Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Synthetic Assembly DNA Cloning of Multiplex Hextuple Luciferase Reporter Plasmids

doi: 10.1007/978-1-0716-2453-1_32

Figure Lengend Snippet: Summary of plasmids described in this work. For each plasmid, the following categories are indicated: plasmid type (Type), plasmid abbreviation (Abbreviation), plasmid description (Description), plasmid function (Function), vector backbone (Backbone), vector resistance (Resistance), and source, including reference.

Article Snippet: , TB:2xP53_RE:MiniP , 2 copies of the p53 DNA binding motif , Transcriptional reporter element , pUPD , Ampicillin , Addgene #133881 ( 12 ).

Techniques: Plasmid Preparation, Control, Luciferase, Binding Assay, Multiplex Assay

Journal: Current protocols in molecular biology

Article Title: Rapid and efficient synthetic assembly of multiplex luciferase reporter plasmids for the simultaneous monitoring of up to six cellular signaling pathways

doi: 10.1002/cpmb.121

Figure Lengend Snippet: (A) DNA parts and destination vectors (Table 1) are ordered from the nonprofit plasmid repository Addgene as bacterial stabs, streaked on bacterial plates, grown up in culture, and prepared according to Basic Protocol 1. (B) DNA parts include pathway response elements consisting of a transcription blocker coupled to transcriptional response elements that can bind transcription factors specific for cellular pathways (e.g., NF-κβ, TGF-β, MAPK/JNK, c-Myc, or p53), five orthogonal luciferases (RedF, FLuc, NLuc, Renilla, and GrRenilla), and the transcriptional terminator/polyadenylation signal from the bovine growth hormone gene (bGHpA). Note that improved TGF-β and c-Myc pathway luciferase reporters were generated that have seven canocical transcription factor binding motifs, instead of four and five as proiviously described (Sarrion-Perdigones et al., 2019). Destination vectors include five positional blue/white destination vectors for position A, B, C, D, and E, located in the final destination vector that provides a blue/white colorimetric screening marker (lacZ), and one pink/white final destination vector that provides a pink/white colorimetric screening marker (tinsel purple). (C) In the first step, novel pathway response elements are built, as described in Basic Protocol 2 or Alternate Protocol 1. (D) In the second step, novel custom luciferase reporter vectors are built in an alpha assembly, using the five positional blue/white destination vectors, pathway response element, luciferase, and transcriptional terminator, following Basic Protocol 3. (E) In the final step, a new multiplex hextuple luciferase reporter vector, consisting of four previously described pathway reporters (coupled to the luciferases RedF, FLuc, NLuc, and GrRenilla, respectively), one novel custom luciferase reporter (coupled to the luciferase Renilla in this case) and one control reporter (coupled to the luciferase ELuc), is generated as described in Basic Protocol 4. The same synthetic assembly pipeline can be tailored to incorporate any five previously described pathway reporters, any five novel custom luciferase reporters, or any combinations thereof, illustrating the versatility of the pipeline. (F-G) Comparison between the assembly cloning pipeline previously published stitching together 5xMyc:Renilla, 2xp53:Nluc, 4xTGFβ:FLuc, 6xMAPK:GrRenilla, CMV:ELuc, and 5xNFκβ:RedF luciferase reporters over five consecutive cloning reactions (Sarrion-Perdigones et al., 2019) (F), and the one presented here, stitching together 5xNFκβ:RedF, 7xTGFβ:FLuc, 3xDBE:Renilla, 2xp53:Nluc, 6xMAPK:GrRenilla, and CMV:ELuc luciferase reporters using a single cloning reaction (G), illustrating a substantial decrease in time and money investment.

Article Snippet: 75 ng of the 2xp53_RE::NLuc alphaD entry vector (Addgene #124533).

Techniques: Plasmid Preparation, Luciferase, Generated, Binding Assay, Marker, Multiplex Assay, Clone Assay

Journal: Current protocols in molecular biology

Article Title: Rapid and efficient synthetic assembly of multiplex luciferase reporter plasmids for the simultaneous monitoring of up to six cellular signaling pathways

doi: 10.1002/cpmb.121

Figure Lengend Snippet: Summary of vectors described in this work.

Article Snippet: 75 ng of the 2xp53_RE::NLuc alphaD entry vector (Addgene #124533).

