Team:Bielefeld-CeBiTec/Collaborations/Lethbridge



Collaborations

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Collaboration - Lethbridge

Summary

  • Discussed practical obstancles in creation of a binding protein library
  • Compared the different approaches of the bacterial two-hybrid system
  • Bielefeld provided Lethbridge with positive control in various strength for characterization of their two hybrid system
  • Lethbridge send the influenza A NS1 peptide as a potential target for generation of Evobodies

Skype Protocol: Lethbridge - Bielefeld

Protocol from 2016-08-26

Participants Lethbridge: Andrew, Taylor, Rhys
Paricipants Bielefeld: Marius, Sebastian, Carsten, Boas
Each team shortly describes their projects

Lethbridge

  • Library: Nanobodies
    • bioinformatic approach to predict good binding amino acids
    • theoretical variability of ~1018 possible sequences accounting for CDR1, CDR2 and CDR3, however only ~105-6 will be sampled during screening.
  • Screen via bacterial two hybrid
    • Nanobody fused to α-subunit of RNA-polymerase on plasmid 1
    • target protein fused to λcI-protein on plasmid 2
    • readout via mRFP1 expression relative to mTagBFP expression and cell sorting
    • system inspired by the invitrogen BacterioMatch II
  • Target
    • Collaboration with local EMS to investigate microbiome in ambulence vehicle
    • Identified microbes via DNA isolation of swabs from various points in ambulences and Nanopore next generation sequencing.
    • Important pathogens identified in sequencing or other studies selected for antibody production via bacteria-two-hybrid selection.

Bielefeld

  • Library of nano- and monobodies
    • randomization of CDR3-loop (nanobodies) respectively three loops (monobodies)
    • theoretical variability of 109 binding proteins
  • Error prone mutation system to further increase the variability
  • Screen via bacterial two hybrid
    • binding protein fused to ω-subunit of RNA-polymerase on plasmid 1
    • target protein fused to 434cI on plasmid 2
    • selection via β-lactamase and cell survival (RFP expression as control)
  • Target
    • contacted experts with respect to possible targets
    • viral envelope proteins (e.g. zika)
    • cellular proteins
Cooperation plans were discussed
  1. Exchange of libraries with detailed cloning strategy
  2. Exchange of controls for the bacterial two hybrid system
  3. Mutual validation of the bacterial two hybrid

Protocol from 2016-08-26

Participants Lethbridge: Andrew, Rhys, Graeme
Paricipants Bielefeld: Marius, Pascal, Carsten, Boas
Each team describes their progress and current obstacles

Lethbridge

  • Library
    • complete randomisation of CDR loops
    • very high library size (Nanopore sequencing)
    • transformation efficiency is limiting (~40,000 clones)
    • transformation strategy: electroporation
  • Two-Hybrid
    • fluorescent protein gBlock contains mutations => use of streptomycin as reporter and selection via survivability
    • measurement of the basal activity of the reporter (weak constitutive promoter)
    • genomic integration of reporter gene is the target => only one copy per cell would be nice => would further decrease basal expression level
    • start selection cycles directly with target protein
  • Target: domain III of Zika E-protein
  • general design
    • two plasmid analog to the Agilent BacterioMatch II Two-Hybrid System
    • initial plan: use two reporters for copy number control
    • monobodies still contain S-S bridges
Mutual discussion about the molecular mechanism of the two hybrid
  • Question: Doesn't a strong antibody-mimetic - target protein affinity hold the RNA-Pol back?
  • No literature addressing this issue was found
  • Possible explanations
    • protein interaction is broken by RNA-Pol
    • DNA is pulled through the enzyme which stays at the promoter position

Bielefeld

  • Library
    • randomisation strategy (leads to 109 variants each)
    • high quality library by using only specifi amino acids
    • relative small library size => potential to use a considerable amount of the whole library
    • transformation strategy: electroporation, large agar plates
  • Two-Hybrid
    • characterization of the two hybrid system by using the HA4 - SH2 binding pair in addition to mutants with decreasing strength
    • => correlation between binding strength and two hybrid output
    • talked about the design of the two hybrid reporter site
      • modified promoter for very low basal expression
      • optimized distance between cI binding site and promoter
    • start selection cycles directly with target protein
  • Target: domain III of Zika E-protein
  • general design
    • two plasmid analog to the Agilent BacterioMatch II Two-Hybrid System
    • initial plan: use two reporters for copy number control
    • monobodies still contain S-S bridges

Protocol from 2016-09-29

Participants Lethbridge: Andrew
Paricipants Bielefeld: Marius, Pascal, Carsten
Current status of each project

Lethbridge

  • Library
    • Optimization of competent cells with cfu ~ 108 cfu/μg
    • currently 40.000 different clones
    • confirmed by deep sequencing
  • Two-Hybrid
    • constitutive fluorescence A as control of plasmid copy number
    • bacterial two hybrid activatable fluorescence B as signal of the bacterial two hybrid
    • currently no strong increase of fluorescence B detectable

Bielefeld

  • Library
    • variable regions are cloned into the scaffolds
    • NGS of the variable regions is planned
  • Two-Hybrid
    • devices are completely assembled but no reporter activity is detected
    • control of the single parts of the two hybrid system
    • control of the DNA binding of cI
    • which cI OR's does Lethbridge use? suggestion that lethbridge checks which OR’s they use for λcI and maybe add missing OR's
    • change of 434 cI to the partsreg λcI + binding site
    • control of the positive control binding by native PAGE and affinity chromatography
Cooperation plans were discussed
  1. Exchange of libraries with detailed cloning strategy
  2. Exchange of controls for the bacterial two hybrid system
  3. Mutual validation of the bacterial two hybrid
  • Discussion points
    • modelling of complete bacterial two hybrid on protein structure level
    • => could yield information about the optimal distance between cI binding site and promoter
    • control of the bacterial two hybrid system
      • positive controls in the HA4-SH2 binding pair from Bielefeld
      • direct fusion between cI and the RNA-Pol subunit as a positive control?
      • GFP inbetween as expression control?
    • target
      • Lethbridge uses the domain III of zika E protein as target
      • Bielefeld uses the complete zika E protein as target