Difference between revisions of "Part:BBa K1655000"

Revision as of 16:35, 11 October 2023

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

Contribution From NJTech-China-B 2023

Group:iGEM NJTech-China-B

Author:Qiming Zou

We are trying to expand the application scenarios of BioBrick BBa_K1655000 and improve its ability to work more efficiently by using optimised related components. The existing composite part BBa_K1655000 was first registered in 2015 by iGEM15_Aalto-Helsinki team. It includes three enzymes of acyl-CoA thioester hydrolase (YciA) from Haemophilus influenzae, Carboxylic acid reductase (CAR) from Mycobacterium marinum and maturation factor phosphopantetheinyl transferase (sfp) from Bacillus subtilis for CAR activation. All coding regions are assembled as an operon after a T7 promoter and RBS from common cloning vectors. It was demonstrated as a part to produce propane in E. coli. We hope to use the part for 1,5-PDO production, and also improve its activity.