Introduction

Synthetic biology involves building novel genes or assembling natural genes from shorter DNA fragments. It generally requires multiple hierarchical levels of assemblies starting from the smallest building blocks of short oligonucleotides to eventually reach a full-length genome. Picky software helps to improve the limitation on the number of fragments that can be assembled in a single step. Given a DNA sequence, Picky predicts the set of short fragments that can be assembled at once and are optimized on the basis of either the cost or fragment count parameters. 

Gibson Assembly

In the Gibson Assembly, three different DNA enzymes are optimally mixed together to assemble double-stranded DNA fragments: 1) a 5’ exonuclease, which shortens the 5’ end of DNA fragments and exposes a single-stranded 3’ overhang that can anneal to the other exposed DNA strands; 2) a DNA polymerase that fills in the missing DNA nucleotides after two strand annealing to repair the gaps; and 3) a DNA ligase that covalently repairs the nicks between two adjacent DNA fragments to make a single DNA molecule.

Using PICKY, one can design the optimum high-number set of fragments to be assembled to a full-length DNA sequence using the Gibson Assembly master mix (NEB #E2011) or the NEBuilder HiFi Assembly master mix (NEB #E2621).

DNA fragment design

Download the special Picky version that allows high-number probe design per gene. If you have not registered yet, register your email to receive your password before you download. Load target genes into Picky and click on its Probe design button. A design parameter window will show up. Picky was originally developed for microarray design, so its parameters were named in that context; we are using Picky for gene assembly fragment design so the parameter settings below are relevant to that purpose. Set the maximum and minimum oligo sizes to 20 or a different value for the preferred fragment junction size, the number of probe candidates to 200, and the number of probes per gene to 100. The latter two parameters should be large enough to instruct Picky to find all probe candidates that qualified as junctions, because generally fewer junctions can be found per gene. Also set minimum match length to 7, minimum trigger similarity to 66% and salt concentration to 500 mM. These parameters increase the sensitivity level of Picky and match Gibson Assembly buffer condition better. Leave the rest of the Picky parameters in their default values. After the computation, save Picky probe design to an output file. Picky will create two files ending in .picky and .report; the .picky file will be used for the next step.

Next step is to download the Perl program (break_up_sequences_adding_restriction_sites) and run it on the .picky file obtained in the previous step. This program takes 5 parameters: 1) the optimization goal for fragment count or synthesis cost (the oligonucleotide cost formula is built into the program and can be modified), 2) the minimum acceptable fragment length, 3) the maximum acceptable fragment length, 4) the .picky filename created from the previous step, and 5) the original gene sequences analysed by Picky. This program either reports an optimal DNA fragment set for each target gene, or reports failure in finding such a set given the limited number of thermodynamically unique junctions on certain genes. Users can adjust some parameters, usually by allowing longer DNA fragments, to try to obtain a working fragment set. Optimization for cost involves exhaustive search and may take a very long time for certain junction distributions, whereas optimization for fragment count can always be efficiently performed.