Abstract
Bacillus subtilis is a model organism for Gram-positive bacteria and widely used in the study of cellular functions and processes including protein secretion, sporulation, and signal transduction. It is also an important industrial host for the production of proteins and chemicals. Generally, genome editing of B. subtilis often needs the construction of integration vectors in Escherichia coli, linearizing the constructed plasmids, and subsequent transformation of the linear deoxyribonucleic acid via natural competence or electroporation. In this work, we examined the feasibility to directly transform and integrate B. subtilis using linear deoxyribonucleic acid from Gibson assembly without the need for cloning in E. coli. Linear deoxyribonucleic acid of 8–10 kb showed the highest transformation efficiency which was similar to that of using linearized plasmids constructed in E. coli. This method shortens the overall process from 1 week to 1 day and allows the integration of multiple genes in one step, providing a simple and fast method for genome editing in B. subtilis.
| Original language | English |
|---|---|
| Pages (from-to) | 471-477 |
| Number of pages | 7 |
| Journal | Engineering in Life Sciences |
| Volume | 19 |
| Issue number | 6 |
| DOIs | |
| State | Published - Jun 2019 |
| Externally published | Yes |
Funding
This work is supported by the research funds from the University of New Hampshire. The authors also wish to acknowledge the Bacillus Genetic Stock Center for providing the integration vectors.
Keywords
- DNA assembly
- gene knockout/knockin
- genome editing mutant libraries
- transformation efficiency