phic31 integrase  (Vollrath)

Journal: ACS synthetic biology

Article Title: A Hot-Swappable Genetic Switch: Building an Inducible and Trackable Functional Assay for the Essential Gene MEDIATOR 21.

doi: 10.1021/acssynbio.5c00085

Figure Lengend Snippet: Figure 1. Engineering and rapid prototyping of the integrase eraser approach. (A) Cloning strategy based on (Guiziou et al.20) for the target integrase target with BsaI adapters highlighted for use in golden gate cloning strategies, see Plasmid Maps. (B) Design of the integrase target. The target is composed of two PhiC31 integrase sites (triangles) surrounding a constitutive promoter (pUBQ10), the rescue coding sequence for MED21:6xHA and the fluorescent reporter (mScarlet). In the absence of integrase, MED21:6xHA is expressed. In the presence of integrase, the integrase mediates inversion of the DNA between the integrase sites, inverting the promoter and leading to mScarlet expression. The expression of the integrase is mediated by the selected promoter selected. (C) Rapid prototyping of the MED21 Eraser target in transient transfections of Nicotiana benthamiana at 2 days after injection. On the left side is a control target that switches from mTurquoise to mScarlet alone (top) and with a p35S:PhiC31 construct (bottom). On the right side shows the MED21 target alone (top) and MED21:HA target with a p35S:PhiC31 construct (bottom). The BFP channel for the MED21:HA is shown to demonstrate the level of background expected for the BFP channel in the negative control. Microscopy images were taken on a 20x objective to allow a wide view of switching efficiency, and the 50-μm scale bar in the RFP channels applies to all paired BFP images.

Article Snippet: The Arabidopsis MED21:HA sequence was amplified by PCR with appropriate Golden Gate restriction sites and the construction of integrase targets was performed by Golden Gate reaction in the modified pGreenIIHygr vector containing compatible Golden Gate sites defined in Guiziou et al.20 Plasmids used for construction of the novel targets are available at Addgene: 5 prime side mScarlet cassette - L0-T4 tRBSC_mScarlet (Addgene #195887), Integrase site flanked pUBQ10 - L0-T1 Target PhiC31−(Addgene https://doi.org/10.1021/acssynbio.5c00085 ACS Synth.

Techniques: Cloning, Plasmid Preparation, Sequencing, Expressing, Transfection, Injection, Control, Construct, Negative Control, Microscopy

Journal: ACS synthetic biology

Article Title: A Hot-Swappable Genetic Switch: Building an Inducible and Trackable Functional Assay for the Essential Gene MEDIATOR 21.

doi: 10.1021/acssynbio.5c00085

Figure Lengend Snippet: Figure 2. Cell-type-specific integrase eraser implemented in lateral root primordium results in MED21 loss of function and increases in lateral root initiation. (A) Schematic of the predicted behavior for the MED21 eraser approach. Lateral root primordium cells (red) will lose expression of the rescue construct, rendering them an effective knockout for MED21. (B−J) Confocal microscopy analysis of the wild-type control switch (from Figure 1C) and MED21 eraser lateral root initiation. All scale bars are 50 μm. (J) Still image from Supplemental Movie 1 of the root growth phenotype of MED21 eraser lines compared to the wild type at day 11. (K) Whole seedling epifluorescent image of MED21 eraser lines at day 14.

Article Snippet: The Arabidopsis MED21:HA sequence was amplified by PCR with appropriate Golden Gate restriction sites and the construction of integrase targets was performed by Golden Gate reaction in the modified pGreenIIHygr vector containing compatible Golden Gate sites defined in Guiziou et al.20 Plasmids used for construction of the novel targets are available at Addgene: 5 prime side mScarlet cassette - L0-T4 tRBSC_mScarlet (Addgene #195887), Integrase site flanked pUBQ10 - L0-T1 Target PhiC31−(Addgene https://doi.org/10.1021/acssynbio.5c00085 ACS Synth.

