M13 redesign, expt'l details

User:Nkuldell:M13 redesign

Vectors

M13K07 plasmid map, from NEB

• M13KE [[1]]
• pUC118 [[2]] or 119 [[3]]
• M13K07 (helper phage)[|sequence][[4]]

File:M13KO7 zerocutters.txt
File:M13KO7 singlecutters.txt
File:M13KO7 2cutters.txt

Possible M13K07 edits

Sequence	code on plasmid map	edit
PS (packing sequence)	?	?
bact ori	"e"	to pSCAN ori
gII and gX	"c" and "g"	unstuff
gV	"i"	different ssBP
gVII	"k"	add loop to be presented on phage head
gIX and gVIII	"l" and "j"	remove overlap of gIX stop with gVIII start
gVIII and gIII	"j" and "b"	add epitope tags
gVI	"f"	add loop to be presented on phage tail
gI and gXI	"d" and "h"	unstuff, and/or add aa substitutions to make maturation PS-indep
gIV	"a"	titrate promoter to vary pore concentration

Primers

For M13K07 manipulations

• First need to delete Pst site from M13K07 polylinker

these oligos should both destroy Pst and add unique EcoRI to position 8083

• NO133: ChangePst2RI_top
  
  • 5'ATAG AAT TCT ATG CA
• NO134: ChangePst2RI_btm
  
  • 5'ACG TTAT CTT AAG AT
    

• Next step is to add PstI to Petrenko site

use site directed mutagenesis to change GCT to GCA (silent change at Ala, first residue in mature protein)

• NO135: AddPst2gVIII_fwd
  
  • CGT TCCGATGCTG TCTTTCGCTG CaGAGGGTGA CGATCCCGCA AAAGCGGCC
  • Tm 74°
• NO136: AddPst2gVIII_rev
  • GGC CGC TTT TGC GGG ATC GTC ACC CTC TGC AGC GAA AGA CAG CAT CGG AAC G
  • Tm 74°

For M13K07 sequencing

• NO137: M13KO7_seq1100_fwd
  • TAAGTAACATG GAGCAGGTCG CGGATTTCGA CAC
  • Tm 65°