Markerless Genome Editing in Bacillus subtilis Guo Wu and Kang Wu Department of Chemical Engineering, University of New Hampshire

Abstract Protein display on B. subtilis spore surface

Bacillus subtilis is the model organism for studying Gram-positive bacteria as well as cell biology including sporulation, bacterial chromosome replication, cell development and differentiation. It is also one of the most advanced model organisms for systems biology study and host for synthetic modules. However, genetic engineering of B. subtilis is very inefficient and time-consuming, relying on E. coli-B. subtilis shuttle vectors. In addition, a resistance marker is often created for either gene knockin or knockout. The limitation of available resistance markers and long turnover time significantly impede the research related to B. subtilis. In this work, we developed an E. coli independent method for marker-less genome editing. Four pieces of DNA for deletion or five pieces for insertion are Fig. 1. Sporulating B. subtilis and protein display on B. subtilis spore surface. assembled using Gibson assembly and then used to transform B. subtilis. Double crossover homologous recombination followed by linearizing the chromosome at a designed I-sceI site will force the removal of the Advantages of displaying proteins on B. subtilis spore surface Applications resistance marker. We have demonstrated this method by deleting the amyE gene or integrating a gfp expression • GRAS bacterium and safe for food and drug processing. • Oral Vaccine delivery: (HA2) universal flu vaccine operon into the amyE site. The development of the E. coli independent marker-less gene replacement method will • Enhanced robustness • Drug delivery: (PAL) treating metabolic diseases greatly facilitate the genome editing in B. subtilis and expedite relevant research including cell biology study, strain • No protein purification is needed. • Bioremediation: (PbrR) heavy metal removal development, and system/synthetic study. • Immobilized enzymes allow for multiple-cycle catalysis. • Biodegradation: (PETase, MHETase) plastic degradation • Biosynthesis: In vitro metabolic engineering

Markerless Gene Knockout: 1 2 3 1 3 Design by Janes and Stibitz, 2006 Original design Improved design

P1 P3 P5 P7 P1 P3 P5 P7 1 2 3 PCR 2 cmR 1 3 2 cmR I-SceI IacI 1 3 1 3 P2 P4 P6 P8 P2 P4 P6 P8

ts ori cmR DNA assembly 2 cmR 1 3 2 cmR I-SceI IacI 1 3 I-SceI cutting site, 18 nt Shift to 37 °C to force recombination 1 2 3 1 2 3 1 3 cmR ts ori 1 2 3

Introduce the enzyme I-SceI 1 2 cmR 1 3 1 2 cmR I-SceI IacI 1 3 1 3 cmR Introduce the enzyme I-SceI Add inducer to produce I-SceI A B ts ori 1 2 3 1 2 1 2

Recombination at A or B cmR 1 3 cmR I-SceI IacI 1 3 1 2 3 A Recombination Recombination

1 3 1 3 1 3 B

Conclusion Future Work • The success rate increases from 50% to 100%. • Further increase the efficiency of assembly and transformation. • No need to do cloning in E. coli. • Use CRISPR/Cas9 to replace I-SceI • Only need one transformation of DNA.