Techniques: Plasmid Preparation, Luciferase, Binding Assay, Multiplex Assay

Journal: Current protocols in molecular biology

Article Title: Rapid and efficient synthetic assembly of multiplex luciferase reporter plasmids for the simultaneous monitoring of up to six cellular signaling pathways

doi: 10.1002/cpmb.121

Figure Lengend Snippet: Restriction Enzyme mixes and expected fragment sizes

Article Snippet: 75 ng of the 2xp53_RE::NLuc alphaD entry vector (Addgene #124533).

Techniques: Plasmid Preparation

Journal: Current protocols in molecular biology

Article Title: Rapid and efficient synthetic assembly of multiplex luciferase reporter plasmids for the simultaneous monitoring of up to six cellular signaling pathways

doi: 10.1002/cpmb.121

Figure Lengend Snippet: Purified plasmid DNA is analyzed by agarose gel electrophoresis two-ways: plasmid fingerprinting after restriction enzyme cutting (to confirm appropriate DNA banding patterns) and uncut (to confirm the absence of unwanted multimerizations). (A) Quality control of domesticator plasmid pUPD3 (#1), destination plasmids AlphaA (#2), AlphaC (#3), AlphaB (#4), AlphaD (#5), and AlphaE (#6), and final destination plasmid Omega-CMV:Eluc (#7). (B) Quality control of luciferase plasmids pFLuc (#8), pRedF (#9), pNLuc (#10) pRenilla (#11), and pGrRenilla (#12), and the transcriptional terminator plasmid pbGH (#13). (C) Quality control of pathway response element plasmids TB:5xNF-κβ_RE:MiniP (#14), TB:7xSMAD_RE:MiniP (#15), TB:7xE-Box:MiniP (#16), TB:2xP53_RE:MiniP (#17), and TB:6xAP-1_RE:MiniP (#18). (D) Quality control of transcriptional response luciferase reporter plasmids 5xNF-κβ_RE::RedF (#19), 7xSMAD_RE::FLuc (#20), 7xE-Box::Renilla (#21), 2xP53_RE::NLuc (#22), and 6xAP-1_RE::GrRenilla (#23).

Article Snippet: 75 ng of the 2xp53_RE::NLuc alphaD entry vector (Addgene #124533).

Techniques: Purification, Plasmid Preparation, Agarose Gel Electrophoresis, Luciferase

Journal: Current protocols in molecular biology

Article Title: Rapid and efficient synthetic assembly of multiplex luciferase reporter plasmids for the simultaneous monitoring of up to six cellular signaling pathways

doi: 10.1002/cpmb.121

Figure Lengend Snippet: (A) The to-be-synthesized sequence of the p53 pathway response element (TB:2xP53_RE:MiniP): both parts of the transcription blocker are indicated in grey, the p53 DNA binding part is indicated in purple, while the minimal promoter is indicated in black. (B) Overview schematic of the p53 pathway response element (TB:2xP53_RE:MiniP) highlighting the three parts: a transcription blocker (TB) part containing a synthetic polyA terminator and the RNA polymerase II transcriptional pause signal from the human α2 globin gene (pause site), a p53 DNA binding part consisting of two tandem p53 response elements (2xP53_RE), and a minimal promoter (MiniP) part. (C) Close-up of the p53 DNA binding part, highlighting two copies of the p53 response element that are both canonical RRRCWWGYYY p53-binding motifs (GAACATGTCT and GGACTTGCCT), and the recognition site for the type II restriction enzyme SphI to perform diagnostic DNA fingerprinting. (D) Detailed schematic of the to-be-synthetized TB:2xP53_RE:MiniP fragment highlighting the appropriate binding sites for the type IIs restriction enzyme BsmBI followed by an overview of the in silico scarless assembly of the fragment in the domesticator plasmid pUPD3 (Universal Part Domesticator plasmid 3) using BsmBI and T4 DNA ligase, resulting in the final plasmid, pTB:2xp53_RE:MiniP. A second type IIs restriction enzyme, BsaI, is used for diagnostic DNA fingerprinting (see G), and will also be used to release the TB:2xp53_RE:MiniP fragment during the next round of assembly when luciferase reporter transcriptional units will be stitched together (see Basic protocol 3 and Figure 5). Assembled plasmids are identified as white colored colonies that are characterized further (see G), while religated pUPD3 plasmids are blue colored due to the presence of the colorimetric LacZ α-fragment. (E) Overview of the cloning reaction. Prepare a reaction mix containing 2 μL obtained synthetized fragment, 75 ng of pUPD3 domesticator plasmid, the type IIs restriction enzyme BsmBI, T4 DNA ligase and 10x T4 DNA ligase buffer. Start the assembly protocol that cycles 25 times between 37°C (favoring cutting) and 16°C (favoring ligation). (F) Extended assembly cycling reaction conditions, with 50 cycles of 37°C and 16°C, followed by 1h at 37°C to favor digestion of uncut plasmids, 20 minutes at 85°C to denature the enzymes and a prolonged incubation at 16°C. (G) Restriction enzyme digestion of 3 white colored colonies using BsaI (3057 and 334 bp), and uncut DNA. All colonies show the correct digestion pattern.

