Difference between revisions of "Help talk:Team experience tutorial"

Revision as of 15:08, 11 June 2007

Written by [http://openwetware.org/wiki/User:Reshma_P._Shetty Reshma Shetty] of [http://openwetware.org OpenWetware] and the [http://parts.mit.edu/wiki/index.php/MIT_2006 MIT 2006 iGEM team]

Assumptions & Overview

Verbal TOC for episodes, larger picture - i.e. making a vanillin producing device - and requirement of rudimentary knowledge of

• Standard Parts
• MB lab experience
• SB: Read Scott's Primer

Coming up with a project

Research previous year's projects

Goals: learn how to find presentations from previous teams, including their videos and wiki pages and their parts on the Registry

Also See: What makes a good project (theory)

1. Go to http://igem2007.com
2. Click on [http://www.igem2006.com/presentations.htm Watch team presentations from iGEM 2006]
   • Here you can watch [http://parts.mit.edu/movies/iGEM2006_MIT.mov team presentations] and view the [http://parts.mit.edu/wiki/images/c/c0/IGEM2006-MIT-Powerpoint.pdf slides]
3. You can also check out more information on previous projects by going to [http://parts.mit.edu/wiki/index.php/Main_Page http://parts.mit.edu/igem]
   1. Click on [http://parts.mit.edu/wiki/index.php/Schools_Participating_in_iGEM_2006 Meet the 37 teams]
   2. Click on [http://parts.mit.edu/wiki/index.php/MIT_2006 MIT] and read about the project

Describe your project

Goal: learn how to find team's wiki page and edit it

1. Decided on an idea for your new project?
   • From the iGEM2007.com site, click [http://parts.mit.edu/igem07/index.php/Main_Page Visit the iGEM07 Wiki]
   • Click [http://parts.mit.edu/igem07/index.php/IGEM2007_Team_List Link to iGEM2007 Team Wiki pages] and find your wiki to record information about your new project

Let's make a new scent! Vanilla.

Design the system

We want a device that takes an input chemical and makes vanilla scent. So we need enzymes that synthesize vanilla.

Search the registry

Goal: text search of the registry

1. Go to the Registry (http://parts.mit.edu)
2. Use the search box to look for existing parts
3. Search for vanilla ... you get no real hits ([http://www.google.com/search?domains=http%3A%2F%2Fparts.mit.edu&num=50&ie=utf-8&oe=utf-8&q=vanilla&btnG=Search&sitesearch=http%3A%2F%2Fparts.mit.edu])
4. Search for vanillin ... ([http://www.google.com/search?domains=http%3A%2F%2Fparts.mit.edu&num=50&ie=utf-8&oe=utf-8&q=vanillin&btnG=Search&sitesearch=http%3A%2F%2Fparts.mit.edu])
5. No one has made a vanilla synthesizing enzyme ... phew!

Search the literature

Goal: learn about pubmed; do a basic literature search on a particular topic;

1. Visit [http://pubmed.org PubMed]
2. Search for vanilla
3. Find a [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17437627&query_hl=1&itool=pubmed_docsum relevant paper]
4. Go read the [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17437627 paper]
5. Identify the useful components described in the paper
6. You seem to need two genes ech and fcs ... let's grab some sequence

Getting the DNA sequence of the gene

Goal: get vanillin DNAs sequences

1. Search for Genbank accession numbers
2. Follow the [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=40788116 Genbank link]

Make a part

Adding and documenting basic part

Goal: see title Also See: Randy describing ideal way to use registry when adding parts

1. Go back to the [http://parts.mit.edu Registry]
2. Login to the Registry
3. Click on the Add a part
4. Click on the Add a basic part now
   • A basic part is a linear sequence of DNA with a fundamental function like a promoter, terminator, ribosome binding site, CDS etc.
5. Choose Allow edits by your iGEM team
6. Choose the next available part number, or some number within your team's allowed naming range.
7. Enter that number into the Selected Part Name field
8. Choose a part type from the drop down menu. (Coding)
   • Find out what the different part types are by clicking the Browse Part Types link to the left. The question marks next to each part type give a description of that part type.
9. Enter a short description ... e.g. feruloyl-CoA hydratase for vanillin biosynthesis (ech)
10. Enter a long description of the part (you can update this more later)
    • Where species is the enzyme from? Pseudomonas fluorescens
    • What reaction does it catalyze? One of two steps in the bioconversion of ferulic acid to vanillin.
    • Does it work in E. coli? Yes. The authors of the paper have shown that it works already.
    • Does it require any other parts? Yes. Both ech and fcs are needed for vanillin biosynthesis
11. Enter the source of this part
    • Include the species, the GenBank accession number and the paper reference
12. Enter design considerations
    • The stop codon is TGA so we changed it to TAATAA to conform with BioBrick standards
13. Click go on to enter the sequence and add feature annotations

Entering Part Sequence and Feature Information

Goal: see title

1. Return to the [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=40788116 Genbank entry]
2. Click on the [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=40788116&from=1994&to=2824&view=gbwithparts cds] link to get the sequence of the coding region
3. Display it in FASTA format
4. Copy the sequence
5. Paste it into the sequence field and click Save in the upper right
6. Change the TGA stop codon to TAATAA
7. The Registry will automatically check your sequence for BioBricks restriction sites.
   • We will have to remove these in fabricating this part
   • Once the sites have been removed we can edit this information in the Registry
   • See [http://www.openwetware.org/wiki/Site-directed_mutagenesis OpenWetWare protocols on site directed mutagenesis]
8. Click the Add a feature link
9. Enter the start codon, stop codons and coding region as features. Click Submit to save after entering each feature. Add any other features that you think are important!

