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Revision as of 22:24, 19 October 2016



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



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)

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: