Arking:JCAOligoTutorial4

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DBBS Method of Composite Part Assembly

The general idea of Biobricks is that you have a collection of basic parts, and then you assemble them into composite parts by some method of general assembly. DBBS (it stands for Digested Biobricks) is a PCR-based method that is very simple and efficient to implement. However, there are many other usable assembly methods. This or the three-antibiotic method are probably you're best choices for doing BBa-type assembly (XbaI/SpeI style). If you are doing BBb (BglII/BamHI), the 1-2-3 method is by far the best option, and will be described in a later tutorial section. The dbbs method assumes you have your basic parts in one of the following plasmids:

 BBa                    BBb
 pSB1A2                 pBca9145
 pSB1A3                 pBca1100
 pSB1AK3                pBca1101
 pSB1AC3                pBca1102
 pSB1AT3
 pBca1020
 pBca1021          (and there will undoubtedly be more made)

All that is really necessary is that oligos ca998 and G00101 have annealing sites outside the Biobrick polylinker, and the Bla gene is present and in the same orientation in both plasmids.

Generally, you're going to PCR amplify half the plasmid for the two parts you want to combine. You then digest the PCR products, ligate them together, and transform.

Implementation

Here is the general construction file for DBBS:

 Oligo1   Oligo2     Template      Digest
 ca1099F  G00101     Left Part     BamHI/BsaI/DpnI
 ca998    ca1099R    Right Part    BglII/BsaI/DpnI

Set up the two Expand PCRs on 25uL scale. Calculate the size of the product and run the appropriate program for that length (usually 4K55).

Run a Zymo column to remove the buffer and polymerase. Elute with 90uL water. Set up 100uL restriction digests as:

 88uL DNA+water
 10uL NEB Buffer 2
 1 uL BamHI or BglII
 1 uL BsaI
 1 uL DpnI

Tap them to mix. It is imperative that you mix them well. If the DpnI doesn't get mixed, parent vector will bleed through resulting in parent vector side products. Incubate the reaction at 37deg for 1hr.

Run another Zymo column, elute the reaction in 30uL of water. These are the "dbbs".

Set up ligations:

 6.5uL water
 1uL 10x T4 DNA Ligase Buffer
 1uL dbbs 1
 1uL dbbs 2
 0.5 uL T4 DNA Ligase

Transform, plate on a carbenicillin plate.

Storage

The dbbs digests will last forever if stored at -20. Since you only used 1 of 30uL of the material, you can do many assemblies with these parts. You'll log the dbbs into a google spreadsheet and place them in a special box for future use.


If you have any comments or want to report a potential error in the tutorial, please email me (Chris Anderson) at JCAnderson2167-at-gmail.com

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