Reviewing your part

Goal: learn about difference between sandbox and favorite parts, explaining difficulty in finding a part you've just added Also See: Randy describing sandboxes and part promotion

Now you can view the part you've created.

1. Go to the [http://parts.mit.edu Registry]
2. Click Browse Parts by School
3. Click on your school
4. You should see the part in your sandbox

Make a device

Protein coding regions by themselves aren't very useful. We need to make composite parts.

Let's make a protein generator to express our vanilla-scent making enzyme.

Finding existing parts

Goal: find existing poorly- and well-documented Promoter, RBS, and Terminator in registry See Also: ...

1. In a new window, click Browse Part Types on the navigation bar to the left.
2. To express a gene, we need a promoter, RBS, and terminator.
3. Browse the promoters by clicking Regulatory
4. Choose a promoter. The green W indicates that the promoter has been shown to work.
5. Enter the part number into the subpart list.
6. Return to browsing parts, choose an RBS and terminator as well.
7. Enter these part numbers into the subpart list.
8. You should now have 4 part numbers in the subparts field.
9. Click Go on to enter other information about the part

Enter a composite part

Goal: adding and documenting a composite part See Also:

• What is a composite part

1. Go back to the [http://parts.mit.edu Registry]
2. Click on the Add a part
3. Click on the Add a composite part now
   • A composite part is composed of two or more basic parts
4. Choose Allow edits by your iGEM team
5. Choose the next available part number, or some number within your team's allowed naming range.
6. Enter that number into the Selected Part Name field
7. Choose a part type from the drop down menu. (Generator)
   • Find out what the different part types are by clicking the Browse Part Types link to the left. The question marks next to each part type give a description of that part type.
8. Enter a short description ... e.g. feruloyl-CoA hydratase for vanillin biosynthesis protein generator
9. Enter a long description of the part (you can update this more later)
   • What does the device do? Helps to biosynthesize vanillin from ferulic acid
   • What are the inputs and outputs to the device? Takes in ferulic acid and produces vanillin.
   • Are any other devices needed? Also requires the other step of the biosynthesis pathway.
   • Does the device have any chassis dependencies?
10. Enter the source of this part
    • Reference the basic part. You can easily link to any existing part on the wiki by typing <partinfo>Part number</partinfo>.
11. Enter design considerations
    • This device uses a strong constitutive promoter to generate a strong vanilla smell. or This device uses an inducible promoter to get a regulatable vanilla smell.
12. Now we need to enter the subparts. Here you enter the list of basic parts that make up the composite part. Enter the part number of the enzyme you entered previously. But you also need a promoter, RBS and terminator to construct a protein generator.

Constructing a part

There are several option for constructing parts. Meagan has already shown you how to obtain existing parts from the distribution.

To make new parts, you have 2 options

1. Find the original genetic material and PCR the part.
2. Order the part from a gene synthesis company.

To get help on how to make a part

1. Click Help on the navigation bar on the lefthandside of the page.
2. Click on Making BioBrick DNA
3. Scroll to the bottom and click on Biobrick Prefix and Suffix can be found here

Or alternatively,

1. Visit the [http://openwetware.org/wiki/BioBricks BioBricks] page on OpenWetWare
2. And click on [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication Part fabrication]

Making your part by PCR

1. Design primers with the [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication#Quick_reference BioBrick prefix and suffix] on them in order to put the part into BioBrick form.
   • Add the [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication#Prefix BioBricks prefix] to the 5' end of the upstream primer
   • Add the [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication#Suffix BioBricks suffix] to the 5' end of the downstream primer
2. Clone it into a BioBricks plasmid.

Making your part by direct synthesis

Each team has 2kb of synthesis from GENEART for 25 cents a base.

Be sure and add the BioBrick prefix and suffix to your part! You can codon optimize the part to improve its expression in certain species. You can also remove restriction sites from the part.

Construct a device

First we should see if any parts of our system have already been made.

Super & Sub part searching

Goal: become familiar with super- and sub-part searching

1. Go to the [http://parts.mit.edu Registry]
2. Click on Part searches on the navigation bar on the left
3. Type your device part number in the Subpart Search field.
4. Click Search.
5. This will tell you whether any subparts of your system already exist.

Or alternatively, you can look for parts that contain your part of interest.