User:Vinoo/Tutorial

Adding a (Basic) Part: Section 1

First, what's a basic part?

  • A basic part is a discrete functional unit of DNA, such as promoter, terminator, ribosome binding site, CDS, etc. It cannot be subdivided into smaller component parts.
  • A sample (physical DNA) for a basic part may be obtained by synthesis, primer extension and PCR, or via other techniques.

Example

We've found the protein coding region for luxC (from Vibrio) on GenBank.

    We can pull out a few details which will be useful as we add this basic part to the Registry
  • Name: luxC acyl-CoA reductase
  • Organism: Vibrio fischeri ES114
  • Gene Type: protein coding
  • Sequence: FASTA

How do I start?

  1. On the Registry's menu, hover over the tools category, and select Add a Part
  2. Select the option to Add a Basic Part Now...
  3. You have now reached the Add a Basic Part page, where you'll need to enter some basic information to add your basic part to the Registry

Adding a (Basic) Part: Section 2

Authorship

  1. Group: Select the group(s) that should have ability to edit this part. You may find that you belong to multiple groups: iGEM team(s), labs, or courses. Each group has a specific part range.
  2. Selected Part Name: Enter a part name within your group's assigned part range. If you belong to multiple groups, be sure to use the part range that belongs to the group you're creating the part for.
  3. Part Type: Choose the part type for your basic part. You can also change this later by going to your part's Hard Information page.

Documentation

  1. Short Description: Enter the short description for your basic part. This is usually the biological or technical short hand for the part's function. You will able to change this by going to your part's Hard Information page.
  2. Long Description: Enter in a detailed description of your part, its functions, and its requirements. The long description will show up on the part's Main Page, and can be edited from there.
  3. Source of this part: Enter the source of this part. You will able to change this by going to your part's Design page.
  4. Design Considerations: Enter in any considerations you may have taken for the part (mutations to remove restriction sites, codon optimization, etc) You will able to change this by going to your part's Design page.
  5. Go on to enter the sequence and add feature annotations: Click this to have your part added to the Registry, but you're not done yet!

Adding a (Basic) Part: Section 3

Sequence & Features





Written by [http://openwetware.org/wiki/User:Reshma_P._Shetty Reshma Shetty] of [http://ginkgobioworks.com Ginkgo BioWorks]

Several [http://2007.igem.org/Podcasts screencasts] illustrating how to add a basic part are available on the [http://2007.igem.org/Podcasts iGEM 2006 wiki]

Coming up with a project

Talk to people

A great way to come up with new ideas is to talk to people. Talk to people in nearby labs about their projects or fun things that they've always wanted to build. Most people have more ideas than they have time so this is a great way to kick off your brainstorming sessions.

Research previous year's projects

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

  1. Watch team presentations from 2009, 2008, [http://2007.igem.org/Presentations 2007], or [http://www.igem2006.com/presentations.htm 2006]
    • You can also download their slides from the same page
  2. You can also check out more information on previous projects by going to [http://2009.igem.org 2009.igem.org]

Make new parts and tools that would make engineering biology easier for future iGEM teams

Some of the most commonly used parts in the Registry are also the most simple. They are the transcriptional terminator B0015, the inducible promoter BBa_F2620, the RBS BBa_B0034 and so on. What do these parts have in common?

Well they

  1. Are available from the Registry. If you don't submit your part, then no one will be able to use it.
  2. Are well-documented. If you take the time to enter all the details re how your part was designed and made, then you make your part more useful to others.
  3. Have some characterization data. The biggest thing missing from the Registry is characterization data on how well each of the parts work. Make some measurements and enter them in so that others can use that info in their own work.

Reuse and characterize existing parts

Engineers often have a slightly different goal than scientists. They are interested in taking a system and making it better -- making it simpler, better understood, more reliable, better characterized etc.

Reusing and characterizing existing parts would make a great project for iGEM 2010.

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 [http://2010.igem.org 2010.igem.org site], click 2010 team wikis
    • In this list you find a link to your team wiki to record information about your team and your chosen project.

