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

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 .

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.

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

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.

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.

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.

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.

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

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.

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.

Further experiments were planned.

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

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 Klenow for filling single strand DNA

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)
• Klenow for filling single strand DNA

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)

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)
• Klenow for filling single strand DNA

Purification of >Klenow via >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

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

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.

Again attempting TOPO cloning of Nanobody fragments.

Oligo nucleotides:

• Annealing (98°C to 37°C, reducing temperature by 1°C per minute)
• Klenow for filling single strand DNA

The target backbone was amplified by 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

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)
• Klenow for overhang filling

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

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

• 

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

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

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

• Cloning of the library
• Plasmid isolation for Phagemid display and MySeq sequencing.
• PCR for generating backbones for Phagemid display

Phagemid display

• Colony PCR
• Sequencing