Team:UCLA/Modeling/Analysis

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Latest revision as of 23:28, 27 September 2013

Analysis of Protein

Software

Analysis of the Mtd protein was conducted using our own 'Protein Calculator' software alongside PyMOL. The Protein Calculator allows users to see the potential effects of polymorphic nucleotides in their DNA library on the protein level. The PyMOL software allows users to upload, visualize, and modify molecules of interest in 3D. Together, these two programs allowed our team to locate variable amino acids in the Mtd, as well as predict the effects on binding affinity that the polymorphic nucleotides can have on the resulting protein.

Instructions to download and use our program are as follows. (Disclaimer! There are still several bugs in the Protein Calculator program that are still in the process of being fixed. The dropbox download link below will be updated with newer versions as the fixes are made.)

1) Click the link dropbox link below and download the files.

Step 1.

2) Your computer may display some type of warning- ignore the warning to download the program.

Step 2.

3) Extract the zip file into the directory/location of your choice.

Step 3.

4) Open Sequence.txt to enter the DNA sequence you wish to analyze. Be sure to keep Sequence.txt in the same directory as Protein Calculator.exe. Important: enter the sequence such that it is within an appropriate reading frame (number of base pairs must be multiple of 3), the nucleotides are in all capital letters, and entered in the 5' -> 3' direction.

Step 4.

5) Once Sequence.txt contains your DNA sequence in the appropriate format, open Protein Calculator.exe. Another warning may appear- click 'Run Anyway' to run the program.

Step 5.

6) A cmd window will open with instructions. A new .txt file should appear in the same directory as Protein Calculator.exe called OutputData.txt. This text file contains information about your polymorphic DNA sequence library.

Step 6.

7) Open OutputData.txt to view resulting data. Before closing the program, copy this data onto a separate file- all text will be deleted upon closing the program (by entering '0' in the cmd window).

Step 7.

8) This data can now be used to accurately locate and modify variable regions in the protein of interest.

Step 8.

https://www.dropbox.com/s/bai70ev5oqksj98/Protein%20Calculator.zip

Retrieved from "http://2013.igem.org/Team:UCLA/Modeling/Analysis"

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          <li><div id="spec"><a href="/Team:UCLA/Modeling"><font color="black">MODELING</font></a></div></li>
 	<li><a href="/Team:UCLA/HumanPractices">HUMAN PRACTICES</a></li>
-	<li><a href="/Team:UCLA/Notebook">NOTEBOOK</a></li>
+	<li><a href="/Team:UCLA/Notebook/Biobrick">NOTEBOOK</a></li>
 	<li><a href="/Team:UCLA/Safety">SAFETY</a></li>
          <li><a href="/Team:UCLA/Attributions">ATTRIBUTIONS</a></li>
@@ Line 63: / Line 63: @@
 == '''Analysis of Protein''' ==
+===Software===
-Our protein of interest, the Major Tropism Determinant (Mtd) protein, is located on the tail-end fibers of the BPP-1 bacteriophage. This protein is important in that it allows the BPP-1 phage to bind and infect its host species of bacteria- <i>Bordetella</i>, the bacterial species that causes whooping cough. Because <i>Bordetella</i> can vary the expression of its surface proteins, the BPP-1 phage in turn has evolved a way to vary the specificity of the Mtd protein. Through targeted mutagenesis from the phages diversity-generating retroelement ([[Team:UCLA/Project/NaturalSystem#Diversity_Generation|DGR]]) system, the Mtd protein has shown to be able to support a massive library of protein variants- theoretically up to 9.22*10<sup>12</sup> possible variants.
+<div>
+Analysis of the Mtd protein was conducted using our own 'Protein Calculator' software alongside PyMOL. The Protein Calculator allows users to see the potential effects of polymorphic nucleotides in their DNA library on the protein level. The PyMOL software allows users to upload, visualize, and modify molecules of interest in 3D. Together, these two programs allowed our team to locate variable amino acids in the Mtd, as well as predict the effects on binding affinity that the polymorphic nucleotides can have on the resulting protein.
-The Mtd gene is a 1146 base pair long sequence that contains two mutagenic islands. There are a total of 22 specific mutagenic nucleotides located in the last ~100 nucleotides on the 3’ end of the sequence, known as the variable repeat (VR). In-vivo, there is a nearly identical sequence of nucleotides known as the template repeat (TR) located downstream of the Mtd gene. The DRG system reverse-transcribes a mutant copy of the TR, with mutations confined only to specific adenine residues, which then replaces the VR that encodes the C terminus of the Mtd.
-On the quaternary level, the Mtd protein assembles into globular trimeric structure comprised of 380 amino acids in each of its monomers. Out of these 380 amino acids, 12 of them can mutate to a range of different amino acids.
+Instructions to download and use our program are as follows. (<b>Disclaimer!</b> There are still several bugs in the Protein Calculator program that are still in the process of being fixed. The dropbox download link below will be updated with newer versions as the fixes are made.)
-Mtd is a multi-domain protein with a C-lectin type fold at its C terminus. The variable regions of the Mtd form a surface on one face of the pyramid-shaped trimer where binding occurs. The rest of the protein forms a stable scaffold that is constant across different Mtd variants. The Mtd protein binds to the phage tail-fiber at the N-terminus.
+) Click the link dropbox link below and download the files.
+[[File:Walkthrough-1.png|thumb|left|Step 1.|800px]]
-Thorough analysis of this protein was conducted through our own 'Protein Calculator' software used concurrently with PyMOL.
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+) Your computer may display some type of warning- ignore the warning to download the program.
-https://www.dropbox.com/s/fanvwnsux1ir8je/Protein%20Variant%20Analyzer.zip
+[[File:Walkthrough-2.png|thumb|left|Step 2.|800px]]
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+) Extract the zip file into the directory/location of your choice.
+[[File:Walkthrough-3.png|thumb|left|Step 3.|800px]]
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+) Open Sequence.txt to enter the DNA sequence you wish to analyze. Be sure to keep Sequence.txt in the same directory as Protein Calculator.exe. <b>Important: </b> enter the sequence such that it is within an appropriate reading frame (number of base pairs must be multiple of 3), the nucleotides are in all capital letters, and entered in the 5' -> 3' direction.
+[[File:Walkthrough-4.png|thumb|left|Step 4.|800px]]
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+) Once Sequence.txt contains your DNA sequence in the appropriate format, open Protein Calculator.exe. Another warning may appear- click 'Run Anyway' to run the program.
+[[File:Walkthrough-5.png|thumb|left|Step 5.|800px]]
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+) A cmd window will open with instructions. A new .txt file should appear in the same directory as Protein Calculator.exe called OutputData.txt. This text file contains information about your polymorphic DNA sequence library.
+[[File:Walkthrough-6.png|thumb|left|Step 6.|800px]]
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+) Open OutputData.txt to view resulting data. Before closing the program, copy this data onto a separate file- all text will be deleted upon closing the program (by entering '0' in the cmd window).
+[[File:Walkthrough-7.png|thumb|left|Step 7.|800px]]
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+) This data can now be used to accurately locate and modify variable regions in the protein of interest.
+[[File:Walkthrough-8.png|thumb|left|Step 8.|800px]]
+</div>
+https://www.dropbox.com/s/bai70ev5oqksj98/Protein%20Calculator.zip
+<!--
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