Team:Bielefeld-CeBiTec/Results/Library/Overview



Library Results

Overview

Overview

We implement a basic diversity of initial binding proteins by creating a high-quality library consisting of over one hundred thousand Monobodies and over one hundred sixty thousand Nanobodies, respectively. Thereby we set up the foundation to build our system on and set a cornerstone for utilizing and generating libraries in iGEM.

We verified the functionality of our library by several approaches.


Assembly

We did not just show that our design and construction of our libraries was successful we also adapted the way to assemble them. Thus we established an unprecedented approach in this competition and provide future iGEM teams a great part collection to build their own libraries.


Figure 1: Gelelectrophoresis of assembled construct for Monobodies. Photography of 1% agarose gel stained with ethidium bromide. Loading of the samples (from left to right): Monobody construct with variable region (MB), 1kb DNA Ladder (by New England Biolabs).


Phagemid Display

We showed that our libraries consists of several initial binders in even small extracts. Thus, the Evobody can get in the directed evolution and can be optimized by mutation and selection.


Figure 2: Example sequence of a Monobody, identified as FGE binder by phagemid display. Top to bottom: Ordered sequence, chromatogram and sequencing result.


Sequencing

Sanger sequencing was applied to check the correctness of assemblies and to examine the appearance of the different chosen bases for positions of the variable regions of our binding proteins, respectively. Also we calculated the actual library sizes by high-throughput sequencing technologies.


Figure 3: Nanobody randomized CDR3 of 24 colonies. Top to bottom: Ordered sequence, chromatogram and sequencing result. Randomized regions show the occurence of the expected bases.