Team:Heidelberg/Part Collection

Discover our Part Collection
Simple Assembly of Accessory Plasmids
We present the first self-contained in vivo evolution toolbox comprising 26 standardized BioBricks. The collection includes several key aspects of our project:

The crucial step of to the cloning of PACE circuits is the generation of the accessory plasmid. These plasmids allow geneIII expression dependent on the evolving protein. The link between the fitness of the protein of interest and the expression of geneIII determines the effectiveness of the directed evolution and the presence of ProteinIII is essential for the production of new phage particles.
Therefore, we provide an accessory plasmid construction kit for phage library selection and propagation in E. coli based on conditional geneIII complementation. The geneIII-driving promotor, RBS and plasmid ori can thereby be fully customized.
If more than one variant of a circuit should be tested at a time, it is necessary to modify the activation region for geneIII and the additional gene that can be located on the AP with a minimum of effort. This is the another reason for a efficient cloning strategy.
To make AP cloning as simple as possible, we defined a new cloning standard that is specifically suited for the assembly of APs, we wrote a BBF RFC that describes our concept in detail (Fig. 1).
Figure 1: In our cloning standard, compatible building blocks are defined by specific functionalities. They are flanked by defined homology regions, indicated by numbers, which are necessary for the assembly of the APs with the Gibson method. This results in a highly customizable plasmid, composed of the desired origin of replication, an antibiotic resistance (4-5), a bicistronic operon with geneIII (2-3)and the desired reporter (3-4), which can be activated by any promoter (1-2)and a second expression cassette for additional genes that are necessary for the respective circuit (1-5).

In Addition, an optogenetic selection stringency modulator comprising the light-dependent EL222 transcription factor and an engineered, hybrid Psp-EL222 promoter.
Furthermore, we provide parts for production of geneIII-deficient M13 phages carrying a custom gene-of-interest to be evolved.

On order to validate our system, we constructed the plasmids for our CRISPR/Cas9 subproject according to the RFC cloning standard. We could constuct several plasmids with help of this standard.

At the heart of our toolbox is an innovative workflow for enzyme engineering, paving the way towards bio-production of novel compounds. It comprises promiscuous enzyme candidates (CYP1A2 and Cytochrome c) and selection-mediating accessory parts such as our riboswitch for detection of an organosilicon (BBa_K2398555). Our simple PREDCEL protocol and accompanying online toolbox guide bring in vivo evolution to future iGEM teams and the broader synbio community.

Finally we created a set of parts, which were used in our different subprojects. These parts were transferred into pSB1C3 in a way that they are still compatible with our standard and submitted them to the registry: