Journal: ACS Synthetic Biology

Article Title: Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants

doi: 10.1021/acssynbio.3c00355

Figure Lengend Snippet: Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic DNA. (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.

Article Snippet: The following plasmids were procured from Addgene and used as template DNA molecules to create the plasmid constructs used in this work: 1066 pBabe puroL PTEN was a gift from William Sellers (Addgene plasmid # 10785; http://n2t.net/addgene:10785 ; RRID:Addgene_10785); pCDNA3_UnaG-Flag-Sec61B was a gift from Hyun-Woo Rhee (Addgene plasmid # 83413; http://n2t.net/addgene:83413 ; RRID:Addgene_83413); pLEX-MCS-ASC-GFP was a gift from Christian Stehlik (Addgene plasmid # 73957; http://n2t.net/addgene:73957 ; RRID:Addgene_73957); pcDNA3.1(+)_SpyCatcher-6aa-sfCherry2(1–10) was a gift from Bo Huang (Addgene plasmid # 117484; http://n2t.net/addgene:117484 ; RRID:Addgene_117484); gRNA_Cloning Vector was a gift from George Church (Addgene plasmid # 41824; http://n2t.net/addgene:41824 ; RRID:Addgene_41824); pSFFV_sfCherry3C(1–10) was a gift from Bo Huang (Addgene plasmid # 117482; http://n2t.net/addgene:117482 ; RRID:Addgene_117482); pGP-CMV-jGCaMP7c variant 1513 was a gift from Douglas Kim & GENIE Project (Addgene plasmid # 105320; http://n2t.net/addgene:105320 ; RRID:Addgene_105320); and pLNCX2-STIM1 was a gift from Shengyu Yang (Addgene plasmid # 89817; http://n2t.net/addgene:89817 ; RRID:Addgene_89817).

Techniques: Sequencing, Plasmid Preparation, Generated, Construct, Control, Flow Cytometry, Fluorescence, Transfection, Illumina Sequencing, Amplification, Binding Assay, DNA Extraction, Transgenic Assay, Derivative Assay, Virus

Journal: Microorganisms

Article Title: Emulsion PCR (ePCR) as a Tool to Improve the Power of DGGE Analysis for Microbial Population Studies

doi: 10.3390/microorganisms8081099

Figure Lengend Snippet: Denaturing gradient gel electrophoresis (DGGE) profiles of mixtures of amplicons obtained from DNA extracted from lactic acid bacteria (A) and DNA extracted directly from bulk-collected coagulase-negative catalase-positive cocci (CNCPC) cells. Panel A: Lane 1, Lc. lactis ; lane 2, Leuc. mesenteroides ; lane 3, P. pentosaceus ; lane 4, Lb. sakei ; lane 5, ePCR (mix 1); lane 6, not emulsified control (NEC), non-emulsified control (mix 1); lane 7, conventional PCR (mix 1); lane 8, ePCR (mix 2); lane 9, NEC, non-emulsified control (mix 2); lane 10, conventional PCR (mix 2). Mix 1: DNA of Lactobacillus sakei (DSMZ 6333), Lactococcus lactis (DSMZ 20481), Leuconostoc mesenteroides (DSMZ 20241) and Pediococcus pentosaceus (DSM 20336) at the same concentration (100 ng·µL −1 ). Mix 2: DNA of the lactic acid bacteria was added at different concentrations: 500 ng·µL −1 Lb. sakei , 300 ng·µL −1 Lc. lactis , 150 ng·µL −1 Leuc. mesenteroides and 50 ng·µL −1 P. pentosaceus . Panel B: PCR, conventional PCR; ePCR, emulsion PCR; NEC, non-emulsified control; 10 −2 , 10 −3 , 10 −4 , 10 −5 , 10 −6 , sample dilution on MSA agar at which the colonies were harvested in bulk and subjected to total DNA extraction and PCR/ePCR analysis.

Article Snippet: The principle is to disperse the template DNA molecules in a thermo-stable water-in-oil emulsion at a concentration where, statistically, a few droplets contain more than one gene amplified in situ by PCR to give >10 3 copies in each droplet [ ].

Techniques: Denaturing Gradient Gel Electrophoresis, Bacteria, Control, Concentration Assay, Emulsion, DNA Extraction