An expanded genetic toolbox to accelerate the creation ofAcholeplasma laidlawiidriven by synthetic genomes

Abstract

Assembling synthetic bacterial genomes in yeast and genome transplantation has enabled an unmatched level of bacterial strain engineering, giving rise to cells with minimal and chemically synthetic genomes. However, this technology is currently limited to members of the Spiroplasma phylogenetic group, mostly Mycoplasmas, within the Mollicute class. Here, we propose the development of these technologies for Acholeplasma laidlawii, which is phylogenetically distant from Mycoplasmas and, unlike most Mollicutes, uses a standard genetic code. Towards this goal, we first investigated a donor-recipient relationship between two A. laidlawii strains through whole-genome sequencing. We then created a multi-host shuttle plasmid and used it to optimize an electroporation protocol. For genome transplantation, we selected A. laidlawii 8195 as the recipient strain and we created a PG-8A donor strain by inserting a Tn5 transposon carrying a tetracycline resistance gene. Our optimized genetic tools will accelerate the creation of Acholeplasma strains driven by synthetic genomes.

Deletion of HindIIR and HindIIIR improves DNA transfer via electroporation toHaemophilus influenzaeRd

“…All ΔHindII/III transformants tested produced the expected banding pattern whereas for WT, 1/10 pSU20 transformants (#5, Figure 1B) and 2/10 pHflu4 transformants (#7 and #9, Figure 1B) did not yield the expected digest products (for additional analysis see Supplementary Figure S4). Interestingly while wild-type H. influenzae did produce some transformant colonies, substantial background growth/biofilm was observed on these plates as early as 24 S5). This background growth was reduced when smaller volumes of recovered cells were plated (Supplementary Figure S5).…”

“…Future work could examine if ΔHindII/III similarly improves plasmid transformation by other methods, such as chemical transformation. In addition, the pHflu plasmid series could be used to evolve ΔHindII/III for greater electroporation efficiency as has been done for Acholeplasma laidlawii [24]. The electroporation method and genetic tools herein will promote exploration of H. influenzae as a synthetic biology chassis organism.…”

“…forest CP and fuGFP were obtained by PCR-amplifying fluorophores from plasmids a gift from Binomica Labs (binomicalabs.org) using primers SH19-20 to afford pHflu6-7. tetM, amplified from pAL1 using SH21-22, was likewise inserted into linearized pHflu1 to create pHflu8 [24]. pHflu4 was assembled using pHflu1 linearized by primers SH15-16 and mRFP amplified by primers SH17-18 from a plasmid obtained from Dr. Thomas Hamilton.…”