Techniques: Expressing, Construct, Knock-Out, Confocal Microscopy, Control

Journal: ACS synthetic biology

Article Title: A Hot-Swappable Genetic Switch: Building an Inducible and Trackable Functional Assay for the Essential Gene MEDIATOR 21.

doi: 10.1021/acssynbio.5c00085

Figure Lengend Snippet: Figure 3. A chemically inducible MED21 eraser. (A) Schematic of the predicted behavior for the MED21 iEraser. The estradiol inducible integrase construct is composed of p35S:XVE (transcriptional activator composed of a DNA-binding domain of LexA, the transcription activation domain of VP16, and the regulatory region of the human estrogen receptor) and pLexA-minimal 35S driving expression of PhiC31. (B,C) Characterization of the iEraser by fluorescence microscopy shows induction as early as 48 h after treatment. The scale bar represents 50 μm (C). (D) Growth phenotypes were visible in estradiol-induced switched plants grown on 1 μM β-estradiol induction plates for 6 days. Seedlings were first grown for 6 days on LS media lacking β-estradiol before being transplanted to induction media (12 days total growth). (E) Protein expression analysis by Western blot for 12 independent iEraser lines. (F) Protein expression analysis by Western blot for roots and shoots isolated from plants grown with or without β-estradiol. Seedlings were grown on LS media for 6 days, and then transplanted to either control or induction media for another 6 days of growth. The experiment was performed in triplicate for each genotype. (G,H) Lengths of lateral (G) and primary (H) roots were quantified at 6 days from the indicated Integrase Eraser type. Seedlings were grown as in (F). Letters indicate significant difference (ANOVA and Tukey HSD multiple comparison test; p < 0.001).

Article Snippet: The Arabidopsis MED21:HA sequence was amplified by PCR with appropriate Golden Gate restriction sites and the construction of integrase targets was performed by Golden Gate reaction in the modified pGreenIIHygr vector containing compatible Golden Gate sites defined in Guiziou et al.20 Plasmids used for construction of the novel targets are available at Addgene: 5 prime side mScarlet cassette - L0-T4 tRBSC_mScarlet (Addgene #195887), Integrase site flanked pUBQ10 - L0-T1 Target PhiC31−(Addgene https://doi.org/10.1021/acssynbio.5c00085 ACS Synth.

Techniques: Construct, Binding Assay, Activation Assay, Expressing, Fluorescence, Microscopy, Western Blot, Isolation, Control, Comparison

Journal: ACS synthetic biology

Article Title: A Hot-Swappable Genetic Switch: Building an Inducible and Trackable Functional Assay for the Essential Gene MEDIATOR 21.

doi: 10.1021/acssynbio.5c00085

Figure Lengend Snippet: Figure 4. Engineering and rapid prototyping of the “hot-swap” isoform switch approach. (A) Schematic of the predicted behavior for the MED21 Swap. Lateral root primordia will switch the expression of the rescue construct from wild-type (blue) to mutant isoform (red). (B) Design of the integrase target. The target is composed of two integrase sites (triangles) surrounding a constitutive promoter (pUBQ10), and each MED21 isoform is fused to the P2A self-cleaving peptide sequence and a fluorescent reporter (mTurquoise or mScarlet). In the basal state, MED21 and mTurquoise are produced. Once the PhiC31 integrase is expressed, it mediates inversion of the DNA between the integrase sites, inverting the promoter and leading to production of mMED21 and mScarlet. (C) Alignment of the AtMED21 N-terminal mutants. (D−G) Epifluorescence microscopy analysis of wild-type and mMED21 swap lateral root initiation in Arabidopsis primary transformants. Microscopy images were taken on a 20x objective, and the scale bar represents 50 μm. (H) Lateral root density (LRD per mm) and (I) lengths of lateral roots (Lateral root length in mm) from primary transformant lines were quantified at 14 days post germination from the indicated Integrase Swap type. At least 15 independent primary transformant lines were tested for each swap type. Lines were transplanted to soil and PCR genotyped for the med21 genotype. In (H), the LRD data points from med21 homozygous plants are colored in blue, while heterozygotes are colored dark gray, and ungenotyped samples are colored light gray. Letters indicate significant difference (ANOVA and Tukey HSD multiple comparison test; p < 0.001). In (I), data are presented as violin plots with nested boxplots to demonstrate that medians and interquartile ranges are comparable across conditions.

Article Snippet: The Arabidopsis MED21:HA sequence was amplified by PCR with appropriate Golden Gate restriction sites and the construction of integrase targets was performed by Golden Gate reaction in the modified pGreenIIHygr vector containing compatible Golden Gate sites defined in Guiziou et al.20 Plasmids used for construction of the novel targets are available at Addgene: 5 prime side mScarlet cassette - L0-T4 tRBSC_mScarlet (Addgene #195887), Integrase site flanked pUBQ10 - L0-T1 Target PhiC31−(Addgene https://doi.org/10.1021/acssynbio.5c00085 ACS Synth.

Techniques: Expressing, Construct, Mutagenesis, Sequencing, Produced, Epifluorescence Microscopy, Microscopy, Comparison