Chapter title |
Synthetic Gene Networks
|
---|---|
Chapter number | 6 |
Book title |
Synthetic Gene Networks
|
Published in |
Methods in molecular biology, January 2012
|
DOI | 10.1007/978-1-61779-412-4_6 |
Pubmed ID | |
Book ISBNs |
978-1-61779-411-7, 978-1-61779-412-4
|
Authors |
Marcel Geertz, Sylvie Rockel, Sebastian J. Maerkl |
Abstract |
Characterizing libraries of mutant proteins is a challenging task, but can lead to detailed functional insights on a specific protein, and general insights for families of proteins such as transcription factors. Challenges in mutant protein screening consist in synthesizing the necessary expression-ready DNA constructs and transforming them into a suitable host for protein expression. Protein purification and characterization are also non-trivial tasks that are not easily scalable to hundreds or thousands of protein variants. Here we describe a method based on a high-throughput microfluidic platform to screen and characterize the binding profile of hundreds of transcription factor variants. DNA constructs are synthesized by a rapid two-step PCR approach without the need of cloning or transformation steps. All transcription factor mutants are expressed on-chip followed by characterization of their binding specificities against 64 different DNA target sequences. The current microfluidic platform can synthesize and characterize up to 2,400 protein-DNA pairs in parallel. The platform method is also generally applicable, allowing high-throughput functional studies of proteins. |
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