Xl10 gold cells

Dulbecco’s modified Eagle medium (DMEM),
fetal bovine serum (FBS), l-glutamine, penicillin/streptomycin
(P/S), Opti-MEM I (OMEM), Simple Blue Stain, Novex 4–12% Tris-glycine
SDS-PAGE gels, Novex Sharp Unstained Protein Standard, GeneRuler 1
kb Plus DNA Ladder, LB Medium Dehydrated Capsules, and the Phusion
High-Fidelity DNA Polymerase were all purchased from Thermo Fisher
Scientific. His-tagged Pfu X7 DNA Polymerase was prepared in-house
by immobilized metal affinity chromatography for routine PCR-based
bacterial colony screening. XL10-Gold Ultracompetent cells were acquired
from Agilent Technologies, Inc. Note XL10-Gold cells have a mutation
in the lacI gene promoter (lacI^q), resulting in higher Lac repressor levels.^{57 (link)} SIGMAFAST EDTA-free Protease Inhibitor cocktail
tablets and IPTG were obtained from Sigma-Aldrich. DpnI, TransIT-LT1 transfection reagent, and 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA) were obtained
from New England Biolabs, Mirus Bio, and Cayman Chemicals, respectively.
Specific-pathogen-free (SPF) eggs and turkey red blood cells (TRBCs)
were purchased from Charles River Labs and the Poultry Diagnostic
and Research Center (Athens, GA), respectively. All cloning and screening
primers (Table S1) used in this study were
synthesized by Integrated DNA Technologies.

Ligation reactions
consisting of 1 μL of the PCR insert and vector mixtures were
transformed into 50 μL of XL10-Gold cells per the manufacturer’s
instructions (Agilent) and cultured overnight at 37 °C on LB-agar
ampicillin plates. Plates were imaged with an Azure C600 and 5–10
individual or pooled colonies were randomly selected for growth on
a master plate and for direct colony screening by PCR. For screening,
colonies were resuspended in 1× PCR reaction buffer (RB) (10×
RB: 200 mM Tris-HCl pH 8.8, 100 mM KCl, 60 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1 mg/mL BSA and 1%
Triton) for lysis and the DNA was amplified over 30 cycles using Pfu
X7 DNA polymerase and a primer pair targeting the plasmid (pHW FWD
screening primer) and the specific insert (NA/HA reverse primer).
The amplified DNA was analyzed by agarose gel (0.8%) electrophoresis.
Overnight liquid cultures (LB broth) were used to amplify the positive
clones for additional studies including virus rescue. Plasmid DNA
was isolated using the QIAprep Spin Miniprep Kit (Qiagen), and all
constructs were sequenced prior to use (Macrogen).

As Spiroplasma have an alternative genetic code, coding sequences for MreB isoforms were codon-optimized and fully synthesized by Genewiz (South Plainfield, NJ, USA). Two constructs for each isoform, each with a different codon optimization, were ordered and indifferently used in constructs after controlling that codon optimization was not affecting the polymerization patterns. Plasmid construction (Table S1) was made by or by restriction/ligation or by Gibson Assembly, using primers listed in Table S2. Constructs were built in competent XL10-Gold cells (Agilent) and subsequently transformed in the appropriate strain for experiments.