Journal: bioRxiv

Article Title: Stepwise Stiffening/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Double Networks

doi: 10.1101/2024.04.04.588191

Figure Lengend Snippet: Double networks can be independently degraded in a stepwise manner. (a) Double networks are first treated with 4S9 to remove the thiol-ene network, and then fully degraded by treatment with 2A9 to yield fully soluble macromolecular building blocks. (b) Peptide recognition sequences for 2A9 and 4S9 included in hydrogel crosslinkers and degradation reaction post sortase treatment. (c) Schematic depicting individual labeling of each network with distinct fluorophores, and the monomeric component released upon each sortase treatment, tracked by increases in supernatant fluorescence. (d) Fluorophore release studies. At time = 0 min, 18 mM GGG, the respective sortase, and 1 mM CaCl 2 were added to the solution the DN hydrogels were in. Hydrogel degradation was tracked by monitoring supernatant fluorescence, with values normalized to those obtained from 100% degraded gels 12 hours post reaction. (e) AFM measurements of DN gels pre- and post-4S9 treatment. DN pre 4S9 treatment: 4648 ± 750 Pa; DN post 4S9 treatment: 908 ± 550 Pa. Unpaired t-test, **p = 0.0024.

Article Snippet: [ , , , ] pET29b expression plasmids for 2A9 and 4S9 were a generous gift from Dr. David Liu at Harvard University (Addgene plasmids #75145 and #75146).

Techniques: Labeling, Fluorescence

Journal: bioRxiv

Article Title: Stepwise Stiffening/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Double Networks

doi: 10.1101/2024.04.04.588191

Figure Lengend Snippet: DNs can be formed and dynamically softened in a cytocompatible manner. (a) Experimental set-up for viability measurements. Static controls of thiol-ene, DN, and SPAAC gels were compared against dynamic DN gels treated with 4S9 on day 3 of culture. (b) Maximum Image Projection (MIP) of representative images (z = 250 µm). Live/Dead staining of encapsulated 10T1/2 fibroblasts shows excellent cytocompatibility of all possible network types on day 7 of culture. Scale bar = 100 µm. (c) Quantification of viability.

Article Snippet: [ , , , ] pET29b expression plasmids for 2A9 and 4S9 were a generous gift from Dr. David Liu at Harvard University (Addgene plasmids #75145 and #75146).

Techniques: Staining

Journal: bioRxiv

Article Title: Stepwise Stiffening/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Double Networks

doi: 10.1101/2024.04.04.588191

Figure Lengend Snippet: Double networks can be reversibly and spatiotemporally patterned to drive changes in encapsulated cell morphology. (a) Schematic depicting stepwise patterning and pattern removal. Soluble monomeric precursors can be mixed together in a one-pot mixture. SPAAC stepwise network formation occurs spontaneously, while thiol-ene polymerization can be spatially controlled photolithographically. Subsequently, thiol-ene patterns are removed with sortase 4S9 treatment. (b) Stiff patterns in a bulk hydrogel are enabled by localized thiol-ene polymerization and can be removed by 4S9 treatment. Insets depict no fluorescence is visible in the FAM channel (thiol-ene network) post enzymatic treatment. Top scale bar = 200 µm, bottom scale bar = 1 mm. (c) AFM measurements of half-patterned gels. “In” denotes a stiff region exposed to light, whereas “out” denotes the covered, non-exposed region. Two-Way ANOVA, ***p = 0.0002. (d) DN design allows for reversible patterning of mechanics. Thiol-ene gel components can be diffused into single network at later time points for mechanical patterning and can be reversibly removed and reinstated by rounds of 4S9 degradation and photopolymerization. Scale bar = 250 µm. (e) Intricate DN formations can be patterned using multiphoton laser-scanning lithography. Scale bar = 100 µm. (f) hMSCs encapsulated in stiffness-patterned hydrogels. Image shows the interface of stiff and soft regions. Scale bar = 100 µm. (g) hMSCs in soft (left) vs stiff (right) regions of patterned hydrogel. (h) Quantification of cell area in soft and stiff regions. Unpaired t-test, ****p < 0.0001.

Article Snippet: [ , , , ] pET29b expression plasmids for 2A9 and 4S9 were a generous gift from Dr. David Liu at Harvard University (Addgene plasmids #75145 and #75146).

