All fragments needed for cloning of pSW036, pSW039, pSW040, pSW068, pSZT025, pSW071 and pSW072 vectors were amplified using Q5® High-Fidelity DNA Polymerase (New England Biolabs) according to the manufacturer’s protocol. All primers and templates used for PCR amplification are listed in Supplementary Table 4 . PCR products were incubated overnight with DpnI restriction enzyme and purified using an EZ-10 Spin Column PCR Products Purification Kit (BioBasic). Fragments were then ligated using NEBuilder® HiFi DNA Assembly (New England Biolabs) according to manufacturer’s protocol and transformed into competent E. coli cells (Stellar, TaKaRa). A synthetic version of the E. coli thioesterase gene tesA lacking its cognate signal sequence (‘tesA) was codon-optimised for Syn7002 (by GenScript, Hong Kong, Ltd). pAcsA_cpt_YFP and pAcsA_cLac143_YFP37 (link) vector templates for PCR were a kind gift from Prof. Brian Pfleger, University of Wisconsin-Madison, USA and pDF-trc was a kind gift from Prof. Patrik Jones, Imperial College London, UK.
Stellar
Manufactured by Takara Bio
Sourced in United States
The Stellar is a highly sensitive and reliable laboratory instrument designed for DNA and RNA amplification. It utilizes advanced thermal cycling technology to precisely control temperature and time, ensuring consistent and accurate results in various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Mach1, by Thermo Fisher Scientific (2 mentions)
Neb5α, by New England Biolabs (2 mentions)
Nebuilder hifi dna assembly, by New England Biolabs (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Ez 10 spin column pcr products purification kit, by Bio Basic (1 mentions)
E coli top10, by Thermo Fisher Scientific (1 mentions)
Oligonucleotide primer, by Merck Group (1 mentions)
Qiaprep spin and midi kits, by Qiagen (1 mentions)
In fusion hd cloning plus, by Takara Bio (1 mentions)
Cloneamp hifi, by Takara Bio (1 mentions)
Gentra puregene yeast bacteria kit, by Qiagen (1 mentions)
Redtaq, by Thomas Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Geneticin g418, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 medium, by Thermo Fisher Scientific (1 mentions)
Lb medium, by Merck Group (1 mentions)
Hek293f suspension cells, by Thermo Fisher Scientific (1 mentions)
18 protocols using stellar
1
Cloning of Synthetic Vectors in E. coli
2
Routine Cloning and DNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Routine cloning was performed using In-Fusion HD Cloning Plus (Takara Bio USA Inc., Mountain View, CA) according to the manufacturer’s instructions. Plasmids were maintained in E. coli Top10 (Life Technologies, Grand Island, NY) or Stellar (TaKaRa, Mountain View, CA) cells and purified using QIAprep spin and midi kits (Qiagen, Valencia, CA). Bacterial genomic DNA was extracted from L. interrogans using the Gentra Puregene Yeast/Bacteria kit (Qiagen) according to the manufacturer’s recommendations. Routine and high-fidelity PCR amplifications were performed using RedTaq (Denville Scientific, Metuchen, NJ, United States) and CloneAmp HiFi (Takara Bio USA Inc., Mountain View, CA), respectively. DNA sequencing was performed by Genewiz, Inc. (Cambridge, MA). Routine sequence analyses were performed using MacVector (version 17.0.1, MacVector, Inc., Cary, NC, United States). Oligonucleotide primers used in these studies were purchased from Sigma-Aldrich (St. Louis, MO); primer sequences are provided in S6 Table .
3
Culturing E. coli and HEK293 Cells
4
Heterologous expression in E. coli and B. subtilis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmid based on pBAD/His (Invitrogen) or pHT01 (MoBiTech) and pMYCO1 were constructed in E. coli DH5α (Takara) and Stellar™ (Takara), respectively. Heterologous expression based on pBAD/His and pHT01 was done in E. coli LMG194 (Invitrogen) and B. subtilis 168 Marburg (MoBiTech), respectively. The restriction-free M. capricolum subsp.
capricolum mutant strain M. capricolum RE(-) [41] (link) was used as recipient for transformation experiments involving pMYCO1-derived plasmids [45] (link).
capricolum mutant strain M. capricolum RE(-) [41] (link) was used as recipient for transformation experiments involving pMYCO1-derived plasmids [45] (link).
5
Cloning of tuf Promoter-mreB5 Construct
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ef-tu (tuf) promoter region from pSTP1 vector [49] was amplified using primers P1 and P2 (Table S3). The mreB5 gene was amplified from genomic DNA of S. citri GII-3 cells using primers P3 and P4. The amplified products of these two PCRs were purified using a PCR clean-up kit (QIAGEN) and eluted in 30 mL MilliQ water separately. An overlap-extension PCR was set up using the purified PCR products to obtain the tuf promoter-mreB5 DNA fragment. The product DNA was extracted from the gel using a gel extraction kit (QIAGEN) and re-amplified using end primers. The amplified product was cleaned up, digested with restriction enzyme EcoRI and cloned into the pSD4 vector [50] by restriction digestion-ligation method. The ligation reaction was transformed into Stellar TM (Takara Bio) electro-competent E. coli cells by electroporation. The obtained transformants were grown in LB (Luria Bertani) broth supplemented with ampicillin (final concentration 100 mg/mL) and plasmid extracted using a QIAprep miniprep kit (QIAGEN). DNA sequence inserted into the plasmid was sequenced using M13 forward and EV13 primers (Table S3) to confirm the sequence.
6
E. coli Protein Expression Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Detailed methods are provided in the online version of this paper and include the following: E. coli cell strains used in this study for cloning, Stellar TM (Takara Bio) electro-competent E. coli, and for protein expression E. coli BL21 (AI), were grown in LB (Luria Bertani) broth supplemented with ampicillin (final concentration 100 mg/mL) at 37 C.
7
Cell Culture Protocols for MRC-5 and HEK293
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human fibroblasts MRC-5 (Biomerieux, France) were cultivated at 37 °C in 5% CO2 and grown in minimal essential medium (MEM) containing 10% fetal bovine serum with antibiotics. HEK293 (ATCC® CRL-1573™) were cultivated at 37 °C in 5% CO2 and grown in minimal essential medium (MEM) containing 10% fetal bovine serum with antibiotics. E. coli strain DH5α and StellarTM (Clontech, USA) were used for cloning procedures. E. coli strain GS1783 was used for BAC mutagenesis33 (link).
8
Generation of ACLY Mutant Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression vector was generated by inserting the coding sequence of homo sapiens ACLY (Genscript, OHu14076) into the EcoRI linearized pCDH-EF-T2A-MCS-Puro EcoRI vector (System Biosciences, CD527A-1). In brief, the coding sequence of ACLY was amplified by polymerase chain reaction (PCR), elongated with the additional bases of 5’-AGCGAATTCGCCACC-3’ on the 5’ end and 5’-GGATCCTTCGAATTC-3’ on the 3’ end, allowing for subsequent recombination with the vector backbone by In-Fusion® HD cloning (Clontech). Next, the serine to alanine mutant was created by Quickchange site-directed mutagenesis of the TCT into GCT codons using PfuII polymerase (Agilent Technologies). The same mutagenesis approach was employed for the generation of the histidine to alanine (CAT to GCT) mutant of ACLY. This resulted in a phospho-mutant (S455A) and catalytically inactive mutant (H760A) of ACLY, respectively. Plasmids were transformed using heat shock competent E. coli bacteria (StellarTM, ST0213, Clontech).
9
Bacterial Propagation and Selection
Check if the same lab product or an alternative is used in the 5 most similar protocols
Escherichia coli XL1-Blue (Stratagene) or Stellar (Clontech), used for plasmid construction, were propagated on Luria-Bertani (LB; Difco) agar or in LB broth supplemented, when necessary, with 20 µg/ml chloramphenicol (Cm) or 100 µg/ml ampicillin (Amp). A. pleuropneumoniae serovar 8 (UK clinical isolates, including MIDG2331) and serovar 15 (reference strain, HS143) were grown at 37°C in 5% CO2 on brain heart infusion agar (BHI; Difco) supplemented with 0.01% β-nicotinamide adenine dinucleotide (BHI-NAD) or in BHI-NAD broth. When required, 1 µg/ml Cm was added for selection of transformants. For sucrose counter-selection, bacteria were plated onto salt-free LB agar consisting of 10 g tryptone, 5 g yeast extract, and 1.5 g agar per L supplemented with 10% filter-sterilised sucrose (LB-S) for E. coli clones, or onto salt-free LB agar supplemented with 10% sucrose, 10% horse serum and 0.01% NAD (LB-SSN) for A. pleuropneumoniae clones.
+ Expand
Escherichia coli XL1-Blue (Stratagene) or Stellar (Clontech), used for plasmid construction, were propagated on Luria-Bertani (LB; Difco) agar or in LB broth supplemented, when necessary, with 20 µg/ml chloramphenicol (Cm) or 100 µg/ml ampicillin (Amp). A. pleuropneumoniae serovar 8 (UK clinical isolates, including MIDG2331) and serovar 15 (reference strain, HS143) were grown at 37°C in 5% CO2 on brain heart infusion agar (BHI; Difco) supplemented with 0.01% β-nicotinamide adenine dinucleotide (BHI-NAD) or in BHI-NAD broth. When required, 1 µg/ml Cm was added for selection of transformants. For sucrose counter-selection, bacteria were plated onto salt-free LB agar consisting of 10 g tryptone, 5 g yeast extract, and 1.5 g agar per L supplemented with 10% filter-sterilised sucrose (LB-S) for E. coli clones, or onto salt-free LB agar supplemented with 10% sucrose, 10% horse serum and 0.01% NAD (LB-SSN) for A. pleuropneumoniae clones.
10
Fungal Strain Cultivation and Induction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fungal strains used in this study are listed in S21 Table . A. nidulans AGB551, which has a WT veA allele, was used for all deletion and epitope taggings. Stellar (Clontech) and MACH-1 (Invitrogen) competent Escherichia coli cells were used for recombinant DNA preparations. The WT and transformed A. nidulans strains were grown in Glucose Minimal Medium (GMM), supplemented with appropriate amounts of vitamins. For vegetative stage experiments, fungal spores were grown submerged in liquid GMM with 180 rpm rotation for 20 or 48h. For induction of development, vegetative mycelia were then filtered through miracloth and placed on solid GMM with 2% Agar. To induce the cultures asexually, cultures were grown vegetatively for 20h and shifted to the plates and were further incubated in the presence of light for 6, 12, 24h. For sexual induction, plates were covered by aluminium foil and incubated in the dark for 6, 12, 24, 48h. E. coli were grown in LB broth agar or liquid LB with Ampicillin (100 μg/ml) at 37°C overnight.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!