UIUC IGEM:Research Papers

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Directed Evolution

Method for Directed Evolution and functional Cloning of Enzymens

Henrik Pedersen, Swen Holder, Daniel P, Sutherlin, Urs Schwitter, David S. King. and Peter G. Schultz. Proc. Natl. Acad. Sci. USA Vol. 95, pp. 10523-10528, September 1998.[1]

• Summary
  • Bacically give the methodology for preforming directed evolution on a specific enzyme, but can probably be adapted for any other protein provided we can isolate the ligand we are trying to isolate
  • The methods are very involved and complex, but are very specific for how to prepare substrates.
    • Create substrate with proteins to be identified and then create conditions so that only very specific bonding can occur. Get rid of elements that do not have a high bond affinity, then un-bond the proteins.
• Note: Some one with more biology background might want to take a look at this, as it deals with really specific topics, that I do not have experience with. Also some relevant over-view things were highlighted.
• --Dan Knipmeyer

Protein Engineering and the Development of Generic Biosensors

Homme W. Hellinga and Jonathan S. Marvin. Trends in Biotechnology Vol. 16, Issue 4 April 1998 pp. 183-189 [2]

• Relevent quotes.
  • Modular molecular engineering system in which the integrated signal transduction site are so constructed so that each can be changed separately with out destroying the communication between them.
  • Maintain constant signal transduction function creating generic biosensor to share same detection instrumentation.
  • Identify protein with particularly well behaved intrinsic signal transduction function and construct appropriate binding site.
    • Use of alpha-haemolysin and GFP
  • Hinge action proteins for FRET analysis.
• --Dan Knipmeyer

Creation of GPCR-based chemical sensors by directed evolution in yeast

Addison D.Ault and James R.Broach. Protein Engineering, Design & Selection [3]

• Relevance to iGEM: High
• Subjects:
  • G protein coupled receptors (GPCRs)specificity vs. sensitiyity
  • Directed evolution in GPCRs
  • Functional expression of heterologous GPCRs in yeast cells
• Summary:
  • The authors took a Human GPCR and used simmilarites between yeast mating response pathways and the human signal transduction pathways to couple a human GPCR to the yeast pheromone respone pathway. They then used random mutagenesis to create hybrids. Ultimately the hybrids were more sensitive, but had the same specificity for the compounds they sere tested on.
• --Dan Knipmeyer

Construction of a Fluorescent Biosensor Family

Robert M. De Lorimier, J. Jeff Smith, Mary A. Dwyer, Loren L. Looger, Kevin M. Sali, Chad D. Paavola, Shahir S. Rizk, Shamil Sadigov, David W. Conrad, Leslie Loew and Homme W. Hellinga Protein Sci. 2002 11: 2655-2675[4]

• Relevance to iGEM: medium/low
• Subjects:
  • bacterial periplasmic (between the cell wall and cell membrane) binding proteins (bPBPs);
  • fluorescence;
  • fusion proteins
• Summary:
  • The authors took bPBPs and did two things to them: they mutated them to change their ligand specificity on a limited scale, and they attached a fluorescent domain to the bPBP so that ligand binding would cause a change in fluorescence without the need for a full signal transduction pathway.
  • The authors' main focus was on the fluorescence. We could conceivably use this type of protein to accomplish the biosensor; however, the authors state that not much is known about the natural bPBPs, so it might be difficult to find a starting point in terms of a specific bPBP.
• --Dave Luedtke