Pathway Assignments

The assignment – Annotating Pathways

http://www.genome.jp/kegg/pathway.html

KEGG Pathway Database

The assignment – Annotating Pathways

KEGG Pathway Database

All teams willbe assigned a

different pathway.This one is providedonly as an example.

Energy

Metabolism

Nitrogenmetabolism

Closely relatedto P. limnophilus

The green boxescorrespond to genesfound in R. baltica

Which genes are found in P. limnophilus?

Hypothesis: If P. limnophilus uses nitrogen as NRG source,then it likely will have the same genes as R. baltica.

http://www.expasy.org/enzyme/

Search by EC number

Field M10 of the Roche Applied Science “Biochemical Pathways” wall chart

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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.

Amino acid sequence

“Click” for FASTA file

3- Obtain amino acid sequence (FASTA format) from KEGG for assigned R. baltica gene

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

4- “COPY/PASTE” entire entry (including description line) into text editing program (i.e., Notepad, Wordpad) for use in subsequent database searches

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

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10- Click “Find Genes” tab

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Get used toright-click thenopen in new tab

11- Click [BLAST] from menu in “Find Genes” tab

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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 downScroll 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

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Joe Bruin joe@ucla.edu

Jenny Bruin jenny@ucla.edu

Jess Bruin jess@ucla.edu

Jeffrey Bruin jeff@ucla.edu

Josie Bruin josie@ucla.edu

- View your Practice Gene by clicking link under “Gene Annotation Assignments”

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- View your team ASSIGNMENT by clicking link under “Pathway/Structure Assignments”

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You should see the “Pathway/Structure Report” page

Notice team memberswith email addresses

(Your TA & instructors also will be listed here)

As a TEAM: set up your team notebook page

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Add pathway & gene information for reference organism

Insert KEGG map: - return to KEGG database - save KEGG map as .gif or .png

Must be in EDIT mode to insert image

Place cursor whereyou want to insert image

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NOTE: Upload entireKEGG map. Only cropthe image if assignedstructure/complex orpathway is portion offull KEGG map

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Result after image inserted and comments added to box:

Each person on the team should enter the informationfor the Reference Organism & Gene used for theirReciprocal BLAST search:

1. This step requires thatyou create additionalboxes and headings.

While in EDIT mode,simply copy/paste thenmodify the text.

2. Include full name ofreference organism.

3. Include amino acidsequence used for thereciprocal BLAST search

(copy/paste from Notepad).

Press SAVE button so you don’t lose your work.

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NOTE: Additional information for MetaCyc will be entered on the page later.

IMPORTANT NOTES!! Only ONE person at a time from each team may modify the team notebook; otherwise you will delete your teammate’s work! Skip MetaCyc section – you may add this later in the quarter.

Return to “Pathway/Structure Report” in imgACT

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Individually add steps or components to your pathway or structure

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 inP. limnophilus genome.

“Pathway/Structure Report” page should be updated

Indicates step creationwas successful

Log out of lab notebook

Sign out of imgACT

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