Revision as of 16:19, 1 April 2009

iGEM 2009 Distribution

This year at iGEM we have made a few important changes to the Registry and the DNA Parts Kit distribution. We have returned to the 384 well format used in 2007, but have taken into consideration the extensive quality control measures used for the 2008 distribution. While we will not be sending out all of the parts in the Registry, we have taken care to create a kit that includes both high-quality parts from the past as well as new parts from 2008 that were designated by teams as favorites. With a smaller number of confirmed quality controlled parts, (3) 384 well plates total, the 2009 kit will be a very effective basis for your synthetic biology projects.

Getting Started with the 2009 DNA Parts Kit

Storage

Make sure to keep the DNA Parts Kit stored with the adhesive foil cover and plastic cover in a -20C freezer when not in use.

Locate the Part

Prior to taking the DNA Parts Kit out for use, it is advised that you know the corresponding well(s) of the part(s) you want. The Registry provides the necessary tools to locate your desired parts within the 384 well plates used in the distribution. It is important to note the necessary information when you find your part, i.e. plasmid and antibiotic resistance(s) of the required part. For more information on locating your part, see below.

Usage

The DNA Parts Kit is very useful tool available to you to perform synthetic biology with standard biological parts. It contains dry DNA of hundreds of parts that are available in the Registry up until 2009. You will transform the DNA into cells and make your own glycerol stocks of any part that you wish, however it does not contain enough DNA to do assembly. To use the DNA in the Parts Kit you may follow these instructions:

1. With a pipette tip, punch a hole through the foil cover into the corresponding well to the Biobrick™-standard part that you want. Make sure you have properly oriented the plate.
2. Add 15uL of diH2O (deionized water)
3. Take 1uL DNA and [http://openwetware.org/wiki/Bacterial_Transformation transform] into your desired competent cells, plate bacteria with the appropriate antibiotic* and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 18 hours.
5. Use the resulting culture to [http://openwetware.org/wiki/Miniprep/Kit-free_high-throughput_protocol miniprep] the DNA AND make your own glycerol stock (for further instruction on making a glycerol see [http://openwetware.org/wiki/Endy:Making_a_long_term_stock_of_bacteria this page]).

*To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.

Locating a Part in the Distribution

Distribution plate orientation

Top view of plates containing dry DNA; red circle indicates well 13H

For the iGEM 2009 distribution, you will receive a set of of 3 Nunc 384-well plates, from which you can extract the part of your choice once you have located it through the Registry repository for iGEM 2009. Unfortunately, the foil cover will obscure both column and well markings. However, you can still find your part by correctly orienting the plate using the two notched corners as markers: well A1 is located at the upper left corner of the plate when the long side of the plate with the notched corners is considered the bottom.

Once you have found the well which your BioBrick™ part of choice is located in in by searching for it through the Registry (for example well 13H in iGEM2007 DNA Parts Kit Plate 1) you want to count across the plate starting with Column 1 until you get to Column 13 and down the plate starting with Row A until you get to Row H.

MAKE SURE that the two notched corners of the plate are oriented at the BOTTOM of the plate (see the top view image at left for correct orientation)

Browsing through the "plates"

1. Go to the Registry.
2. Click on DNA Repositories in the toolbox.
3. Click on Spring 2009 Source Plates. The Spring 2009 Distribution Plates are currently unavailable.
4. Here you will find a list of contents for the plates that will comprise the 2009 Spring DNA Distribution. You can browse through these plates and take a look at all of the Quality Control information.

