Transcript source file

Pathway Assignments
The assignment – Annotating Pathways
KEGG Pathway Database
http://www.genome.jp/kegg/pathway.html
The assignment – Annotating Pathways
KEGG Pathway Database
Metabolism
Energy
Nitrogen
metabolism
All teams will
be assigned a
different pathway.
This one is provided
only as an example.
Which genes are found in P. limnophilus?
Closely related
to P. limnophilus
The green boxes
correspond to genes
found in R. baltica
Hypothesis: If P. limnophilus uses nitrogen as NRG source,
then it likely will have the same genes as R. baltica.
Search by EC number
http://www.expasy.org/enzyme/
Field M10 of the Roche Applied Science “Biochemical Pathways” wall chart
What do you do with assigned gene?
1- Go to KEGG Pathway
Database
2- Click on “green box”
corresponding to the
gene that was assigned
to you.
“Click”
3- Obtain amino acid sequence
(FASTA format) from KEGG
for assigned R. baltica gene
“Click” for FASTA file
Amino acid sequence
4- “COPY/PASTE” entire entry
(including description line)
into text editing program
(i.e., Notepad, Wordpad)
for use in subsequent
database searches
FASTA format:
• Description line preceded by greater than sign
• Amino acid sequence for protein encoded by corresponding gene
• NOTE: Nucleotide sequences also can be depicted in FASTA format
5- In the text editor (Notepad), save the file
containing the amino acid sequence in
FASTA format as plain text file (.txt)
6- Return to imgACT
9- Right click on img/edu icon and open Organism Details page
for Planctomyces limnophilus DSM 3776
“Click”
Get used to
right-click then
open in new tab
“Click”
10- Click “Find Genes” tab
11- Click [BLAST] from menu in “Find Genes” tab
“Click”
12- Retrieve amino acid sequence for R. baltica gene.
Copy/paste into query box. Select blastp program with
E-value cutoff of 1e-2 and P. limnophilus genome as the
database to be searched. Then hit <Run BLAST>.
Referred to as a
“Reciprocal BLAST”
search in instructions
13- Results of BLAST search will be displayed in format
similar to what is generated with NCBI database
Inspect “hit” list
14- Select best “hit” and add to “Gene Cart”
Scroll down
Note Gene Object ID (OID)
14- Return to imgACT
- imgACT “Student Teams” will be created by the
instructor once all students have registered for course
- Find your course “Home” page on imgACT
Select MIMG 103L, Winter 2010
- View your team(s) by clicking on Student Team name
“Click”
“Click”
Josie Bruin
[email protected]
Joe Bruin
[email protected]
Jenny Bruin
[email protected]
Jess Bruin
[email protected]
Jeffrey Bruin
[email protected]
- View your Practice Gene by clicking link under
“Gene Annotation Assignments”
“Click”
- View your team ASSIGNMENT by clicking link
under “Pathway/Structure Assignments”
“Click”
You should see the “Pathway/Structure Report” page
Notice team members
with email addresses
(Your TA & instructors also will be listed here)
As a TEAM: set up your team notebook page
“Click”
Add pathway & gene information for reference organism
Insert KEGG map:
- return to KEGG database
- save KEGG map as .gif or .png
“Click”
Must be in EDIT mode to insert image
“Click”
Place cursor where
you want to insert image
“Click”
NOTE: Upload entire
KEGG map. Only crop
the image if assigned
structure/complex or
pathway is portion of
full KEGG map
“Click”
Result after image inserted and comments added to box:
Each person on the team should enter the information
for the Reference Organism & Gene used for their
Reciprocal BLAST search:
1. This step requires that
you create additional
boxes and headings.
While in EDIT mode,
simply copy/paste then
modify the text.
2. Include full name of
reference organism.
3. Include amino acid
sequence used for the
reciprocal BLAST search
(copy/paste from Notepad).
Press SAVE button so you don’t lose your work.
“CLICK”
IMPORTANT NOTES!!
 Only ONE person at a time from each team may modify the team notebook;
otherwise you will delete your teammate’s work!
NOTE:
formay
MetaCyc
willlater
be entered
on the page later.
 SkipAdditional
MetaCyc information
section – you
add this
in the quarter.
Return to “Pathway/Structure Report” in imgACT
Individually add steps
or components to your
pathway or structure
“Click”
Enter the “Step name” and “Gene Object ID”.
Then press <SAVE>.
Your TA may have suggestions
for “step name” (or component
name for cell structures) if you
are not sure what to call it.
The Gene Object ID corresponds
to top reciprocal BLAST hit in
P. limnophilus genome.
“Pathway/Structure Report” page should be updated
Indicates step creation
was successful
Log out of lab notebook
Sign out of imgACT
“CLICK”
Close all tabs
and windows
before exiting
the browser