found at 3690 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1379
Illegal NgoMIV site found at 3608 - 1000COMPATIBLE WITH RFC[1000]
Contribution From NJTech-China-B 2023
Group:iGEM NJTech-China-B
Author:Qiming Zou
The biobrick BBa_K1655000 contains a acyl-CoA thioester hydrolase (YciA) from Haemophilus influenzae, a carboxylate reductase (CAR) from Mycobacterium marinum and its maturation factor phosphopantetheinyl transferase (sfp) from Bacillus subtilis. However, all genes are operated by a T7 promoter, which limited the application scenarios of the part.
Unfortunately, the chassis of E. coli NT1003 we engineered for 1,5-PDO production is a strain that does not carry the lambda DE3 lysogen, so we optimized the part in several ways and created a new part BBa_4800008.
We suppose that replacing T7 promoter with trc promoter expanded the application scenarios of BioBrick BBa_K1655000 on 1,5-PDO production.To further improve its ability to work more efficiently, we employed the mutation of carboxylate reductase and the immobilization by a self-assembling protein scaffold system. We evaluate its activity towards the bioconversion of 5-hydroxyvalerate to 1,5-PDO by a whole-cell process, comparing to the the wild type control.
In summary, we successfully designed and obtained a new part BBa_K4800008 on the basis of the original part BBa_K1655000. Various of data proofed that our part do has a obvious improvement and success.
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