Synthetic Biology:BioBricks/3A assembly

Introduction

3A assembly (which stands for three antibiotic assembly) is a method for assembling two BioBrick parts together. It differs from the more common assembly approach and the Silver lab approach in that it relies on three way ligation (between the two parts and the backbone vector) for assembly rather than two way ligation between a part and a part + vector molecule. It relies on both positive and negative selection to reduce/eliminate the number of incorrect assemblies that give rise to colonies after transformation.

Motivation

3A assembly was designed such that gel purification of the digested parts is unnecessary. Incorrect assemblies are selected against via either positive or negative selection. The motivations for eliminating the ge purification step are as follows

1. Gel purification is slow and not easily susceptible to automation and scaling. As the registry moves to larger scale assembly of parts, gel purification can be a limiting step in terms of how many assemblies can be done in parallel.
2. Gel purification tends to be inefficient especially on small pieces of DNA. It can be difficult to isolate small pieces of DNA (< 200 bp) from a gel. Thus, when assembling two small pieces of DNA, 3A assembly might prove advantageous.

The primary disadvantages of 3A assembly are that currently the vectors necessary for doing 3A assembly are only available at high copy. Thus, if your assembly generates a construct that places a large burden on the cell at high copy, it may be difficult to assemble using this technique until new vectors are available.

Materials

• Two parts in BioBricks format to be assembled
• A destination vector with a different resistance marker from each of your two parts
  • pSB1AK3-1
  • pSB1AT3-1
  • pSB1AC3-1

Procedure

1. Miniprep your two parts and a destination plasmid.
2. Digest your two parts and destination vector with the following enzymes
   • Prefix part with EcoRI and SpeI
   • Suffix part with XbaI and PstI
   • Destination vector with EcoRI and PstI
3. Purify the restriction digest.
   • For parts < 200 bp in length, do a Micropure EZ and Microcon purification. All other parts and the destination vector can be purified using a QIAquick PCR purification kit.
4. Ligate the two parts and destination vector together.
5. Transform the ligation product.
6. Analyze the transformation with single colony PCR followed by agarose gel electrophoresis.
   • In rolling, large scale assembly, this step is omitted.
7. Miniprep clones that generated a band of the appropriate size.
8. Sequence the clone.

Notes

• This page is intended as a replacement for the original static webpage describing 3A assembly.

Contact

This assembly technique was formulated by Randy Rettberg and implemented by Reshma Shetty.