In ipb-halle/GoldenMutagenesis: A tool to calculate and validate primers for Golden Gate Cloning

Point Mutagenesis

You can find the lastest version of this file at https://github.com/ipb-halle/GoldenMutagenesis/blob/master/vignettes/Point_Mutagenesis.md

knitr::opts_chunk$set(echo = TRUE)

Experimental Workflow

Target sequence

open reading frame mCherry in pet28a (Kanamycin resistance)

Clone into

pAGM9121

Genomic sequence mCherry

ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCG TGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGT GACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGT GAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTC GAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCG GCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACGATGGGCTGGGAGGCCTCCTCCGAGCGGATGT ACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGT CAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCC CACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGT ACAAGGTCGACAAGCTTGCGGCCGCACTCGAGTGA

Restriction Enzyme

BbsI

Recognition site: GAAGAC

Envisioned Mutation

Leucine66 to Valine (point mutation)

CTG -> GTG

R Workflow

library("GoldenMutagenesis")

input_sequence<-"ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACGATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGGTCGACAAGCTTGCGGCCGCACTCGAGTGA"
recognition_site_bbsi<-"GAAGAC"
recognition_site_bsai<-"GGTCTC"
cuf<-"e_coli_316407.csv"

The domesticate function checks for internal cleavage sites. If corresponding sites are present silent mutations are introduced to destroy the recognition sites. The functions returns a list containing the position of the choosen amino acid residue for silent mutation.

mutations_bbsi<-domesticate(input_sequence, recognition_site_bbsi, cuf)
mutations_bbsi
mutations_bsai<-domesticate(input_sequence, recognition_site_bsai, cuf)
mutations_bsai

The mutate_spm function designs the necessary set of primers for the desired mutations.\ \ The function has the following parameters:\ prefix: Additional nucleobases in 5' position of the recognition site [default: TT]\ restriction_enzyme: Recognition site sequence of the respective restriction enzyme [default: GGTCTC]\ suffix: Spacer nucleotides matching the cleavage pattern of the enzyme [default: A]\ vector: Four basepair overhangs complementary to the created overhangs in the acceptor vector [default: c("AATG", "AAGC")]\ replacements: The desired substitutions\ binding_min_length: The minimal threshold value of the template binding sequence [default: 4]\ binding_max_length: Maximal length of the binding sequence [default: 9]\ target_temp: Melting temperature of the binding sequence in °C [default: 60]\ cuf: The Codon Usage Table which is being used to select the codon for an exchanged amino acid. [default: e_coli_316407.csv]\ fragment_min_size: The minimal size of a generated gene fragment in base pairs [default: 100]\ \ It will return an object of the class primer_set.

mutations<-c(list(c(66, "V")), mutations_bbsi)
primers<-mutate_spm(input_sequence, prefix="TT", restriction_enzyme = recognition_site_bbsi, suffix = "AA", vector=c("CTCA", "CTCG"), replacements = mutations, binding_min_length=4 ,binding_max_length = 9, target_temp=60, fragment_min_size = 60, cuf=cuf)
primers

Objects of the classes "primer", "primer_msd" and "primer_set" can have a slim textual output by using the function print_primer.

print_primer(primers)

The textual output can be printed into a file.

sink("primers.txt", append=FALSE, split=FALSE)
print_primer(primers)
sink()

ipb-halle/GoldenMutagenesis documentation built on Sept. 1, 2020, 3:24 p.m.