Abstract
One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.
Highlights
Biological engineering faces unique challenges that must be addressed before it will be as rapid, reliable and robust as traditional engineering disciplines [1,2]
Robust protocols for engineering the genome of ADP1 typically require assembling linear double-stranded DNA cassettes with at least 500 bp of flanking genomic homology added on each end, and using even longer flanking homology regions further increases the efficiency of transformation [53,54]
The PCR product is added to a growing culture of ADP1, allowing DNA uptake and homologous recombination to replace the native sequence between the homology regions
Summary
Biological engineering faces unique challenges that must be addressed before it will be as rapid, reliable and robust as traditional engineering disciplines [1,2]. The main approaches to genome simplification fall into two categories: a bottomup approach in which DNA pieces are assembled into a minimal genome that is tested to see if it can successfully ‘boot up’ self-replication in a host cell and a top-down approach in which chromosomal regions found to be dispensable are removed in a step-wise fashion to create a streamlined genome. These efforts have led to more efficient and reliable microbial cell factories and blurred the line between chemical and living systems [5,6,7,8]
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