Part:BBa_K2398024

Hybrid phage shock- tet operator promotor for application in Phage-assisted continous evolution (PAC

This part provides a phage shock promoter with an engineered tet-operator binding site, flanked by two homology regions for the usage due to the cloning standard of the iGEM Team Heidelberg 2017 (http://2017.igem.org/Team:Heidelberg/RFC). Figure one gives a short overview of our standard. Our BioBricks from the registry can easily be used for the assembly of blasmid with the standard (Fig.: 2).

The part was designed on the basis of the constructs designed by Hubbard etal.[1]. It gets activated upon phage infection and is in addition regulated by the tet-operator

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).

Figure 2: Compatibility of our cloning stadard with the RFC10;Any AP building block can be cloned into RFC[10] standard by inserting BglII sites between the homology regions and the biobrick prefix or suffix, respectively. To use such a part for AP assembly, it has to be digested with BglII. The resulting fragment should be purified and can subsequently used for Gibson assembly with other parts.

References

[1]Hubbard, Basil P.; Badran, Ahmed H.; Zuris, John A.; Guilinger, John P.; Davis, Kevin M.; Chen, Liwei et al. (2015): Continuous directed evolution of DNA-binding proteins to improve TALEN specificity. In: Nature methods 12 (10), S. 939–942. DOI: 10.1038/nmeth.3515.