Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis

Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here, we introduce morphogen‐age measurements as a novel approach to test models of morphogen gradient formation. Using a tandem fluorescent timer as a protein age sensor, we find a gradient of increasing age of Bicoid along the anterior–posterior axis in the early Drosophila embryo. Quantitative analysis of the protein age distribution across the embryo reveals that the synthesis–diffusion–degradation model is the most likely model underlying Bicoid gradient formation, and rules out other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bicoid gradient formation and demonstrate how morphogen‐age information can complement knowledge about movement, abundance, and distribution, which should be widely applicable to other systems

Standard for Synthesis of Customized Peptides by Non-Ribosomal Peptide Synthetases

The purpose of this RFC is to introduce a standardized framework for the engineering of customizable non-ribosomal peptide synthetases (NRPS) and their application for in vivo and in vitro synthesis of short non-ribosomal peptides (NRPs) of user-defined sequence and structure

HiCT: High Throughput Protocols For CPE Cloning And Transformation

The purpose of this RFC is to provide instructions for a rapid and cost efficient cloning and transformation method which allows for the manufacturing of multi-fragment plasmid constructs in a parallelized manner: High Throughput Circular Extension Cloning and Transformation (HiCT). Description of construct libraries generated by the HiCT method can be found at http://2013.igem.org/Team:Heidelberg/Indigoidine. This RFC also points out further optimization strategies with regard to construct stability, reduction of transformation background and the generation of competent cells

Data from: PCR duplication: a one-step cloning-free method to generate duplicated chromosomal loci and interference-free expression reporters in yeast

Here, we report on a novel PCR targeting-based strategy called ‘PCR duplication’ that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning

Data from: PCR duplication: a one-step cloning-free method to generate duplicated chromosomal loci and interference-free expression reporters in yeast

Here, we report on a novel PCR targeting-based strategy called ‘PCR duplication’ that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning

Mapping degradation signals and pathways in a eukaryotic N-terminome [data set]

Data accompanying the paper "Mapping degradation signals and pathways in a eukaryotic N-terminome"

scverse/spatialdata-io: v0.0.9

<h3>Fixed</h3> <ul> <li>(Xenium) bug when converting feature_name #81, from @fbnrst</li> <li>(Visium) visium() supports file counts without dataset_id #91</li> </ul&gt

Images, masks and tables for quantitative microscopy measurements of FAR1 induction

This file contains all original and processed images as well as segmentation masks and tables that underlie the experiment as described under "Time course and quantitative light microscopy after pheromone induction" of this publication. The top directory contains the images, "Results" contains all masks and quantification results and "src" contains the source code used for the analysis. Images should be viewed using a scientific image analysis software such as Fiji. The .dv files contain images and metadata produced from the microscopy system we used, DeltaVision

Duplication of the <i>TUB4</i> gene.

<p>(<i>a</i>) PCR duplication of the TUB4 gene including the associated intergenic regions 5′- and 3′- to the TUB4 ORF. (<i>b</i>) Western blot and detection of Tub4 using 4 biological replicates of the strain containing the duplication, as well as 4 clones of the wild type strain. Tub4 and, as a reference, Pgk1 were detected using specific antibodies. Quantifications are shown in c. A GFP-tagged version of Tub4 shows the specificity of the antibody. (<i>c</i>) Western blot quantification, RT-qPCR and qPCR quantification of TUB4 protein, mRNA and the chromosomal gene copy number, respectively, in the different strains. qPCRs were performed on 1 biological replicate done in 5 technical replicates each. Error bars denote S.D. In all cases, differences between wild type and duplications are statistically significant (two sample t-test, unequal variance, p<0.05).</p