A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction

Abstract

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 augment a mini-Mu transposon-based screening approach and devise the intein-assisted bisection mapping (IBM) method. IBM robustly reveals clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further show that the use of inteins expands functional sequence space for splitting a protein. We also demonstrate the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins, and that basal activities of highly active proteins can be mitigated by splitting them. Our work offers a generalizable and systematic route towards creating split protein-intein fusions for synthetic biology.

Protein Splicing of Inteins: A Powerful Tool in Synthetic Biology

“…Another challenge lied in identifying insertion sites of inteins. Besides sharing the similar difficulties in searching a general split site, the introduction of inteins brought an extra layer of complexity since the splicing efficiency largely depends on the extein junction sequences ( Ho et al, 2021 ). To address this issue, Ho et al used a mini-Mu transposon-based screening approach (intein-assisted bisection mapping (IBM) method) to reveal the split sites for a given protein.…”

“…The clones fulfilling the standard were then sequenced to reveal the split sites at the fusion joints. Using this method, they discovered clusters of split sites on five proteins ( Calles and Lorenzo, 2013 ; Ho et al, 2021 ). Their work established a generalizable methodology to create split protein-intein fusions for synthetic biology.…”

“…TetR or ECF20 were reconstituted only when both the N-lobes and the C-lobes were present under the induction (induced by arabinose and DAPG (2,4-Diacetylphloroglucinol) respectively). The full protein either repressed or activated the expression of mScarlet (output), and effectively generated a NAND (TetR) or AND (ECF 20) logic ( Ho et al, 2021 ).…”

The Future Potential of Biosensors to Investigate the Gut-Brain Axis

“…New studies will be needed to demonstrate how integrated sensing of multiple cues correlates with mental health disease states. Towards the goal of signal integration, there has been success in both protein-based (Ho et al, 2021;Vishweshwaraiah et al, 2021) and transcriptional logic gates to enhance specificity by processing multiple inputs (Cubillos-Ruiz et al, 2021).…”

Efficient gene editing through an intronic selection marker in cells

“…To assist in the retrieval of successfully repaired clones, the target gene coding sequence (CDS) can be tagged with a fluorescent protein at the N- or C-terminus as a selectable marker [ 11 ]. To remove these tags, seamless repair using piggyBac or sleeping beauty is available [ 12 , 13 ]. However, this kind of selection can be used only when the target sites are near the terminus, and it changes the open reading frame (ORF), which may have unpredictable negative effects on genetic regulation.…”