Team:Washington/Future

From 2009.igem.org

(Difference between revisions)
(The Trench Work)
(Overview)
 
(42 intermediate revisions not shown)
Line 2: Line 2:
{{Template:Team:Washington/Templates/Header}}
{{Template:Team:Washington/Templates/Header}}
-
==Overview==
+
='''Overview'''=
*Target Construct
*Target Construct
# Attempt to add additional proteins into the vector and test for functionality
# Attempt to add additional proteins into the vector and test for functionality
Line 14: Line 14:
# Transfer to a Chlor resistance, p15A origin vector as used in the original papers
# Transfer to a Chlor resistance, p15A origin vector as used in the original papers
# Add original upstream DNA (50bp) before the native RBS to ensure proper function
# Add original upstream DNA (50bp) before the native RBS to ensure proper function
-
# Add an arabanose inducible promoter for better control over secretion system activation
+
# Add an arabinose inducible promoter for better control over secretion system activation
# Combine with target vector so entire secretion system is contained in one plasmid.
# Combine with target vector so entire secretion system is contained in one plasmid.
   
   
*Display System
*Display System
-
# Test additional proteins in the new custom display construct (GFP, OpdA, etc)
+
# [https://2009.igem.org/Team:Washington/Project/CDS Construct a new modular display system:  CDS]
-
# Test designed proteins from FoldIt
+
# [https://2009.igem.org/Team:Washington/Project/FoldIt Use computational protein design to create a monomeric protein that binds tightly to biotin:  FoldIt]
-
==Current Focus==
 
-
====New Display Vector====
 
-
===[http://www.fold.it Fold-It]===
+
'''Continue to [https://2009.igem.org/Team:Washington/Accomplishments Accomplishments and & Submitted BioBricks]'''
-
====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.[[ http://www.ncbi.nlm.nih.gov/pubmed/18287646?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum|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.
+
-
<br><br>
+
-
[[Image:Foldit_2.png | 325px|left]][[Image:Foldit_4.png | 325px|right]]
+
-
<br><br><br><br><br><br><br><br><br><br><br><br>
+
-
 
+
-
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 ===
+
-
#
+
-
{{Template:Team:Washington/Templates/Footer}}
+

Latest revision as of 04:09, 20 October 2009

Uw title logo.png

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 arabinose 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. Construct a new modular display system: CDS
  2. Use computational protein design to create a monomeric protein that binds tightly to biotin: FoldIt


Continue to Accomplishments and & Submitted BioBricks