The open reading frame of human mtPAP (1749 bp) was PCR amplified from WT-LCLs using the following primers: Xba-mtPAP forward (5′-gacTCTAGAATGGCGGTTCCCGGCGTGGGGCTCT-3′) and Mlu-mtPAP reverse (5′-gacACGCGTTCATGTCTGAGTACTAATTGTTCTC-3′) and subcloned into a TA-vector using the manufacture's protocol (Invitrogen) to generate the WT mtPAP plasmid. The resulting product was sequenced to confirm WT mtPAP cDNA sequence. The TA-cloned mtPAP cDNA was then digested with XbaI and MluI restriction enzymes and the purified fragment was ligated into XbaI-MluI-digested (New England Biolabs, Ipswich, MA, USA), SAP-treated (Clontech, Mountain View, CA, USA), lenti 308 plasmid vector containing GFP (UCLA Vector Core, Los Angeles, CA, USA). Once the plasmid was generated, ∼15 × 106 LCLs were transiently transfected with 15 μg of Lenti-mtPAP/GFP (WT mtPAP) or Lenti-GFP control (CTL) expression constructs using electroporation (250 V, 1180 μF) in a Cell-Porator (Invitrogen). Cells were harvested for experiments two days after transfection. Transfection efficiencies were estimated at ∼80% by fluorescent microscopy imaging of GFP-positive cells.
Cell porator
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Cell-Porator is a laboratory instrument designed for the electroporation of cells. Electroporation is a technique used to introduce foreign molecules, such as DNA, RNA, or proteins, into cells by creating temporary pores in the cell membrane. The Cell-Porator provides controlled electric pulses to facilitate this process.
Automatically generated - may contain errors
10 protocols using cell porator
1
Cloning and transfection of human mtPAP
2
Construction of a BAC Library
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A BAC library using pESAC13-Apramycin (Bio S&T, Canada) was constructed. The vector DNA was digested with BamHI. The partially digested high-molecular-weight DNA was size-selected on 1% (w/v) pulsed-field agarose gels in 0.5X TBE on a CHEF DRIII (Bio-Rad, Canada). Partially digested size-selected DNA fragments were ligated to the vector DNA in a volume of 50 μl with 1X ligase buffer and three units of ligase (USB, Canada) at 14°C for overnight incubation. Two microliters of ligation mix were used to transform 20 μl of E. coli DH10B cells (Invitrogen, USA) by electroporation using a CellPorator equipped with a voltage booster (Invitrogen). The cells were then selected on LB medium supplemented with 5% sucrose plus Apramycin by incubation at 37°C overnight. The clones were selected and arrayed in 6 × 384-well plates in a medium containing freezing medium supplemented with 10% (v/v) glycerol and Apramycin. The average insert size was evaluated using randomly selected clones. The miniprep of PAC clones and digestion of PAC DNA with DraI was done according to standard procedure. Pulsed-field gel electrophoresis (PFGE) was conducted to check the insert sizes.
3
Cloning and Expressing Target Gene in P. pastoris
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The full-length target gene was amplified with primers p-1 and p-40 (Table 1 ). Then, the amplified product was treated with T4 DNA polymerase supplemented with 1 mM dTTP to form overhangs compatible with the sticky ends of the pHBM905A vector digested with CpoI and NotI as previously described [33 (link)]. These fragments were ligated and transformed into E. coli XL10-gold to generate recombinant vectors. The recombinant plasmid (10 μg) was linearized with SalI and transformed into P. pastoris GS115 by electroporation (7000 V/cm, 25 μF, 400 × Bio-Rad Gene Pulser, USA) using the Life Technologies Cell Porator (USA). The positive transformants were selected on MD plates and confirmed by colony PCR.
4
Electroporation of Jurkat, J1.1, CHME5/HIV, and U1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Cell-Porator™ [Life Technologies, Inc; Bethesda Research Labs (BRL)] was used to transfect cells per the manufacturer’s instructions. Briefly, Jurkat, J1.1, CHME5/HIV, and U1 cell lines were electroporated in RPMI 1640 media containing 10% FBS and 5% l -glutamine. The cell lines were transfected with DNA (20 µg) at the following parameters: a capacitance of 800 µF, low resistance, pulse voltage of 230 V for cell lines and 210 V for primary cells, and fast charge rate.
5
Constructing Deletions and Complementing Mutations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Deletion mutants (Supplementary Table S7 ) were constructed according to the method described by Datsenko and Wanner55 (link) using the primer sets listed in Supplementary Table S6 to amplify a FRT-flanked-CmR-gene with homology extensions to the respective gene region to be deleted. After selecting the CmR-positive deletion mutants, the CmR gene was eliminated using the helper plasmid pCP2055 (link). After the CmR-gene was eliminated, the strains were tested for the correct gene deletion by Sanger sequencing using the primer sets indicated in Supplementary Table S6 . To transfer the pAFP12-05829 plasmid into a recipient strain, a transposase located on the plasmid was replaced by a CmR gene as mentioned above. The pAFP12-05829 Δtransposase::CmR plasmid was purified and introduced into the K12 C600 recipient strain by electroporation with a Life Technologies Cell-Porator according to the manufacturer’s specifications as previously described56 (link). After elimination of the CmR gene, the afp deletion was introduced, resulting in K12 C600 (pAFP12-05829 Δtransposase Δafp::CmR). To complement the afpA deletion, the afp operon of strain 12-05829 was cloned into pBeloBac11 via Gibson Assembly using the primer set indicated in Supplementary Table S6 57 (link).
6
Molecular Cloning and Bioinformatics Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
E. coli BL21 was used for the propagation of recombinant plasmid DNA with backbones of the following vectors: pGP17251 (link) and pet28a(+) (Novagen). Genomic and plasmid DNA were prepared, amplified, and sequenced according to standard protocols. Primers were obtained from Eurofins MWG Operon. Restriction enzymes were purchased from New England Biolabs. Foreign DNA was introduced into E. coli strains by electroporation with a Life Technologies cell porator according to the manufacturer’s specifications as described earlier28 (link). Nucleotide and translated protein sequences were analyzed using the Geneious 10.0.0552 (link), the NCBI website (http://www.ncbi.nlm.nih.gov/ ), DiANNA 1.1 (http://clavius.bc.edu/~clotelab/DiANNA/ ), SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/ ) and ExPASy (http://www.expasy.ch ).
+ Expand
E. coli BL21 was used for the propagation of recombinant plasmid DNA with backbones of the following vectors: pGP17251 (link) and pet28a(+) (Novagen). Genomic and plasmid DNA were prepared, amplified, and sequenced according to standard protocols. Primers were obtained from Eurofins MWG Operon. Restriction enzymes were purchased from New England Biolabs. Foreign DNA was introduced into E. coli strains by electroporation with a Life Technologies cell porator according to the manufacturer’s specifications as described earlier28 (link). Nucleotide and translated protein sequences were analyzed using the Geneious 10.0.0552 (link), the NCBI website (
7
Optimized PETase Expression in Pichia pastoris
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IsPETase coding sequence including the signal peptide region was optimized based on the usage preference of P. pastoris and cloned into pHBM905BDM vector for secretory expression (Supplementary Table 1 , pHBM905BDM-IsPETase, Addgene ID 195561). The multicopy expression vectors bearing 2, 3, and 4 copies of the IsPETase expression cassettes were constructed with the bio-brick method as previously reported36 (link). To express Fast-PETase (Supplementary Table 1 ), the ORF of Fast-PETase generated through overlapping PCR with IsPETase ORF as the template, followed by cloning into pHBM905BDM (pHBM905BDM-Fast-IsPETase, Addgene ID 195561). To generate the recombinant P. pastoris strains, the recombinant plasmids were linearized with SalI and transformed into P. pastoris GS115 by electroporation (7000 V/cm, 25 mF, 400 Ω; Life technologies cell porator, USA). Transformants were selected on MD plates (without histidine). The recombinants were identified with whole-cell PCR.
8
Optimized Production of Human Lysozyme in P. pastoris
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The constructed p905M-opt-hLYZ-1C, p905M-opt-hLYZ-3C, p905M-opt-hLYZ-6C, and p905M-opt-hLYZ-12C plasmids were linearized by SalI, followed by transformation into P. pastoris GS115 through electroporation (Life Technologies Cell Porator, Carlsbad, CA, United States), which generated the recombinant strains of opt-hLYZ-1C, opt-hLYZ-3C, opt-hLYZ-6C, and opt-hLYZ-12C, respectively. The positive transformants were selected on MD plates without histidine and confirmed by colony PCR. Subsequently, the plasmids containing different molecular chaperones, including pGAZB-Bip, pGAPZB-Ero1, pGAPZB-Pdi1, pGAPZM-Bip-(GAP∗)-Ero1, pGAPZM-Bip-(GAP∗)-Pdi1, and pGAPZM-Ero1-(GAP∗)-Pdi1, were linearized by AvrII and transformed into the opt-hLYZ-6C competent cells, generating recombinant strains of opt-hLYZ-6C-B, opt-hLYZ-6C-E, opt-hLYZ-6C-P, opt-hLYZ-6C-BE, opt-hLYZ-6C-BP, and opt-hLYZ-6C-EP, respectively. These strains were screened on YPDZ plates with Zeocin antibiotics (100 μg/mL).
9
Heterologous Protein Expression in Pichia pastoris
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The recombinant plasmids were linearized using SalI and transformed into P. pastoris GS115 by electroporation (7000 V/cm, 25 μF, 400×; Life Technologies Cell-Porator, USA). Transformants were selected on MD plates without histidine, followed by identification on BMMY plates supplemented with 1% casein [29 (link), 30 (link)].
10
Transfecting HEK293T Cells via Electroporation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Cell-Porator™ [Life Technologies, Inc.; Bethesda Research Labs (BRL), Frederick, MD, USA] was used to transfect HEK293T cells per the manufacturer’s instructions. Briefly, HEK293T cells (5 × 106 cells per sample) were electroporated in DMEM media containing 10% FBS and 5% L-glutamine. The cell lines were transfected with DNA (20 µg) at the following parameters: a capacitance of 800 µF, low resistance, a pulse voltage of 230 V, and a fast charge rate.
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!