Help:3A Assembly Kit

Revision as of 16:55, 2 May 2012 by Kitwa (Talk | contribs)

We're currently hard at work on the 3A Assembly Kit manual. We've added preliminary protocols for all of the necessary steps so you can read through them for now!

The 3A Assembly Kit
You've just received the 3A Assembly Kit! We hope your team will enjoy using the kit and in the process learn about synthetic biology based on standard parts and 3A assembly. Before you start please make sure to...
  • check the contents of the box with the inventory, to make sure you have everything
  • inspect the materials to make sure nothing has been damaged during shipping
  • refrigerate the agar plates and any materials labeled as requiring storage at 4C.
  • order the NEB reagent kit. You'll need to use the NEB enzymes and reagents for 3A assembly. See the High School Division sponsor page for more information.


If you're interested in using the 3A Assembly Kit, and would like to order one, please contact hq (at) igem (dot) org.


Introduction

The 3A Assembly Kit and following protocols will take you through the process of 3A Assembly, and by the end you will have assembled your own composite part in the lab. The kit includes two parts: Part A (BBa_J04500) and Part B (BBa_J04650), which when assembled together will form a RFP (red fluorescent protein) generator. Your cells will turn red!

Note: The 3A Assembly Kit also includes prepared plasmid DNA for Part A and Part B, so you can skip directly to Step #3: Restriction Digest if you'd like.


Step #1: Growing

Grow up the E. coli that contains your parts!
Your kit includes agar stabs for Part A and Part B. The agar stabs contain live bacteria (E. coli) that house your parts. The parts themselves belong to a plasmid which replicates within the E. coli cell.

Before working with your parts, you'll need to make sure you have enough copies of them. You can do this by growing up cell cultures from the agar stabs: more E.coli cells means more plasmids, which means more copies of your parts. Use the provided agar stabs to streak out the bacteria containing your parts onto agar plates. Then pick a single colony to grow up a cell culture.

The 3A Assembly Kit also includes an agar stab with the NEB 10-beta strain that you'll need to make competent cells for '''Step #5: Transformation.''' Please be sure to follow the growing instructions for the NEB 10-beta strain, as you'll need to prepare these two days in advance to '''Step #5: Transformation.'''
Step #2: Miniprep

Miniprepping cell cultures to extract the DNA for your parts.
You now have cell cultures of E. coli containing lots of copies of Part A and Part B. In order to access the plasmid DNA containing your part, you'll need to lyse the cells (make them burst open), separate the DNA from the cellular material, and purify the sample to make sure you only have the plasmid DNA and not the E. coli's own genomic DNA. The 3A Assembly Kit includes the reagents and tubes required for the Miniprep step, however, a centrifuge is required.

If you would simply like to skip the Miniprep step, the 3A Assembly Kit includes prepared plasmid DNA for '''Part A''' and '''Part B''', so you can skip directly to '''Step #3: Restriction Digest.'''

Step #3: Restriction Digest
Cutting the DNA for your parts and the pSB1C3 linearized plasmid backbone in a restriction digest
Now that you have Part A and Part B, you'll want to cut them out from their pSB1AK3 plasmid backbones. You'll use restriction enzymes (provided by NEB) to cut the parts out. This will leave small overhangs at the end of each part which will eventually act like connectors to assembled them together.
Step #4: Ligation
Ligating your two parts together into the pSB1C3 linearized backbone
After your restriction digest you'll have Part A, Part B, and the pSB1C3 linearized plasmid backbone cut. Now you'll need to assemble all of these components together (Part A + Part B, into pSB1C3). You'll do this through the use of the overhangs (connectors) you've created by cutting the DNA with restriction enzymes and ligase, which will keep everything glued together.
Step #5: Transformation
Transforming the ligation to get your newly assembled composite part. At the end you should see red colonies!
You now have an assembled product through ligation (Part A + Part B, in pSB1C3). To get this new part into E. coli cells, you'll need to use a process called transformation. You'll need to make the provided NEB 10-beta strain competent, so it can uptake your ligated product (the plasmid containing your new part). Once the cells have taken in your plasmid, you'll grow them on a chloramphenicol plate (only cells containing the pSB1C3 plasmid backbone will live), and from those cells that live, there will be red colonies that have the correct new part.
Step #6: Results
Post your results!
Hopefully you've learned quite a bit while using the 3A Assembly Kit. Use the Results page to post what happened during your assembly.