Article Snippet: 75 ng of the 2xp53_RE::NLuc alphaD entry vector (Addgene #124533).

Techniques: Synthesized, Sequencing, Binding Assay, Diagnostic Assay, DNA Profiling, In Silico, Plasmid Preparation, Luciferase, Clone Assay, Ligation, Incubation

Journal: Current protocols in molecular biology

Article Title: Rapid and efficient synthetic assembly of multiplex luciferase reporter plasmids for the simultaneous monitoring of up to six cellular signaling pathways

doi: 10.1002/cpmb.121

Figure Lengend Snippet: (A) Overview of the in silico scarless assembly of a multiplex luciferase reporter in the destination vector Omega Destination-CMV:ELuc:bGH. Five “transcriptional reporter” plasmids, AlphaA 5xNF-KB:RedF:bGHpA (Addgene #124530) reporting on NF-κβ pathway signaling using red firefly luciferase (RF), AlphaB 7xSMAD:FLuc:bGHpA (Addgene #124531) reporting on TGF-β signaling using firefly luciferase (FL), AlphaC 3xDBE:Renilla:bGHpA (Addgene #124535) reporting on FoxO pathway signaling using renilla luciferase (Re), AlphaD 2xp53:NLuc:bGHpA (Addgene #124533) reporting on p53 pathway signaling using nano luciferase (NL), and AlphaE 6xAP1_RE:GrRenilla:bGHpA (Addgene #124534) reporting on MAPK/JNK pathway signaling using green renilla luciferase (GR), as well as the destination vector Omega Destination-CMV:ELuc:bGH containing the control enhanced beetle luciferase (EL) for normalization purposes are incubated together with BsmBI and T4 DNA ligase. Correct multipartitie stitching of all five BsmBI-released transcriptional luciferase reporter units into BsmBI-opended Omega Destination-CMV:ELuc:bGH, results in the final multiplex luciferase vector MLRV2:NF-kb-SMAD-DBE-P53-AP1 (Addgene #124536), consisting of five transcriptional luciferase reporter units stitched together in a specified order, and one control luciferase reporter unit (constitutively expressed ELuc luciferase). Assembled plasmids are identified as white colored colonies that are characterized further (see C), while religated Omega Destination-CMV:ELuc:bGH plasmids are pink to purple colored due to the presence of the colorimetric marker, tinsel purple. (B) Overview of the cloning reaction. Prepare a reaction mix containing 75 ng of the final pink/white destination vector (Omega Destination-CMV:ELuc:bGH), 75 ng of each of the luciferase entry vectors, the type IIs restriction enzyme BsmBI, T4 DNA ligase and 10x T4 DNA ligase buffer. Start the assembly protocol that cycles 25 times between 37°C and 16°C. (C) Extended assembly cycling reaction conditions, with 50 cycles of 37°C and 16°C, followed by 1h at 37°C to favor digestion of uncut plasmids, 20 minutes at 85°C to denature the enzymes and a prolonged incubation at 16°C. (D) Restriction enzyme digestion of 3 white colored colonies using ScaI (6510, 2219, 1790, 1543, 1088 and 486 bps) and XhoI (6500, 2263, 1508, 1098, 979, 794 and 494 bps), and uncut DNA. All colonies show the correct digestion pattern.

Article Snippet: 75 ng of the 2xp53_RE::NLuc alphaD entry vector (Addgene #124533).

Techniques: In Silico, Multiplex Assay, Luciferase, Plasmid Preparation, Incubation, Marker, Clone Assay