clone

Documentation

Overview

Package clone provides functions for cloning DNA sequences.

Since 1973, the most common way to make recombinant DNA has been restriction enzyme cloning (though lately, homologous recombination based methods like Gibson assembly have attracted a lot of use). The cloning functions here allow for simulation of restriction enzyme cloning.

For a historical review leading up to the discovery: https://doi.org/10.1073/pnas.1313397110

The idea of restriction enzyme cloning is that you can cut DNA at specific locations with restriction enzyme and then glue them back together in different patterns using ligase. The final product is (99.9% of the time) a circular plasmid that you can transform into a bacterial cell for propagation.

While simulation is simple for simple cases, there are a lot of edge cases to handle, for example:

• Which input sequences are circular? How do we handle their rotations?
• Is the enzyme that is cutting directional? How do we handle that directionality?
• Are there multiple possible outputs of our ligation reaction? For example, ligations may be able to create a "library" of plasmids, in which there are millions of valid combinations.
• How do we handle sequences that get ligated in multiple orientations?

These cloning functions handle all those problems so that they appear simple to the end user.

In particular, there is a focus here on GoldenGate Assembly: https://en.wikipedia.org/wiki/Golden_Gate_Cloning https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/golden-gate-assembly

GoldenGate is a particular kind of restriction enzyme cloning reaction that you can do in a single tube and that is extraordinarily efficient (up to 50 parts) and is popular for new modular DNA part toolkits. Users can easily simulate GoldenGate assembly reactions with just their input fragments + the enzyme name.

Let's build some DNA!

Keoni

PS: We do NOT (yet) handle restriction enzymes which recognize one site but cut in multiple places (Type IIG enzymes) such as BcgI.

Index

• func CircularLigate(fragments []Fragment) ([]string, []string, error)
• func GoldenGate(sequences []Part, enzymeStr string) ([]string, []string, error)
• type Enzyme
• type Fragment
  • func CutWithEnzyme(seq Part, directional bool, enzyme Enzyme) []Fragment
  • func CutWithEnzymeByName(seq Part, directional bool, enzymeStr string) ([]Fragment, error)
• type Overhang
• type Part

Examples

• GoldenGate

Functions

func CircularLigate

func CircularLigate(fragments []Fragment) ([]string, []string, error)

CircularLigate simulates ligation of all possible fragment combinations into circular plasmids.

func GoldenGate

func GoldenGate(sequences []Part, enzymeStr string) ([]string, []string, error)

GoldenGate simulates a GoldenGate cloning reaction. As of right now, we only support BsaI, BbsI, BtgZI, and BsmBI.

Example

package main

import (
	"fmt"

	"github.com/TimothyStiles/poly/clone"
	"github.com/TimothyStiles/poly/seqhash"
)

// pOpen plasmid series (https://stanford.freegenes.org/collections/open-genes/products/open-plasmids#description). I use it for essentially all my cloning. -Keoni
var popen = clone.Part{"TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGGCCTACTATTAGCAACAACGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACCTGCACCAGTCAGTAAAACGACGGCCAGTAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTTGGTTCAGGTGGAGTGGGAGTAgtcttcGCcatcgCtACTAAAagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtatgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGgcgatgttgaagaccaCGCTGAGGTGTCAATCGTCGGAGCCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCATGGTCATAGCTGTTTCCTGAGAGCTTGGCAGGTGATGACACACATTAACAAATTTCGTGAGGAGTCTCCAGAAGAATGCCATTAATTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG", true}