Let's make go make a bacterial lava lamp! We're breaking new ground with iGEM home decor!

Design the system

We want to design a bacterial lava lamp. How do we start?

Search the registry

Goal: learn how to search the registry

  1. Go to the Registry (https://parts.igem.org)
  2. Click "Search parts" under Tools
  3. Search for "lava lamp" ... you get no hits
  4. Try a different search term "light" ... now some stuff comes up. The top hit is BBa_I712019 Firefly luciferase
  5. BBa_I712019 is pretty interesting. It produces light when a particular precursor is present.
  6. Hmm, this part is just a protein coding sequence. Let's see if anyone has tried expressing it.
  7. Go back to the "Search parts" page.
  8. Use the third search box, superpart search to find parts that contain BBa_I712019.
  9. It looks like there are only parts with a T7 or CMV promoter, so I'll need to make my own parts to express this luciferase.

Browse the Registry

Goal: learn how to browse for parts

  1. Go to the Registry (https://parts.igem.org)
  2. Click "Catalog of parts & devices"
  3. Here you can browse parts in many ways: by type, by function, by chassis, and by contributor.
  4. Check out

Find parts that are available

  1. To determine how to obtain any particular part, click "Get this Part" on the top right hand side of the part page.
  2. Get BBa_I750016
  3. There are multiple ways that you can obtain particular parts.

Randy will discuss the Registry star system.

Make a part

Annoyingly, the firefly luciferase only works if we supply an exogenous substrate luciferin to the cells. So unless I want to keep adding luciferin to my lava lamp, it's not going to produce light. So I need a different luciferase that makes its own substrate.

After doing some literature searches, I find that there is a bacterial lux operon luxCDABE capable of producing light itself from natural E. coli metabolites, no exogenous substrate needed. Not finding the individual genes in the Registry, let's go make a new part out of luxC.

Find the DNA sequence of a gene

Goal: get the luxC DNA sequence

  1. Go to the [http://www.ncbi.nlm.nih.gov/sites/entrez?db=nuccore&itool=toolbar NCBI nucleotide database]
  2. Search for luxC
  3. Let's check out the [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=retrieve&dopt=full_report&list_uids=3280765&log$=databasead&logdbfrom=nuccore Vibrio fischeri luxC gene]
  4. Click on the [http://www.ncbi.nlm.nih.gov/protein/59482354 protein sequence]
  5. Depending on how you want to make the part, get the nucleotide sequence.
    • If you are going to synthesize the part, use [http://baderlab.bme.jhu.edu/gd/ Gene Design] to reverse translate the amino acid sequence into a nucleotide sequence
      • UPDATE: The Gene Design software doesn't minimize repeats in the coding sequence, it just uses the same codon for each amino acid. So [http://helixweb.nih.gov/dnaworks/ DNAWorks] or DNA2.0's software tends to be a better bet for gene synthesis.
    • If you are going to construct the part by PCR, obtain the natural nucleotide sequence by clicking the [http://www.ncbi.nlm.nih.gov/nuccore/171902372?from=1047306&to=1048745&report=gbwithparts CDS link].
      • Note that the sequence is on the reverse strand. So you'll need to [http://www.bioinformatics.org/sms/rev_comp.html take the reverse complement of it].

