Neb 5 alpha competent e coli

Plasmids expressing RFP-WT Tmod2 and Tmod2 mutants (ED and A2) were constructed by moving the gene, which encodes Tmod2 (with and without the mutations), from ClFP-Tmod2 constructs to pCAGGS destination vectors containing N-terminal RFP, using two-step Gateway cloning protocol [31 (link)] provided in the manual (ThermoFisher Scientific, Waltham, MA, USA). For this, attb sites were added to the insert using primers:

Tmod2_attb1 forward:

5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGCTCCCCTTTCAAAAAGG-3′

Tmod2_attb2 reverse:

5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACCTCCTGTCTCCTTCAACTC-3′

Tmod2 A2_attb2 reverse:

5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAAACTCTCTTCTTTCGAACCAGG-3′

Sequence-verified constructs were transformed into NEB 5-alpha-competent E. coli (New England Biolabs, Ipswich, MA, USA) cells for amplification. To obtain transfection-grade plasmid DNA, transformed E. coli cells were grown in 250 mL culture flasks, and plasmids were isolated using PureLinkTM HiPure Plasmid Midiprep Kit (ThermoFisher Scientific, Waltham, MA, USA).

Plasmids were constructed using Gateway Technology (Invitrogen). Target gene promoters were PCR amplified from genomic DNA using Phusion polymerase (Thermo Scientific), gel purified, A-overhangs added by incubating with Taq RED Master Mix Kit (Apex Bioresearch Products), cloned into the pCR8/GW/TOPO vector (Thermo Scientific) following the manufacturer’s specifications then transformed into NEB 5-alpha Competent E. coli (New England BioLabs). Plasmid digestion was used to confirm the forward orientation of the amplicon. Promoters were recombined into the promoterless Gateway GFP expression vector pCZGY32 vector (a kind gift from Yishi Jin, UC San Diego) using Gateway LR Clonase II Enzyme Mix (Invitrogen) following the vendor’s instructions and transformed into NEB 5-alpha Competent E. coli. Plasmid digestion was used to confirm identity. Constructs were microinjected into the germlines of either wild type or stIs10055[ cnd-1p::his-24::mCherry unc-119(+)] worms at 20ng/μL in conjunction with either unc-122::GFP (coelomocyte::GFP) or ttx-3p::RFP co-injection plasmids and pBlueScript II to a final concentration of 50ng/μL. F1 hermaphrodites that expressed the co-injection marker were single plated and screened to obtain stable lines.

To inactivate mouse KGDH, sgRNAs were cloned into LentiCRISPR2 vector (Addgene) using the GoldenGate protocol:^{68 (link)}- KGDH_1: Fw 5′-caccgCAGCATCCAAAATCCCCAG-3′;
Rv 5′-aaacCTGGGGATTTTGGATGCTGc-3′;
- KGDH_2: Fw 5′-caccgGTGAACTGCATGATCCCAG-3′;
Rv 5′-aaacCTGGGATCATGCAGTTCACc-3′;
Specific CRISPR/Cas9 transfection was compared to a non-targeting control (NTC) sgRNA:
- NTC: Fw 5′-caccgTTCCGGGCTAACAAGTCCT-3′;
Rv 5′-aaacAGGACTTGTTAGCCCGGAAc-3′.
The obtained plasmids were transformed into NEB 5-alpha Competent E. coli (High Efficiency –New England Biolabs), then DNA was purified by EZNA Fastfilter Endo-Free Plasmid DNA Maxi Kit (Omega Bio-Tek), and a concentration of 6 μg was used for transfection in 4T1 cells; the transfection was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. All experiments were performed by using the polyclonal population emerging after a round of puromycin selection showing residual KGDH expression, specifically, after 48 h 4T1 cells were selected by 1.5 μg/mL puromycin recovered from selection and tested for KGDH knock-out by western blot.

ALI1 ORF sequence (At5g14470) was amplified from cDNA of Col-0 Arabidopsis using ALI1-specific primers (Additional file 2: Table S1). Subsequently, ALI1 ORF sequence was supplemented with attB sites using sequence-specific primers containing attB sites (Additional file 2: Table S1). Thereafter, the PCR product was purified using PEG precipitation protocol [56 (link)]. BP clonase recombination reaction was performed to subclone ALI1 into the pDONR207 vector using Gateway® BP Clonase™ II Enzyme Mix following the manufacturer’s protocol. NEB 5-alpha Competent E. coli (New England Biolabs) cells were transformed with the product of recombination reaction using the heat shock method. Colony PCR was performed using DNR3 and DNR5 primers and amplicons were digested with BamHI and ran on a gel for further analysis. Positive constructs were sent for sequencing. LR clonase recombination reaction was performed to insert ALI1 ORF into different destination vectors (pGWB21 or pGWB441) using Gateway™ LR Clonase™ II Enzyme Mix following the manufacturer’s protocol. Different destination vectors were used for the study. For protein expression and purification, pDEST17 was used whereas pGWB441 was used for cellular localization studies.