Team:Bielefeld-CeBiTec/Notebook/Library

Lab Notebook

Documentation is like sex; when it's good, it's very, very good, and when it's bad, it's better than nothing. - Dick Brandon

Library

Week 1: (04.04.16-10.04.16):

First get-together and initiation of a phase for pure literature research on issues involving (synthetic) genetic libraries.

Week 2: (11.04.16-17.04.16):

Reverberate results of the outcome of first literature research. Achieved knowledge suggesting orientation on somatic recombination (VDJ recombination ) to create the binding protein library. Further research on ScFv fragments, ScFab fragments and single domain antibody fragments .

Week 3: (18.04.16-24.04.16):

Increased knowledge of the topic leads to choose of DARPins and Monobodies as antibody mimetics, hence initiated the search for DNA sequences and additionally collected further information on the se proteins.

Week 4: (25.04.16-01.05.16):

Intensified the search for DNA sequences and collected further information on protein scaffolds.

Week 5: (02.05.16-08.05.16):

Got in touch with the inventors of Monobodies, Shohei and Akiko Koide from the Koide Laboratory which inter alia led to receiving information on sequences for Monobodies.

Week 6: (09.05.16-15.05.16):

Library was started with two leading threads , one following antibody mimetics (Monobodies) and another single domain antibodies (Nanobodies).
Again: searching for DNA sequences, this time for the constant parts of both scaffolds.

Week 7: (16.05.16-22.05.16):

Several hints in literature lead to the choice of minimalistic schemes for variable regions. Sorting thoughts and further research on well-suited schemes.
Yet focusing on finding DNA sequences for constant regions.
Introducing the thought to order constant regions as IDT gBlocks and the variable regions as oligo-nucleotides.

Week 8: (23.05.16-29.05.16):

Design of sequences with gained knowledge of constant regions.
Worked out a control method for supervising the correct folding of protein scaffolds containing disulifde bonds. Using bimolecular fluorescence complementation (BiFC) for the control seemed suited. Search of suitable sequences for a BiFC is initialised.

Week 9: (30.05.16-05.06.16):

Design of sequences for constant regions of the protein scaffolds.
Focused work on sequences, which are possible for the BiFC control.

Week 10: (06.06.16-12.06.16):

Finishing of the design of the constant regions and ordering of gene fragments at IDT.
Desig of oligo nucleotides for variable regions with several minimalistic schemes.

Week 11: (13.06.16-19.06.16):

Ongoing design of oligo sequences variable regions and also construction of several primers.
Achieving a theoretical variety of about one billion per protein scaffold by choosing three different variants for nucleic acid schemes for variable regions.
Finally, ordering the oligo synthesis for variable regions and also fitting primers.

Week 12 (20.06.16-26.06.16):

Further experiments were planned.

Week 13 (27.06.16-03.07.16):

Start of practical work on library.

Backbone generation of BBa_J04450 with primers MB-psb-1-fw and MB-psb-1-rev: (without success)

• Transformation of BBa_J04450 into DH5α
• Colony PCR with VF and VR
• Plasmid Isolation
• PCR with primers MB-psb-1-fw and MB-psb-1-rev
• Gel extraction

Oligo nucleotides:

• Fusion PCR without primers for overhang filling

Week 14: (04.07.16-10.07.16):

Development of a method to anneal the ordered oligo nucleotides, also how to fill single strand DNA to double strand DNA to be able to continue working with them.

Oligo nucleotides:

• Annealing of oligo nucleotides (98°C to 4°C, reducing temperature by 1°C per minute)
• Using Week 15: (11.07.16-17.07.16):
  
  Backbone generation of BBa_B0030: (without success)

  • Transformation of BBa_B0030 into DH5α
  • Colony PCR with VF and VR
  • Plasmid Isolation
  • Enzymatic digestion
  • Gel extraction

  
  Oligo nucleotides:

  • Oligo annealing (98°C to 4°C, reducing temperature by 1°C per minute)
  • Week 16: (18.07.16-24.07.16):
    
    Backbone generation of BBa_J04450: (without success)

    • Plasmid Isolation
    • Restriction digestion
    • Gel extraction

    
    Backbone generation of BBa_J04450:

    • Q5 PCR
    • DPN1 digestion
    • Gel extraction

    
    Oligo nucleotides:

    • Purification from agarose gel (without success)
    
    Week 17: (25.07.16-31.07.16):
    
    Backbone generation of BBa_J04450:

    • Plasmid Isolation
    • PCR
    • DPN1 digestion
    • Gel extraction

    
    Oligo nucleotides:

    • Oligo annealing (98°C to 4°C, reducing temperature by 1°C per minute)
    • Week 18: (01.08.16-07.08.16):
      
      Purification of >native PAGE :

      • Preparation of solutions
      • Native page (12%)
      • Gel extraction

      
      Nanobody BiFC:

      • Gibson-Assembly of BiFC Binder/ BiFC GFP and BBa_J04450 backbone
      • Transformation to DH5α
      • Colony PCR with VF and VR
      
      Week 19: (08.08.14-14.08.16):
      
      Nanobody BiFC GFP:

      • Colony PCR with VF and VR
      • Plasmid isolation
      • Sequencing

      
      Nanobody BiFC Binder:

      • Gibson-Assembly of BiFC Binder and psb1k3 https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers#library RFP backbone
      • Transformation into DH5α
      • Colony PCR with VF and VR
      • Pasmid isolation
      • Sequencing

      
      Monobodies:

      • Gibson-Assembly of Monobody constant regions and BBa_B0030 backbone
      • Transformation to DH5α
      • Colony PCR with VF and VR
      
      Week 20: (15.08.16-21.08.16):
      
      After Colony PCR with VF and VR showed seemingly correct results sequencing of Monobodies was ordered.
      To save genes fragments without altering sequences an attempt for TOPO cloning of Nanobodies was made. (without success)
      Design and order primers for target backbone.
      