Adding and documenting basic part

Goal: Add a and lightly document a basic part to the registry

  1. Go back to the Registry
  2. Click on the Add a part
  3. Click on the Add a basic part now
    • You may be asked to log-in at this point but you can log in and then return to the page where you had been previously.
    • A basic part is a linear sequence of DNA with a fundamental function like a promoter, terminator, ribosome binding site, CDS etc.
  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. (Coding)
    • Find out what the different part types are by visiting the Catalog and browsing parts by type. The question marks next to each part type give a description of that part type.
  8. Enter a short description ... e.g. Long-chain-fatty-acyl-CoA reductase luxC; first enzyme in bacterial luciferase operon
  9. Enter a long description of the part (you can update this more later)
    • Which species is the enzyme from? Vibrio fischeri
    • What reaction does it catalyze? One of five steps in the production of light.
    • Does it work in E. coli? Yes, several groups have used this operon as a reporter in E. coli.
    • Does it require any other parts? Yes. The full luxCDABE operon is needed for light production.
  10. Enter the source of this part
    • Include the species, the GenBank accession number and the paper reference
  11. Enter design considerations
    • The stop codon is TGA so we changed it to TAATAA to conform with BioBrick standards
  12. Click go on to enter the sequence and add feature annotations

Entering part sequence and feature information

Goal: Add the sequence and feature information to a part in the registry

  1. Copy the sequence that you generated previously
  2. Paste it into the sequence field and click Save in the upper right
  3. Change the TGA stop codon to TAATAA
  4. 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]
  5. Click the Add a feature link
  6. 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!

NOTE: that you did not include the BioBrick prefix and suffix in the part sequence that you entered. The prefix and suffix should not be in the part sequence!

Add categories

In the Registry, categories are used to mark parts for inclusion in tables. Many parts will occur in more than one category. For example, luxC can be used in both biosynthesis and as a reporter of gene expression. Thus, you should categorize it both ways by selecting each category in turn from the menu and saving the result.

Reviewing your part

Goal: learn about the difference between sandbox and favorite parts, explaining the difficulty in finding the part you just added

Now you can view the part you've created.

  1. Go to the Registry and click "Browse parts and devices by contributor"
  2. Click on iGEM 2009
  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 LuxC enzyme. Note that entering a composite part is very similar to adding a basic part. However, instead of entering a DNA sequence, you will enter a sequence of parts.

Enter a composite part

Goal: adding and documenting a composite part

  1. Go back and browse the Registry to find the part numbers of a ribosome binding site and terminator of your own selection
  2. Go back to the Registry
  3. Click on the Add a part
  4. Click on the Add a composite part now
    • A composite part is composed of two or more basic parts
  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. (Generator)
  9. Enter a short description ... e.g. luxC for light production protein generator
  10. Enter a long description of the part (you can update this more later)
    • What does the device do? Helps to produce light
    • What are the inputs and outputs to the device? Takes in a long-chain aldehyde + CoA + NADP(+) and produces a long-chain acyl-CoA + NADPH.
    • Are any other devices needed? Also requires the other genes of the bacterial luciferase pathway.
    • Does the device have any chassis dependencies?
  11. 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>.
  12. Enter design considerations
    • This device uses a strong RBS to drive translation of luxC.
  13. Now we need to enter the subparts. Here you enter the list of basic parts that make up the composite part. Enter the part numbers of the RBS, the luxC enzyme you entered previously and a terminator.

Constructing a part

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

Making your part by PCR

Ginkgo offers a [http://ginkgobioworks.com/cgi/primer.cgi primer design tool] for designing primers to your part of interest.

Synthesize your primers, PCR the part and then clone it into one of the plasmid backbones available from the Registry.

Making your part by direct synthesis

Alternatively, DNA synthesis companies can synthesize your part for you. This is particularly useful if you don't have access to the source DNA or need to codon optimize your part.

Before you order your part, 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.

Assembling parts

Standard assembly

  • Ginkgo has a [http://ginkgobioworks.com/support/ step by step manual] for doing BioBrick Assembly. It uses the [http://www.neb.com/nebecomm/products/productE0546.asp BioBrick Assembly Kit from NEB and Ginkgo].
  • Alternatively, you can [http://ginkgobioworks.com/bricklayer.html outsource assembly to Ginkgo].

Plasmid backbones

There are many plasmid backbones available from the Registry. You can learn more about them here. To comply with iGEM requirements, teams must send their parts to the Registry in a Registry-supported plasmid backbone. Keep this in mind as you plan your work with standard parts.

Alternative assembly standards

Measuring parts

To be added.

Sending parts to the Registry

To be added.