Intein_assisted_Bisection_Mapping
Intein_assisted_Bisection_Mapping_collection Version 1 (Collection)
Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augmented a mini-Mu transposon-based screening approach and devised the intein-assisted bisection mapping (IBM) method. IBM robustly revealed clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further showed that the use of inteins expands functional sequence space for splitting a protein. We also demonstrated the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins. Furthermore, the intein inserted at an identified site could be engineered by the transposon again to become partially chemically inducible, and to some extent enabled post-translational tuning on host protein function. Our work offers a generalizable and systematic route towards creating split protein-intein fusions and conditional inteins for protein activity control.

BBa_I733007
BBa_I733007 Version 1 (Component)
Weight cells either turn blue or die in response to both inputs and HSL level

BBa_I741109
BBa_I741109 Version 1 (Component)
Lambda Or operator region

BBa_K549004
BBa_K549004 Version 1 (Component)
LacI promotor fused with the iron dependent regulator fur

BBa_K228013
BBa_K228013 Version 1 (Component)
(pSal PO) OR Gate - GFP

BBa_K415011
BBa_K415011 Version 1 (Component)
PtetR : RBS : LuxR : Term : PluxR/cI-OR : RBS : mCherry : Term : Plux/cI-OR : RBS : LuxI

BBa_K415005
BBa_K415005 Version 1 (Component)
pLux/cI-OR : RBS-mCherry : Term : p(tetR) : RBS-luxR : Term

SEGA
SEGA_collection Version 1 (Collection)
In the Standardized Genome Architecture (SEGA), genomic integration of DNA fragments is enabled by λ-Red recombineering and so-called landing pads that are a common concept in synthetic biology and typically contain features that i) enable insertion of additional genetic elements and ii) provide well-characterized functional parts such as promoters and genes, and iii) provides insulation against genome context-dependent effects. The SEGA landing pads allow for reusable homology regions and time-efficient construction of parallel genetic designs with a minimal number of reagents and handling steps. SEGA bricks, typically synthetic DNA or PCR fragments, are integrated on the genome simply by combining the two reagents (i.e. competent cells and DNA), followed by incubation steps, and successful recombinants are identified by visual inspection on agar plates. The design of the SEGA standard was heavily influenced by the Standard European Vector Architecture (SEVA). SEGA landing pads typically hosts two major genetic “control elements” that influence gene expression on the transcriptional (C1), and translational (C2) level. Furthermore, landing pads contain gadgets such as selection and counterselection markers.