func main() {
	// Fragment 1 has a palindrome at its start. This isn't very common but
	// can occur. These two fragments are real DNA fragments used in the
	// FreeGenes Project. They are used because they were on my computer
	// - Keoni
	fragment1 := clone.Part{"GAAGTGCCATTCCGCCTGACCTGAAGACCAGGAGAAACACGTGGCAAACATTCCGGTCTCAAATGGAAAAGAGCAACGAAACCAACGGCTACCTTGACAGCGCTCAAGCCGGCCCTGCAGCTGGCCCGGGCGCTCCGGGTACCGCCGCGGGTCGTGCACGTCGTTGCGCGGGCTTCCTGCGGCGCCAAGCGCTGGTGCTGCTCACGGTGTCTGGTGTTCTGGCAGGCGCCGGTTTGGGCGCGGCACTGCGTGGGCTCAGCCTGAGCCGCACCCAGGTCACCTACCTGGCCTTCCCCGGCGAGATGCTGCTCCGCATGCTGCGCATGATCATCCTGCCGCTGGTGGTCTGCAGCCTGGTGTCGGGCGCCGCCTCCCTCGATGCCAGCTGCCTCGGGCGTCTGGGCGGTATCGCTGTCGCCTACTTTGGCCTCACCACACTGAGTGCCTCGGCGCTCGCCGTGGCCTTGGCGTTCATCATCAAGCCAGGATCCGGTGCGCAGACCCTTCAGTCCAGCGACCTGGGGCTGGAGGACTCGGGGCCTCCTCCTGTCCCCAAAGAAACGGTGGACTCTTTCCTCGACCTGGCCAGAAACCTGTTTCCCTCCAATCTTGTGGTTGCAGCTTTCCGTACGTATGCAACCGATTATAAAGTCGTGACCCAGAACAGCAGCTCTGGAAATGTAACCCATGAAAAGATCCCCATAGGCACTGAGATAGAAGGGATGAACATTTTAGGATTGGTCCTGTTTGCTCTGGTGTTAGGAGTGGCCTTAAAGAAACTAGGCTCCGAAGGAGAGGACCTCATCCGTTTCTTCAATTCCCTCAACGAGGCGACGATGGTGCTGGTGTCCTGGATTATGTGGTACGCGTCTTCAGGCTAGGTGGAGGCTCAGTG", false}
	fragment2 := clone.Part{"GAAGTGCCATTCCGCCTGACCTGAAGACCAGTACGTACCTGTGGGCATCATGTTCCTTGTTGGAAGCAAGATCGTGGAAATGAAAGACATCATCGTGCTGGTGACCAGCCTGGGGAAATACATCTTCGCATCTATATTGGGCCACGTCATTCATGGTGGTATCGTCCTGCCGCTGATTTATTTTGTTTTCACACGAAAAAACCCATTCAGATTCCTCCTGGGCCTCCTCGCCCCATTTGCGACAGCATTTGCTACGTGCTCCAGCTCAGCGACCCTTCCCTCTATGATGAAGTGCATTGAAGAGAACAATGGTGTGGACAAGAGGATCTCCAGGTTTATTCTCCCCATCGGGGCCACCGTGAACATGGACGGAGCAGCCATCTTCCAGTGTGTGGCCGCGGTGTTCATTGCGCAACTCAACAACGTAGAGCTCAACGCAGGACAGATTTTCACCATTCTAGTGACTGCCACAGCGTCCAGTGTTGGAGCAGCAGGCGTGCCAGCTGGAGGGGTCCTCACCATTGCCATTATCCTGGAGGCCATTGGGCTGCCTACTCATGATCTGCCTCTGATCCTGGCTGTGGACTGGATTGTGGACCGGACCACCACGGTGGTGAATGTGGAAGGGGATGCCCTGGGTGCAGGCATTCTCCACCACCTGAATCAGAAGGCAACAAAGAAAGGCGAGCAGGAACTTGCTGAGGTGAAAGTGGAAGCCATCCCCAACTGCAAGTCTGAGGAGGAAACCTCGCCCCTGGTGACACACCAGAACCCCGCTGGCCCCGTGGCCAGTGCCCCAGAACTGGAATCCAAGGAGTCGGTTCTGTGAAGAGCTTAGAGACCGACGACTGCCTAAGGACATTCGCTGCGTCTTCAGGCTAGGTGGAGGCTCAGTG", false}

	Clones, _, _ := clone.GoldenGate([]clone.Part{fragment1, fragment2, popen}, "BbsI")

	fmt.Println(seqhash.RotateSequence(Clones[0]))
}

Output:

AAAAAAAGGATCTCAAGAAGGCCTACTATTAGCAACAACGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACCTGCACCAGTCAGTAAAACGACGGCCAGTAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTTGGTTCAGGTGGAGTGGGAGAAACACGTGGCAAACATTCCGGTCTCAAATGGAAAAGAGCAACGAAACCAACGGCTACCTTGACAGCGCTCAAGCCGGCCCTGCAGCTGGCCCGGGCGCTCCGGGTACCGCCGCGGGTCGTGCACGTCGTTGCGCGGGCTTCCTGCGGCGCCAAGCGCTGGTGCTGCTCACGGTGTCTGGTGTTCTGGCAGGCGCCGGTTTGGGCGCGGCACTGCGTGGGCTCAGCCTGAGCCGCACCCAGGTCACCTACCTGGCCTTCCCCGGCGAGATGCTGCTCCGCATGCTGCGCATGATCATCCTGCCGCTGGTGGTCTGCAGCCTGGTGTCGGGCGCCGCCTCCCTCGATGCCAGCTGCCTCGGGCGTCTGGGCGGTATCGCTGTCGCCTACTTTGGCCTCACCACACTGAGTGCCTCGGCGCTCGCCGTGGCCTTGGCGTTCATCATCAAGCCAGGATCCGGTGCGCAGACCCTTCAGTCCAGCGACCTGGGGCTGGAGGACTCGGGGCCTCCTCCTGTCCCCAAAGAAACGGTGGACTCTTTCCTCGACCTGGCCAGAAACCTGTTTCCCTCCAATCTTGTGGTTGCAGCTTTCCGTACGTATGCAACCGATTATAAAGTCGTGACCCAGAACAGCAGCTCTGGAAATGTAACCCATGAAAAGATCCCCATAGGCACTGAGATAGAAGGGATGAACATTTTAGGATTGGTCCTGTTTGCTCTGGTGTTAGGAGTGGCCTTAAAGAAACTAGGCTCCGAAGGAGAGGACCTCATCCGTTTCTTCAATTCCCTCAACGAGGCGACGATGGTGCTGGTGTCCTGGATTATGTGGTACGTACCTGTGGGCATCATGTTCCTTGTTGGAAGCAAGATCGTGGAAATGAAAGACATCATCGTGCTGGTGACCAGCCTGGGGAAATACATCTTCGCATCTATATTGGGCCACGTCATTCATGGTGGTATCGTCCTGCCGCTGATTTATTTTGTTTTCACACGAAAAAACCCATTCAGATTCCTCCTGGGCCTCCTCGCCCCATTTGCGACAGCATTTGCTACGTGCTCCAGCTCAGCGACCCTTCCCTCTATGATGAAGTGCATTGAAGAGAACAATGGTGTGGACAAGAGGATCTCCAGGTTTATTCTCCCCATCGGGGCCACCGTGAACATGGACGGAGCAGCCATCTTCCAGTGTGTGGCCGCGGTGTTCATTGCGCAACTCAACAACGTAGAGCTCAACGCAGGACAGATTTTCACCATTCTAGTGACTGCCACAGCGTCCAGTGTTGGAGCAGCAGGCGTGCCAGCTGGAGGGGTCCTCACCATTGCCATTATCCTGGAGGCCATTGGGCTGCCTACTCATGATCTGCCTCTGATCCTGGCTGTGGACTGGATTGTGGACCGGACCACCACGGTGGTGAATGTGGAAGGGGATGCCCTGGGTGCAGGCATTCTCCACCACCTGAATCAGAAGGCAACAAAGAAAGGCGAGCAGGAACTTGCTGAGGTGAAAGTGGAAGCCATCCCCAACTGCAAGTCTGAGGAGGAAACCTCGCCCCTGGTGACACACCAGAACCCCGCTGGCCCCGTGGCCAGTGCCCCAGAACTGGAATCCAAGGAGTCGGTTCTGTGAAGAGCTTAGAGACCGACGACTGCCTAAGGACATTCGCTGAGGTGTCAATCGTCGGAGCCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCATGGTCATAGCTGTTTCCTGAGAGCTTGGCAGGTGATGACACACATTAACAAATTTCGTGAGGAGTCTCCAGAAGAATGCCATTAATTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG

Types

type Enzyme

type Enzyme struct {
	Name            string
	RegexpFor       *regexp.Regexp
	RegexpRev       *regexp.Regexp
	Skip            int
	OverhangLen     int
	RecognitionSite string
}

Enzyme is a struct that represents restriction enzymes.

type Fragment

type Fragment struct {
	Sequence        string
	ForwardOverhang string
	ReverseOverhang string
}

Fragment is a struct that represents linear DNA sequences with sticky ends.

func CutWithEnzyme

func CutWithEnzyme(seq Part, directional bool, enzyme Enzyme) []Fragment

CutWithEnzyme cuts a given sequence with an enzyme represented by an Enzyme struct.

func CutWithEnzymeByName

func CutWithEnzymeByName(seq Part, directional bool, enzymeStr string) ([]Fragment, error)

CutWithEnzymeByName cuts a given sequence with an enzyme represented by the enzyme's name. It is a convenience wrapper around CutWithEnzyme that allows us to specify the enzyme by name.

type Overhang

type Overhang struct {
	Length   int
	Position int
	Forward  bool
}

Overhang is a struct that represents the ends of a linearized sequence where Enzymes had cut.

type Part

type Part struct {
	Sequence string
	Circular bool
}

Part is a simple struct that can carry a circular or linear DNA sequence. In the field of synthetic biology, the term "DNA Part" was popularized by the iGEM competition http://parts.igem.org/Main_Page , so we use that term here.