...Facilitating the search of phages for therapy Phage Directory started out as a directory of phage researchers and...patients.   A phage directory is crucial because it's hard to find the right phage for the job. Phages are viruses...correct phages will test. These includes labs with a large number of different phages or with phages targeting...successful phage therapy, Sacher and Zheng also saw the need to archive phage reagents. If a phage researcher...teamed up to create Phage Directory in a whirlwind three days in November 2017. #Phage researchers! I am...antibiotics. We need lytic non-lysogenic phage URGENTLY to find suitable phage matches. Email [email protected]... to do so, the phage must recognize a specific part on the bacterium making each phage is very specific...

...second lytic phage. Bacteria that receive this phage are now antibiotic sensitive, but lytic phage-resistant... Figure 1: Single-phage strategies for bacterial CRISPR targeting. Phage-derived vectors carry CRISPR...them with both replication-competent phages and antibiotics. Phage therapy fell out of fashion with the... two-phage CRISPR system to resensitize antibiotic-resistant bacteria (Figure 2). The first phage introduced...Figure 2: Two-phage strategies to resensitize bacteria to antibiotics. A lysogenic phage infects antibiotic...lytic phage through the introduction of a crRNA (shown in blue). Infection with the lytic phage kills ...discussed above, with the caveat that phages are very diverse, and each phage has a narrow bacterial host range...

...recognition factors of phages) constitute one of the most popular classes of phage proteins to study. These...find Sequencing and cloning phage DNA can be difficult, in part because phages frequently alter their DNA...random fragment library of phage DNA This first step involves digesting phage genomic DNA into 1-3 kb (...Szymanski lab. Reasons to study how a phage recognizes its host Bacteriophages (viruses that prey on bacteria...industry, which is in the process of capitalizing on phage RBPs as diagnostic tools and therapeutics. In addition...addition, the strategic use of whole phages as therapeutics, which is also gaining a lot of new traction...depends on knowledge of the structure(s) a given phage will recognize on a host cell. To make matters ...

...The Liu lab developed the Phage-Assisted Continuous Evolution (PACE) and Phage-Assisted Non-Continuous ...T7 RNAP. A phage encoding the deaminase TadA-7.10 A mutagenesis plasmid controlling the phage mutation ...mutation rate Phage infection results in a complete ABE protein. If the phage delivers an active variant of ...infect bacterial cells. If a phage does not deliver an active variant, the phage is not infectious. The editing...overall dilution and less stringent phage selection. Figure 2: Phage-assisted evolution of base-editing.... Evolving better base editors with the help of phages! The latest ABE from the Liu lab is ABE7.10 which...editing speed dictates which phage variants can propagate faster than the rate of dilution (passage from...

...more useful to your colleagues. Technique: Probe Phage Genomes for Host Binding Proteins & Running for ... posts this month. The first, "Technique: Probe Phage Genomes for Host Binding Proteins," was contributed...Alberta and describes a technique you can use to find phage proteins that bind to your bacterium of interest.... For those of you who are not microbiologists, phages are viruses that infect bacteria and that can do...with Michael Koeris to learn how his company uses phage to test for Listeria in food products). I picked...picked this post for a few reasons: 1) I think phage are super cool and have considered getting a tattoo of... of my favorite molecular biology tool, P1 phage. 2) The post describes an ingenious but simple technique...

... inside, phages can follow one of two different life cycles: lytic or lysogenic. Lytic phages hijack the...properties. The phagemids are packaged into replication-incompetent phage particles with assistance from a...infect other bacteria. In the lysogenic cycle, the phage’s genetic material is incorporated into the host’...at a particular integration site. The integrated phage remains dormant until it is triggered to enter the...be accidentally packaged into the newly created phages. Transfer of this DNA to another cell is referred... a ‘helper’ phage prior to transduction. Scientists also use transduction to introduce foreign DNA into...scientists commonly use phagemids, a DNA cloning vector that contains both bacteriophage and plasmid properties...

...only to store a record of invading phages but to also destroy the phage upon re-exposure (2, 3). More specifically...against invading phages. Rising to the challenge - Evolution of anti-CRISPR genes in phages The CRISPR/Cas...experiments exposed S. thermophilus to predatory phages to test if the exogenous DNA would be incorporated...data highlight an interesting "arms race" between phages and prokaryotes; the ultimate victor will be the...repeat sequences operated as a safeguard against bacteriophages did not come to light until 2007. Initial experiments...

...sequences of hybridomas, but phage display is a popular way to generate scFvs.  For phage display, a pooled library... coats of bacteriophages, a process which links genotype and phenotype. Incubating the phage display library...screened for with in vitro display methods such as phage display (Bradbury et al., 2011). In vitro selection...antigen helps select for high affinity scFvs. Bound phage are then proliferated by infecting bacteria for ... and scFv characterization.    Figure 2: Phage display starts with a pooled library of scFvs that...select for scFvs with high affinity binding. Bound phage are then proliferated by infecting bacteria for ...screening. Created with Biorender.com.   Some phage display libraries screen antibody genes from B cells...

...xCas9 through directed evolution Hu et al. used phage-assisted evolution to generate and select for Cas9... the gene of interest. They created a selection phage (SP) to carry catalytically dead SpCas9 (dCas9) ...transcription from the accessory plasmid (AP) encoding phage gene III. The accessory plasmid library contained...would induce transcription of gene III, permitting phage propagation and keeping that dCas9 sequence in the...Created with BioRender.com. After 24 days of phage propagation, Hu et al. isolated xCas9 1.0-1.4 and...and subjected them to another 72 hours of phage-assisted evolution, generating xCas9 2.0-2.6. This second...round of evolution included continuous outflow of phage, increasing stringency and promoting survival of...

...islandicus CRISPR-Cas system. 5. Phage therapy Phage therapy uses phage to treat bacterial infections as...proteins are phage-derived small protein inhibitors of CRISPR-Cas systems that help phages evade the CRISPR-Cas...bacterial pathogens possess CRISPR-Cas systems, phage therapy might not be as effective. This is where...they could be engineered to “armor” therapeutic phages to protect them from pathogenic bacteria’s CRISPR-Cas...

...recombination reactions that take place when lambda phage infects bacteria. In vivo, these recombination reactions... the recombination of attachment sites from the phage (attP) and the bacteria (attB). As a result of recombination...recombination between the attP and attB sites, the phage integrates into the bacterial genome flanked by ...sites can recombine, leading to the excision of the phage from the bacterial chromosome and the regeneration...attP and attB sites.   Figure 1: Lambda phage integration and excision reactions. Recombination....   Figure 2: The Gateway system adopts phage integration into the BP and LR reactions. The BP...18429245. 3. Ptashne, M. (1992). A Genetic Switch: Phage (Lambda) and Higher Organisms (Cambridge, MA: Cell...

...helped change Novophage's focus. The company went from developing therapeutics to developing phage-based tools...develop bacteriophage for use as antibiotics 8:26 - 10:30 : How Dr. Koeris' decided to develop phage for ...the time was not right for the development of phage-based therapeutics. After a brief stint at the venture... phage... changed its name from Novophage to Sample 6. To learn more about Novophage's successful transformation...' lab at the time) got the idea to engineer bacteriophage (viruses that infect bacteria) for use as antibiotics...Koeris and Lu founded the biotech startup company, Novophage. As you'll hear Dr. Koeris explain however, the...

...Once the phagemids are constructed, they are used to produce a phage library consisting of phages that display...members) in comparison to other display systems using phage and yeast (109-1010 members). This selection step...protein can be recovered for the subsequent steps. Phage display mRNA from the enriched pool of sybody candidates... protocol describes going through two rounds of phage display to increase the enrichment of target-specific...assay (ELISA) The enriched sybody pools from the phage display screens are subcloned (using FX cloning)...fragments that are efficiently subcloned into the phagemid pDX_init plasmid, using the fragment exchange ...

...a plasmid. Phage A final option for expressing the Red system is to use a lambda red phage, λTetR, that...integrated defective prophage 2) from a plasmid, 3) from mini-λ or 4) from the lambda red phage itself (each ... defective prophage is to use mini-λ, a defective non-replicating, circular piece of phage DNA, that when...genes due to integration of a defective lambda red phage. One such strain is DY380, which is derived from... and gam is tightly regulated by the endogenous phage promoter pL and repressor CI. For recombineering...arabinose-inducible pBAD promoter and the endogenous phage pL promoter.  It is a good idea to choose plasmids...because it results in stable integration of the phage into the E. coli genome. Selection and confirmation...

...*F1 is a phage-derived ori that allows for the replication and packaging of ssDNA into phage particles...particles. Plasmids with phage-derived ori's are referred to as phagemids.   Other factors that affect plasmid...

...Mol Biol. 2018;1827:313-334. Daniel Christ pComb3 phage display vector system for producing human antibodies...103 (1995). Carlos Barbas Variant of the pComb3 phage display vector system for isolation of scFv, diabody...generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods. 2000 Aug 28;242(1-2)...antibody fragments selected from Tomlinson I+J phagemid libraries. Immunol Lett. 2015 Oct;167(2):95-102...

...recombineering. In recombineering, bacteria express phage recombination machinery, which facilitates easy ...recombination. In most recombineering strategies, only 3-4 phage proteins are needed and these can be provided via...making your own electrocompetent cells in which the phage recombination machinery is induced using L-arabinose...

... but scientists have devised means of co-opting phage genetic systems to facilitate homologous recombination... with recombineering, a technique that utilizes phage recombination machinery to promote homologous recombination... and kanamycin resistance E. coli carrying the phage recombineering machinery are first electroporated...Jiang et al., 2015). pCas contains Cas9 and the phage recombination gene lambda Red. pTargetF contains... transcription including transcription factors, phage proteins, and RNAP components (Dong et al., 2018...Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials. mBio 11: ....number of ways but success has been observed with Phagemids (Selle et al., 2020, Citorik et al., 2015, Bikard...

...like PIK3CA (Plasmid 116771: pHAGE-PIK3CA) and EGFR (Plasmid 116731: pHAGE-EGFR), are represented in this...

...same tools, they successfully assembled a 40 kb T7 phage genome from 52 (!!) fragments in one very Golden...above to complete a 52-piece assembly of a 40 kb T7 phage genome. They first compared 10-piece linear (the... was possible to select for correctly assembled phage genomes, it required a very stringent selection ...