Xl10 gold

Plasmids from E. coli XL10-Gold (Agilent) cells were extracted and purified with the “NucleoSpin Plasmid” kit (Macherey-Nagel).

The cloning vector pKU6Cas9ccdB (1 μg) was digested and dephosphorylated using Fast Digest SapI (LguI) (Thermo, FD1934) and Alkaline phosphatase (Thermo, EF0654) and ran on a 1% agarose gel followed by purification using agarose gel purification kit (Macherey and Nagel). The Guide RNAs were synthesised from Sigma and reconstituted by mixing 10 μM of the forward and reverse strands for each guide with 1 μl of 10X ligation buffer (NEB), 0.5 μl T4 polynucleotide kinase (NEB) and 6.5 μl nuclease free PCR water. Annealing was carried out by incubating at 37 °C for 30 min, then increasing to 95 °C for 5 min before cooling at 25 °C at a ramp speed of 0.1 °C/sec. Annealed primers were then diluted to 1 μl in 200 μl and ligated to the digested and dephosphorylated pKU6Cas9ccdB (50 ng/μl). Two gRNAs were constructed for knocking out PVP01_0000100 orthologue PKNH_1300500. The details of the reaction mixtures and primers for repair template and gRNA vector assemblies are provided in the Supplementary Table 5.
The assembled pUC19 Repair template and gRNA/Cas9 vector were transformed in E. coli Ultracompetent cells (XL-10 gold, Agilent) and plated onto Amp⁺ LB plates. Colonies positive for the insertion were sequence verified (Genewiz) before preparation using a midiprep purification kit (Macherey and Nagel) for transfection.

Primers for site-directed mutagenesis were designed using Agilent QuikChange Primer design tool. PCR reactions were carried out using QuikChange II Site-Directed Mutagenesis Kit (Agilent, 200523-5) following the manufacturer’s protocol. After PCR, non-mutated template vector was removed from the PCR mixture through digestion for 1 h at 37°C by the enzyme Dpn1. Following digestion by Dpn1, 4 μL of the mixture was transformed into XL10 Gold (Agilent, 200315) chemically competent cells and plated on suitable antibiotic-containing agar plates. Sequencing of purified plasmid DNA established whether the desired mutation had been introduced.

Point mutations were introduced into the pNIC28-Bsa4 and pTT5 constructs using the QuikChange II XL Site-directed Mutagenesis kit (Agilent Technologies) following the manufacturer's instructions. Mutagenesis primers and their reverse sequence were synthesized by Eurofins MWG Operon and TIB MOLBIOL. The generated products were digested with DpnI for 1 h at 37 °C and transformed into XL10-Gold or XL1-Blue competent bacteria (Agilent Technologies). Plasmid DNA was purified from individual colonies as described above. The presence of the desired mutations was confirmed by DNA sequencing (SMB Services in Molecular Biology).
For the fluorescence recovery studies, mutant coding sequences were introduced into pcDNA6.2/N-EmGFP-BRD4 using the megaprimer PCR method (50 (link)) to amplify a BamHI/KpnI-flanked region (BD1) or KpnI/EcoRI-flanked region (BD2) of the wild-type expression plasmid using AccuPrime Pfx (Invitrogen). The expression plasmid and PCR products were digested with the appropriate restriction enzymes (New England Biolabs) followed by gel purification, dephosphorylation of the cut expression plasmid (Antarctic phosphatase, New England Biolabs), and ligation (T4 ligase, New England Biolabs) of the fragments to generate mutant GFP-tagged expression clones.

A previously reported E. coli strain with enhanced amino acid utilization was used for the conversion of proteins into biofuels (Table 1). The derivative strains with gene deletions including gdhA, gltB, gltD, or lsrA were created by using P1 transduction or λ phage recombination. Two adjacent promoters were located upstream of glnA: the σ⁵⁴-dependent glnAp2 and the σ⁷⁰-dependent glnAp1. The latter was located between the two NtrC binding sites at the 5′ end of glnAp2. To eliminate the interference from glnAp1 on σ⁵⁴-mediated transcription, only the − 1 to − 99 region that encompasses the core glnAp2 promoter and its first three NtrC binding sites from the 5′ end was cloned from E. coli MG1655 genomic DNA. Other σ⁵⁴-dependent promoters were also cloned from the genomic DNA and inserted into the 5′ end of the two gene cassettes comprising the biofuel biosynthetic pathway [3 (link)] using Gibson assembly (Additional file 1: Tables S1 and S2). Cloning was carried out using E. coli strain XL10-Gold (Agilent Technologies, Santa Clara, CA, USA). The E. coli strains were routinely cultured in Luria–Bertani (LB) broth or LB agar supplemented with 50 μg mL^− 1 kanamycin or 100 μg mL^− 1 ampicillin.

For each of the variants made in this work a Bio-Rad C1000 Thermal Cycler and the Agilent QuickChange Lightning Site-Directed Mutagenesis Kit were employed. In brief, mutagenesis reactions were prepared on a 50 µL reaction scale and required the addition of 200 ng of the needed plasmid (pET-28a(+)-TEV-tsaM, pMCSG9-pdo, or pMCSG9-vanA) and 125 ng of each oligonucleotide primer (synthesized by IDT). Each DNA and protein sequence is provided in a Supplementary Data 1 File and each primer sequence is supplied in Supplementary Table 2. Following PCR, digestion was accomplished using 2 µL of DpnI, and the reaction mixtures were transformed into either XL10-Gold or JM109 (Agilent) competent cells. Successful mutations were individually verified by Sanger sequencing (Genewiz).