Gibson Assembly in pydna

Visit the full library documentation here

Gibson Assembly is a powerful method to assemble multiple DNA fragments into a single, continuous sequence in a seamless, one-step reaction. Developed by Daniel Gibson and colleagues in 2009, this method has been widely applied to work in molecular cloning, biotechnology, and synthetic biology.

pydna provides the Assembly class to simulate the assembly of DNA sequences. Below is an example fpr performing Gibson Assembly with pre-existing DNA fragments, followed by primer design for generating these fragments via the pcr method, if needed.

The Assembly takes the following arguments:

  • frags: list of DNA fragments as Dseqrecord objects

  • limit: the minimum sequence homology required.

  • algorithm: the function used to find homology regions between DNA fragments. For Gibson Assembly, we use the terminal_overlap function, which finds homology regions only at the terminal regions. By default, the Assembly class uses the common_sub_strings function to find homology regions, which finds homology anywhere, as it could happen in a homologous recombination event.

Open In Colab 
# Install pydna (only when running on Colab)
import sys
if 'google.colab' in sys.modules:
    %%capture
    # Install the current development version of pydna (comment to install pip version)
    !pip install git+https://github.com/BjornFJohansson/pydna@dev_bjorn
    # Install pip version instead (uncomment to install)
    # !pip install pydna


from pydna.dseqrecord import Dseqrecord
from pydna.assembly import Assembly
from pydna.common_sub_strings import terminal_overlap

#Creating example Dseqrecord sequences
fragment1 = Dseqrecord("acgatgctatactgCCCCCtgtgctgtgctcta")
fragment2 = Dseqrecord("tgtgctgtgctctaTTTTTtattctggctgtatc")
fragment3 = Dseqrecord("tattctggctgtatcGGGGGtacgatgctatactg")

#Creating a list of sequences to assemble
fragments = [fragment1, fragment2, fragment3]

#Performing Gibson assembly, with a minimum shared homology of 14bp
assembly = Assembly(fragments, limit=14, algorithm=terminal_overlap)

#Displaying the assembled product
print(assembly)

Assembly
fragments..: 33bp 34bp 35bp
limit(bp)..: 14
G.nodes....: 6
algorithm..: terminal_overlap

The printed output shows the length of each fragment provided to the assembly, the minimum length required for sequence homology search, the number of nodes (number of overlapping regions), and the algorithm used for sequence homology search. Please refer to the full Assembly module documentation for more information on the algorithm applied.

To make a circular sequence from an Assembly, pydna provides the assemble_circular method. The assembled sequence can be printed as normal, as Dseqrecord objects. Note that the assemble_circular method returns a list, where the two elements are reverse complement of each other.

from pydna.contig import Contig

#Circularizing the assembled sequence
assembly_circ = assembly.assemble_circular()

#Printing the sequence records
print(assembly_circ[0])
print()
print(assembly_circ[1])


Dseqrecord
circular: True
size: 59
ID: id
Name: name
Description: description
Number of features: 0
/molecule_type=DNA
Dseq(o59)
acga..GGGt
tgct..CCCa

Dseqrecord
circular: True
size: 59
ID: id
Name: name
Description: description
Number of features: 0
/molecule_type=DNA
Dseq(o59)
taga..AAAA
atct..TTTT

Please refer to the Example_Gibson page for an example of a completed workflow for modelling Gibson Assembly using pydna.