      Week 21: (22.08.16-28.08.16):
      
      Again attempting TOPO cloning of Nanobody fragments.
      
      Oligo nucleotides:

      • Annealing (98°C to 37°C, reducing temperature by 1°C per minute)
      • PCR with primers Target_bb_fw and Target_bb_rev and extracted from agarose gel.
        Constant Monobody regions with overlaps for selection plasmid was involved in PCR with primers MB-Insert-fw and MB-Insert-rev and extracted from gel.
        
        Selection plasmid and Monobodies:
        • Gibson-Assembly of target backbone and constant Monobody regions
        • Transformation in to electro competent DH5α
      
      Week 22: (29.08.16-04.09.16):
      
      Selection plasmid with Monobodies:

      • Colony PCR with VF and VR
      • Sequencing
      • Backbone amplification by PCR of selection plasmid with Monobody constant regions with primers MB-bb-fw and MB-bb-rev

      
      BiFC:

      • Competent cells of GFP BiFC
      • Transformation of Binder BiFC
      • Overnight cultures

      
      Primer ordering.
      
      Oligo nucleotides:

      • Ordering of new F2.2-fragment
      • Annealing (98°C to 37°C, reducing temperature by 1°C per minute)
      • Week 23: (05.09.16-11.09.16):
        
        BiFC:

        • Measuring fluorescence with TECAN , no success
        • Double Transformation of both plasmids into DH5α

        
        Good sequencing results of Nanobody and Monobody constant regions.
        
        Selection plasmid with Nanobodies:

        • PCR with Nanobody gBlocks (constant regions of Nanobodies) using primers NB-Insert-fw and NB-Insert-rev
        • Gibson-Assembly
        • Transformation into DH5α
        • Colony PCR with primers VF and VR
        • Sequencing
        
        Week 24: (12.09.16-18.09.16):
        
        BiFC:

        • Overnight cultures
        • Measuring fluorescence with TECAN fluorometer - bad results
        • Transformation of gBlocks to DH5α
        • Plasmid isolation

        
        Target backbone and Nanobodies:

        • Backbone generation of the selection plasmid with Nanobody constant regions using primers NB-bb-fw and NB-bb-rev

        
        Monobodies and Nanobodies:

        • Gibson assembly of generated backbones with constant regions and filled-up oligos (variable regions)
        • Plasmid isolation
        • Sequencing: bad sequencing results

        
        Oligos:

        • New polymerases were tested: Q5, KOD, Phusion, GoTaq G2
        • Oligo nucleotide annealing (95°C to 50°C , reducing temperature 1°C per minute) with polymerases
        • Separation in a 3% agarose gel

        • 

          Beschreibung

        
        Monobodies and Nanobodies:

        • Gibson-Assembly of selection plasmid backbones with constant regions and differently filled up oligos
        • Transformation into DH5α
        • Highest efficiency was observed with Q5 polymerase.
        • Colony PCR with VF and VR
        • Plasmid isolation
        
        Week 25: (19.09.16-25.09.16):
        
        As a proof of concept for our library we started a phagemid display. Design of the phagemid display and primer ordering.
        
        Monobodies and Nanobodies:

        • Sequencing of colonies with Q5 oligos showed good results!
        • Generating more library backbone with primers MB-bb-fw and MB-bb-rev for Monobodies and primers NB-bb-fw and nb-bb-rev for Nanobodies
        • Plasmid isolation of library parts

        
        BiFC:

        • Sequencing
        • Proof of fluorescence in TECAN fluorometer

        
        Target Zika e protein:

        • Backbone generation with primers Zk_cMyc_BB_fw and Zk_cMyc_BB_rev
        • Gibson assembly with backbone and gene synthesis of Zika E protein
        • Colony PCR with VF and VR
        
        Week 26: (26.09.16-02.10.16):
        
        Monobodies and Nanobodies:

        • Sequencing of library parts in one run
        • Preparing devices

        
        Target Zika E protein:

        • Gibson assembly with backbone and gene synthesis of Zika E protein
        • Transformation into DH5α
        • Plasmid isolation

        
        Phagemid display:

        • Plasmid isolation
        • Sfi1 digestion over night
        • Gel extraction

        
        We decided to change the ori gin of replication and the antibiotic resistance of the mutation plasmid, so the library has to be cloned into pSB1K3.
        
        Monobodies and Nanobodies:

        • Digestion with EcoRI and PstI of the Nanobody/Monobody Inserts (rpoZ and binding protein constant regions)
        • Gel extraction
        • Ligation with pSB1K3 backbone

        
        
        
        Week 27: (03.10.16-09.10.16):
        
        

        • Cloning of the library
        • Plasmid isolation for Phagemid display and MySeq sequencing.
        • PCR for generating backbones for Phagemid display
        
        Week 28: (10.10.16-16.10.16):
        
        Phagemid display

        • Colony PCR
        • Sequencing