Overview

• Target Construct

1. Attempt to add additional proteins into the vector and test for functionality
2. Vary linker lengths
3. Make a simpler version for trouble shooting the secretion system: [6x-His]-[NheI]-[prtB]
4. Transfer to a vector with the same origin and resistance as described in the original secretion system (pBR322+Carb).
5. Add a lacI into the expression and target vectors so repression is hard-coded into the vector and expression can be induced regardless of the cell line.

• Secretion System

1. Transfer to a Chlor resistance, p15A origin vector as used in the original papers
2. Add original upstream DNA (50bp) before the native RBS to ensure proper function
3. Add an arabanose inducible promoter for better control over secretion system activation
4. Combine with target vector so entire secretion system is contained in one plasmid.

• Display System

1. Test additional proteins in the new custom display construct (GFP, OpdA, etc)
2. Test designed proteins from FoldIt

Current Focus

Custom Display Vector

Problem

The underlying design of the streptavidin display constructs in the registry has the displayed protein bound close to the cell membrane. This constraint could be preventing streptavidin on the surface of the cell from forming tetramers lowering its effectiveness at binding biotin. Furthermore these existing display systems prevent the addition of another protein into them, preventing the user from displaying other proteins.

Idea

The first goal of making a new display vector was to incorporate a GS linker between the displayed protein and the ompA protein anchoring in in the cell wall. However when reviewing the construct we decided to add some other useful features as well. Maintaining the Lpp tag (to direct the protein to the periplasm) and the OmpA trans-membrane regions (for anchoring the construct) of the original 2006 parts we added:

1. GS Linker (Gly4Ser)4 - allowing for more space between the protein and the cell wall
2. TEV (Tyrosine-Glutamate-Valine) - protease site allowing for cleavage of displayed proteins
3. NheI restriction site - allowing for the insert of any BioBrick protein into the display construct

Current Status

We have built the Custom Display Vector and have inserted streptavidin into it, however we ran out of time and were not able to characterize this part. It was subbmited to the registry with streptavidin (BBa_K215210 and BBa_K215211) and without a displayed protein(BBa_K215200 and BBa_K215200).

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[http://www.fold.it Fold-It]

Problem

Streptavidin in its native form exists as a homotetramer, where adjacent subunits interact allowing for a strong interaction with biotin. This interaction is strong (Kd = 1.5E-15 M at pH 5.0) and can withstand most strong denaturing agents [1]. However when in its monomeric form, streptavidin does not maintain this strong interaction and its usefulness as a strong binder diminishes. For our system we needed a protein that could: be easily displayed on the surface of the cell, specifically bind a ligand, and release this ligand in the presence of biotin. The ability to display a protein on the cell surface is trivial, however there is difficulty in trying to get a protein to be functional on the surface of the cell. In the case of streptavidin the ability of the protein to form tetramers on the cell surface seems to be hindered, due to the poor ability of cells displaying streptavidin to bind biotinylated fluorophore (observed above). From this issue the idea of using a monomeric protein to bind biotin arose.

The Idea

There are engineered forms of streptavidin that have mutations preventing the formation of tetrameric structures. However as mentioned before, as a monomer streptavidin has a weaker affinity to biotin than would be desired. Instead of screening proteins from the literature for ability to bind biotin our group approached the Baker lab at our university. After mentioning our problem, it was recommended that we design a biotin binding protein using the [http://boinc.bakerlab.org/rosetta/ Rosetta software] they developed. Rosetta in conjunction with [http://www.folt.it Fold-It] (also developed at the University of Washington) would allow use to design and optimize proteins for binding biotin.

The Trench Work

The first step in designing our protein was looking at the native biotin-streptavidin interaction and taking measurements between key amino acids and the biotin molecule. From here we entered the constraints into Rosetta where it matched our measurements into proteins from a protein scaffold library. This produced a large set of scaffolds with different ways each one could be used to bind biotin. These scaffolds must be screened manually, and the scaffolds that look the most promising can be placed into Fold-It. Once in Fold-It, the public has access to your protein design and can tweak and tune the protein to optimize its interaction with biotin. This allows anyone (with or without prior protein knowledge) to optimize your protein scaffold.

As can be seen below Fold-It uses an easily learned user interface and uses a score board to show the players who is the best folder.

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The Wiggle function in Fold-It allows the user to fine tune the protein structure. Finding a local-minima for the amino acid conformations.

The Mutate Function in Fold-It allows the user to sample many amino acids at a particular site, or the whole protein. Mutate looks for global-minima while sampling amino acids.

The Shake Function in Fold-It performs coarse sampling of the amino acid conformations, looking for global-minima.

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This accessible format has allowed over 100,000 users to help design proteins. Currently we have published protein puzzles on Fold-It and are screening though the top scoring designs. An undergrad in our group will be active though out the next year testing the designs and looking for biotin binding proteins.

References

1. Haugland RP, "Coupling of antibodies with biotin". http://www.ncbi.nlm.nih.gov/pubmed/18287646?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum