IGEM:Help/Team experience tutorial

Coming up with a project

Research previous year's projects

1. Go to http://igem2007.com
2. Click on Watch team presentations from iGEM 2006
   • Here you can watch team presentations and view the slides
3. You can also check out more information on previous projects by going to http://parts.mit.edu/igem
   1. Click on Meet the 37 teams
   2. Click on MIT and read about the project

Describe your project

1. Decided on an idea for your new project?
   • From the iGEM2007.com site, click Visit the iGEM07 Wiki
   • Click 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

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 ([1])
4. Search for vanillin ... ([2])
5. No one has made a vanilla synthesizing enzyme ... phew!

Search the literature

1. Visit PubMed
2. Search for vanilla
3. Find a relevant paper
4. Go read the 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
7. Search for Genbank accession numbers
8. Follow the Genbank link

Make a part

Enter a basic part

1. Go back to the 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
14. Return to the Genbank entry
15. Click on the cds link to get the sequence of the coding region
16. Display it in FASTA format
17. Copy the sequence
18. Paste it into the sequence field and click Save in the upper right
19. Change the TGA stop codon to TAATAA
20. 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 OpenWetWare protocols on site directed mutagenesis
21. Click the Add a feature link
22. 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

Now you can view the part you've created.

1. Go to the 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.

Enter a composite part

1. Go back to the 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.

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.

Finding existing parts

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

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 BioBricks page on OpenWetWare
2. And click on Part fabrication

Making your part by PCR

1. Design primers with the BioBrick prefix and suffix on them in order to put the part into BioBrick form.
   • Add the BioBricks prefix to the 5' end of the upstream primer
   • Add the 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.

1. Go to the 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.