Locating a particular part

1. Go to the Registry.
2. In the search box on the upper right, enter the part number that you are interested in (e.g. BBa_B0015) and click GO.
3. In the part navigation bar at the top of the page, click on Physical DNA.
4. You will see all of the locations for this part. You are looking for the location in the Spring 2009 Distribution Library
   • Note that we only have physical DNA for parts whose part status (box in the upper right hand corner) reads Available

   

Availability

If you decide to do a targeted search for a particular part that you think you might want to use, you need to make sure that the part is actually available. There are many parts in the Registry that people are still working on, or decided not to continue working on anymore, therefore we never received or don't yet have the physical DNA for them. This of course means that the DNA is not available in 2009 DNA Parts Kit. The simplest way to tell whether the part is available is to look at the top right of the part's Main Page. If the part is available the top part of the box will be green and say "DNA available."

Ordering a part

We've taken care to create a very diverse and extensive DNA Parts Kit this year, including the high quality parts from previous years, and the 2008 parts deemed as favorites by all of the teams. However, should you find the part, or parts, that you require are not available in the distribution but available in the Registry, feel free to send us an email in order to request it. Just include the part name, the plasmid it's located in, and the source, and we'll send it out to you.

Quality Control Process

For this year's distribution we have carried over our extensive quality control measures conducted in 2008, in order to better ensure the accuracy of DNA parts sent to the iGEM 2009 teams. The QC process from 2008 examined each part in the Registry to determine if the plasmid and the part within are correct according to the documentation provided with each submission. The results for each part were then posted on the Registry's DNA repository page, allowing iGEM teams to access this information before using the submissions to create their own parts. This year's DNA Parts Kits contains hundreds of these high quality parts from previous years, as well as the team favorites from 2008, which have all undergone the same quality control measures before inclusion into the 2009 kit.

Streaking for Single Colonies

Once it was decided which part would be assigned to each plate, bacterial stocks containing the parts were streaked for single colonies.

Parts in the Registry prior to iGEM 2008 were kept in glycerol stocks in the Registry's -80C freezer, where they were directly streaked onto petri dishes with appropriate antibiotic and grown overnight at 37C.

The rest of the parts - ones submitted by iGEM 2008 teams - were received as DNA and transformed into competent cells. Once transformed, they were also plated onto petri dishes with their appropriate antibiotic and allowed to incubate overnight at 37C.

Preparation and Inoculation from Staging Plate

The next morning (16 hours of incubation), the petri dishes were removed from the incubator and a single colony was picked from each plate. Using a pipette tip, the chosen colony was put into a corresponding well of a 96 deep well plate filled with the appropriate antibiotic broth, creating a staging plate for these grouped bioparts.

Once all 96 wells of the staging plate were inoculated with single colonies, a 5.0ul pin tool replicator was used to then inoculate from the staging plate to antibiotic testing plates,miniprep plates, and glycerol plates, and the plates were grown for 16 hours, and 10 hours for the glycerols.

Antibiotic testing plates

Four antibiotic testing plates were prepared, testing known bacterial resistance of ampicillin, kanamycin, chloramphenicol, and tetracycline. The plates were grown overnight at 37C, pelleted and examined for resistant growth in wells. The plates are then photographed and added to the DNA repository for 2009 as part of our open quality control process. For the 2009 DNA Parts Kit, this step was applied to the favorite parts of the 2008 teams, but was forgone for the high quality confirmed parts from previous years, all of which had already gone through this process last year.

Glycerol plates

As well as the antibiotic testing plates, two glycerol plates containing 10% glycerol broth and appropriate antibiotic were prepared for the long term storage of the bacterial stocks. One of these plates will remain in the Registry -80C freezer while the other will be stored offsite.

Miniprep plates

Lastly, two miniprep plates were inoculated, and incubated overnight at 37C. The following day, the plates were pelleted, and photographed for entry into the DNA repository for 2009 as part of our open quality control process. Finally the plates underwent miniprepping and were then used to produce all the kits for the 2009 distribution. However, for the favorite parts of the 2008 teams, a portion of the DNA extracted from the cultures was resuspended in TE and 15.0ul of each sample digested and run on an electrophoresis get to examine the size of plasmid and part. In addition, 24.0ul was sent to the Broad Institute for DNA sequencing. The high quality parts from previous years had already gone through this process.