Difference between revisions of "Part:BBa K3846050"

Revision as of 17:34, 19 October 2021

modified pAM5411 (phytobrick acceptor backbone)

An important feature of useful golden-gate assembly (MoClo) compatible backbones is the possibility to easily recognise successful integration of fragments inside the vector to avoid the necessity to test multiple clones. iGEM provides the universal acceptor plasmid BBa_P10500 for creating new phytoparts containing lacZ-α for blue-white screening. Substrates for blue-white screening are not cheap and a better way to also reduce costs would be using fluorescent proteins as visible markers as was shown to be feasible by the iGEM Marburg Team 2018 with their improvement of BBa_P10500 the Phytobrick Entry Vector BBa_K2560002 K2560002. In our project, we are using cyanobacteria and wanted to have a universal acceptor plasmid compatible with both species. Therefore, we modified an RFS1010-based broad-host range vector [1]by introducing BsaI and BsmbI restriction sites flanking an eYFP expression cassette to obtain a cyanobacteria compatible fluorescence dropout acceptor plasmid. Successful integration of golden gate assembly fragments leads to the removal of the eYFP coding sequence from the plasmid and it is then possible to distinguish between wrong (yellow/green) and correct (white) colonies. An additional feature of this vector is that it can be used for conjugation of cyanobacteria by triparental mating.

The characterisation of this part is unfortunately still pending.

Figure 1: Plasmid map of BBa_K3846050.

Sequence and Features

iGEM Hamburg 2021 part collection

Terpenoids are an important group of natural products used as biofuels, drugs or fragrances. Naturally occuring in plants it has been shown that microbial terpene production in microorganisms like yeast, E. coli or cyanobacteria is possible. Nevertheless iGEM projects seem to rarely focus on this interesting class of natural products which is correlated with a lack of useful parts inside the iGEM registry.

Fortunately we were able to change that and designed a novel golden gate based toolbox which allows.

1. production of terpenoid precursors and simple terpenoids
2. creation of CYP P450-reductase fusion enzymes to optimise processing of terpenoid precursors and production of bioactive target products
3. modularity of the system to enable exchange of linker sequences/promoters/etc. (MoClo-compatible toolbox)

MoClo-based Part Design 2.0

<p>To improve the usefulness of our parts, we then aimed to make our parts compatible with the MoClo standard of goten gate based IIS restriction enzyme assembly. Thereby we expanded the Common Genetic Syntax for fusion sites to allow the creation of a) fusion proteins connected by linker sequences and b) multiple CDS expressed in an operon. More useful information and an overview of all our parts can be found on our wiki.

Figure 2: MoClo syntax of the part collection.