Difference between revisions of "Team:Bielefeld-CeBiTec/Results/Library/Overview"
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<figcaption class="figure-caption"><b>Figure 3: Nanobody randomized CDR3 of 24 colonies.</b> Top to bottom: Ordered sequence, chromatogram and sequencing result. Randomized regions show the occurence of the expected bases. </figcaption> | <figcaption class="figure-caption"><b>Figure 3: Nanobody randomized CDR3 of 24 colonies.</b> Top to bottom: Ordered sequence, chromatogram and sequencing result. Randomized regions show the occurence of the expected bases. </figcaption> | ||
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Revision as of 03:43, 20 October 2016
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. By this 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.
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. By that 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 dyed with ethidium bromide. Loading of the samples (from right to left): Monobody construct with variable region (MB), 1kb DNA Ladder (by New England Biolabs).
Phagemid Display
We could show 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.
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.
