BEGIN:VCALENDAR
VERSION:2.0
PRODID:-//wp-events-plugin.com//6.6.4.4//EN
TZID:Asia/Jerusalem
X-WR-TIMEZONE:Asia/Jerusalem
BEGIN:VEVENT
UID:1225@biotech.technion.ac.il
DTSTART;TZID=Asia/Jerusalem:20261012T140000
DTEND;TZID=Asia/Jerusalem:20261012T143000
DTSTAMP:20261004T110944Z
URL:https://biotech.technion.ac.il/events/%d7%a1%d7%9e%d7%99%d7%a0%d7%a8-%
 d7%a1%d7%99%d7%95%d7%9d-%d7%9e%d7%92%d7%99%d7%a1%d7%98%d7%a8-%d7%97%d7%92%
 d7%99-%d7%a9%d7%9b%d7%98%d7%a8-%d7%9e%d7%94%d7%9e%d7%a2%d7%91%d7%93%d7%94-
 %d7%a9%d7%9c/
SUMMARY:סמינר סיום מגיסטר: חגי שכטר\, מהמעבדה 
 של פרופ' סימה ירון
DESCRIPTION:Research Topic: Leveraging URS-Induced Host Stress Cascades for
  thePhage-Assisted Evolution of Antimicrobial Peptides\nAbstract: ** Lectu
 re will be given in English**\nThe global escalation of antimicrobial resi
 stance (AMR) demands novel therapeutic strategies\, including the developm
 ent of RNA- and peptide-based antimicrobial agents derived from underrepre
 sented sequences (URSs). URSs are short amino acid triplets (such as CMY) 
 naturally rare in bacterial proteomes that induce irreversible translation
  arrest within the ribosomal exit tunnel. To optimize and enhance URS pept
 ide potency from a genomic perspective\, this thesis aimed to engineer an 
 indirect phage-assisted directed evolution system in Escherichia coli. By 
 coupling the internal toxic effect of URS expression to the indirect trans
 criptional activation of host stress promoters driving the essential phage
  coat protein gene III (gIII)\, this platform seeks to establish a positiv
 e selection loop for evolving toxic peptide variants.\nInitial optimizatio
 n of induction parameters demonstrated that low inducer (IPTG) concentrati
 ons (0.1–0.25 mM) achieved significant URS-mediated bactericidal activit
 y—reducing cell viability by up to four orders of magnitude—without in
 ducer-related growth interference. Western blot analysis confirmed time-de
 pendent intracellular accumulation of the 32-amino-acid CMYWKx5 peptide (~
 5 kDa) over two hours post-induction. High-throughput RNA sequencing (RNA-
 seq) of E. coli BL21 expressing CMYWKx5 revealed distinct transcriptomic a
 lterations\, identifying ibpA (ibpAB operon) and pspA (pspACDG operon) as 
 the most strongly upregulated host promoters based on normalized transcrip
 t abundance (TPM). Dual-plasmid fluorometric and cell viability assays con
 firmed that ibpA promoter activation (measured as relative fluorescence un
 its normalized to OD600 and log(CFU/ml)) is specifically triggered by acti
 ve URS peptides (CMYWx5 and CMYWKx5)\, whereas non-toxic URS-like control 
 peptides (SMYWKx5) failed to activate the promoter.\nTo construct the cont
 inuous evolution vector\, the CMYWKx5 cassette under an arabinose-inducibl
 e pBAD promoter was cloned into a gIII-deleted fth1 (Fd) filamentous bacte
 riophage backbone. Evaluation of this selection phage construct across mul
 tiple E. coli strains demonstrated an absence of arabinose-induced toxicit
 y or viability reduction\, highlighting key architectural and expression b
 ottlenecks when porting toxic URS sequences into bacteriophage vectors. Ov
 erall\, while this study successfully identified endogenous host stress pr
 omoters capable of linking internal peptide toxicity to indirect gene acti
 vation\, full operational assembly of the phage-assisted continuous evolut
 ion system requires further vector engineering to maintain selection press
 ure within the phage framework.
CATEGORIES:סמינרים
END:VEVENT
BEGIN:VTIMEZONE
TZID:Asia/Jerusalem
X-LIC-LOCATION:Asia/Jerusalem
BEGIN:DAYLIGHT
DTSTART:20260327T030000
TZOFFSETFROM:+0200
TZOFFSETTO:+0300
TZNAME:IDT
END:DAYLIGHT
END:VTIMEZONE
END:VCALENDAR