Techniques: Fluorescence

Journal: Cell reports

Article Title: Nonreciprocal and Conditional Cooperativity Directs the Pioneer Activity of Pluripotency Transcription Factors

doi: 10.1016/j.celrep.2019.07.103

Figure Lengend Snippet:

Article Snippet: Sfp enzyme in pET29B vector , Addgene , Cat# 75015.

Techniques: Recombinant, Plasmid Preparation, Mutagenesis, Software

Journal: Nature Chemical Biology

Article Title: Structural basis of colibactin activation by the ClbP peptidase

doi: 10.1038/s41589-022-01142-z

Figure Lengend Snippet: a , Sequence similarity network (SSN) for 730 ClbP homologs, colored by identified BGC (if any). Peptidases involved in amicoumacin, edeine, paenilamicin, and zwittermicin biosynthesis cluster together, along with the newly identified probable Gram-positive colibactin producers. The Gammaproteobacterial ClbPs are split into two distinct subsets, one comprising close relatives of the sequences found in Pseudovibrio strains – and the other representing ClbPs from BGCs with canonical architecture in Escherichia species (with homologs from Erwinia , Frischella , Gilliamella , and Serratia strains , among others). Similarly, AmiB homologs in Xenorhabdus strains cluster with XcnG sequences rather than Gram-positive AmiBs. Intriguingly, the genomes of some strains – such as the edeine-producing B. formosus NF2 – appear to have multiple biosynthetic gene clusters containing authentic prodrug peptidases with potentially distinct activities. b , SSN colored to highlight proximity (within a ±10 gene neighborhood) of the peptidase gene to a gene containing both NRPS A and C domains, as a proxy for the presence of a NRPS module playing a ClbN-like role. The only SSN clusters in which this condition was met were clusters containing homologs of known prodrug peptidases (circled in black). All our sequence conservation analyses were performed using these clusters. c , SSN colored by phylum, highlighting that prodrug-activating peptidases are most common among Firmicutes, with some spread into Proteobacteria (as seen with the ami , clb , and xcn BGCs) and into Actinobacteria. d , The prodrug-activating peptidase SSN is colored by amino acid sequence length to emphasize that a large subset of sequences (including EdeA, PamJ, and ZmaM homologs) are much longer. This can be attributed to fusion with a second domain with homology to components of an ABC transporter, commonly annotated as a cyclic peptide transporter. However, Gram-positive AmiB and ClbP sequences (and other unidentified but related proteins) lack this additional domain and more closely resemble E. coli ClbP, X. bovenii AmiB, and XcnG. e , Sequence logos built from the alignment of 271 candidate prodrug-activating peptidases detail sequence conservation of the catalytic triad and of periplasmic-TMD interface and intra-TMD positions discussed in the main text.

Article Snippet: ClbP constructs were derived from a previously described plasmid (Addgene, 48244 ) containing the Escherichia coli CFT073 clbP sequence (GenBank ID: NP_754344.1) inserted between the NdeI and XhoI sites of pET29b.

Techniques: Sequencing

Journal: Nature Chemical Biology

Article Title: Structural basis of colibactin activation by the ClbP peptidase

doi: 10.1038/s41589-022-01142-z

Figure Lengend Snippet: a , Sequence logo representing conservation of dimer interface regions highlighted in panel b on the structure and in Supplementary Figure 3 on the sequence among 15 ClbP homologs from colibactin biosynthetic clusters. Residues predicted to be important for dimerization are not strongly conserved, suggesting that the mode of dimerization we observe in E. coli ClbP may be an adaptation of Proteobacterial ClbP. b , ClbP dimer interface highlighting the α8-β11 loop region (residues 296-324; blue) and α11 helix (357-372; red). c , Unrooted sequence similarity tree of S12 homologs with structures deposited in the PDB. The inset details the homologs in the same clade as ClbP. d , Equivalent views of the α8-β11 loop region (blue) in the structures of homologs in the same clade as ClbP. The α8-β11 loop region are highly variable in structure in the S12 homologs. These regions only mediate formation of a dimer in ClbP and FmtA (PDB ID: 5ZH8), but the dimer geometry is different and only the ClbP dimer has the active sites of the two subunits facing each other on either side of the substrate-binding cavity. The catalytic triad and product analog of ClbP are shown as sticks for context.

Article Snippet: ClbP constructs were derived from a previously described plasmid (Addgene, 48244 ) containing the Escherichia coli CFT073 clbP sequence (GenBank ID: NP_754344.1) inserted between the NdeI and XhoI sites of pET29b.

Techniques: Sequencing, Binding Assay