From 2008.igem.org

Revision as of 16:01, 23 May 2008

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.[http://igem-illini.googlegroups.com/web/Method%20for%20Directed%20Evolution%20and%20functional%20Cloning%20of%20Enzymens.pdf]

• 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

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

Addison D.Ault and James R.Broach. Protein Engineering, Design & Selection [http://igem-illini.googlegroups.com/web/Creation%20of%20GPCR-based%20chemical%20sensors%20by%20directed%20evolution%20in%20yeast.pdf]

• 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

Bio-sensors

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[http://igem-illini.googlegroups.com/web/Construction%20of%20fluorescent%20biosensor.pdf]

• 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

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 [http://igem-illini.googlegroups.com/web/Genetic%20Biosensors.pdf]

• 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

Heterologous Expression of the human D2s dopamine receptor in protease-deficient Saccharomyces cerevisiae strains

Peter Sander, Sylvia Grünewald, Marion Bach, Winfried Haase, Helmut Reiländer, Hartmut Michel (1994) Heterologous Expression of the Human D2S Dopamine Receptor in Protease-Deficient Saccharomyces cerevisiae Strains European Journal of Biochemistry 226 (2) , 697–705 [http://igem-illini.googlegroups.com/web/heterologous%20expression%20of%20receptor%20in%20yeast%20strain.pdf]

• Functional expression of human receptor in yeast
  • Overcome problem of protein degradation during growth or during protein preparation

-Aleem Zafar

Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening

Jasmina Minic, Marie-Annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Edith Pajot-Augy (2005) Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening FEBS Journal 272 (2) , 524–537 [http://igem-illini.googlegroups.com/web/Functional%20expression%20of%20olfactory%20receptors%20in%20yeast.pdf]

• Relavent quotes
  • functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening.
  • The aim of this study was to optimize the baker's yeast Saccharomyces cerevisiae as a host system for properly expressing an OR at the plasma membrane, and for its efficient coupling to a signaling pathway that produces a measurable response to odorant stimulation.
  • The yeast system was chosen for several reasons. Firstly, S. cerevisiae has been successfully used for functional expression of many GPCRs [22–28]. Secondly, yeast constitutes an attractive system to study membrane receptors providing a null background for mammalian GPCRs and G proteins. Finally, yeast cells may provide a means for detailed investigation of receptor pharmacology in vivo through the use of sensitive reporter systems that take advantage of the functional homologies between yeast pheromone and mammalian GPCR signaling pathways.

-Aleem Zafar

Foreign Gene Expression in Yeast: A Review

Michael A. Romanos, Carol A. Scorer, Jeffrey J. Clare. Foreign gene expression in yeast: a review. Yeast Volume 8 (6) pg 423-488. 1992 [http://igem-illini.googlegroups.com/web/foreign%20gene%20expression%20in%20yeast.pdf]

• A great review
• Table of Contents
  • Introduction, Transformation and selectable markers, Episomal vectors, Integrating vectors, Transcriptional promoters and terminators, Factors affecting intracellular expression, Secretion of foreign proteins, Expression in non-Saccharomyces yeasts, Physiology of foreign gene expression

• Recent changes
• What links here
• Related changes
• Special pages
• My preferences

• Printable version
• Permanent link
• Privacy policy
• Disclaimers