User:Nkuldell:M13 redesign

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(Due Diligence)
Line 74: Line 74:
====to delete Pst site from M13K07 polylinker====
====to delete Pst site from M13K07 polylinker====
Adds unique EcoRI to position 8083
Adds unique EcoRI to position 8083
-
*ChangePst2RI_top
+
ChangePst2RI_top
-
*5'TAGAATTCTATGCA
+
*5'ATAG AAT TCT ATG CA
-
 
+
ChangePst2RI_btm
-
*ChangePst2RI_btm
+
*5'ACG TTAT CTT AAG AT
-
*5'ACGTATCTTAAGAT
+
-
 
+
====to add Pst site to Petrenko site====
====to add Pst site to Petrenko site====
For site directed mutagenesis for GCT to GCA (silent at Ala, first residue in mature protein)
For site directed mutagenesis for GCT to GCA (silent at Ala, first residue in mature protein)
-
*AddPst2gVIII_fwd
+
AddPst2gVIII_fwd
*CGT TCCGATGCTG TCTTTCGCTG CaGAGGGTGA CGATCCCGCA AAAGCGGCC
*CGT TCCGATGCTG TCTTTCGCTG CaGAGGGTGA CGATCCCGCA AAAGCGGCC
* Tm 74°
* Tm 74°
-
*AddPst2gVIII_rev
+
AddPst2gVIII_rev
*GGC CGC TTT TGC GGG ATC GTC ACC CTC TGC AGC GAA AGA CAG CAT CGG AAC G  
*GGC CGC TTT TGC GGG ATC GTC ACC CTC TGC AGC GAA AGA CAG CAT CGG AAC G  
*Tm 74°
*Tm 74°
====M13K07 sequencing primers====
====M13K07 sequencing primers====
-
*M13KO7_seq1100_fwd
+
M13KO7_seq1100_fwd
*TAAGTAACATG GAGCAGGTCG CGGATTTCGA CAC
*TAAGTAACATG GAGCAGGTCG CGGATTTCGA CAC
*Tm 65°
*Tm 65°

Revision as of 11:10, 24 October 2006

Contents

Due Diligence

Life Cycle of the Phage

M13 phage proteins

NCBI link to seq: [[1]]
Source: Phage Display of Peptides and Proteins: a laboratory manual. Kay, Winter, McCafferty. Ch 1. [[2]]

protein aa (mature) function notes
I 348 assembly includes gXI within, the two form structure in cytoplasmic membrane for phage maturation
II 410 replication of DNA + strand includes gX within
III 406 phage tail protein (5 copies) tag at N-term
IV 405 assembly organizes to pore in OM
V 87 binds ssDNA recognizes DNA PS
VI 112 phage tail protein (5 copies) packages with pIII
VII 33 phage head protein (5 copies) packages with p9
VIII 50 phage coat protein (2700 copies) seg seq cleaved, matured form at aa 23 = clones to N-term, "Petrenko" site [[3]]
IX 32 phage head protein (5 copies) stop codon overlaps start of gVIII
X 111 DNA replication internal reinitiation inside gII
XI 108 assembly translation reinitiation inside gI

Vectors for M13 protein expression

Primers for phage redesign

to delete Pst site from M13K07 polylinker

Adds unique EcoRI to position 8083 ChangePst2RI_top

  • 5'ATAG AAT TCT ATG CA

ChangePst2RI_btm

  • 5'ACG TTAT CTT AAG AT

to add Pst site to Petrenko site

For site directed mutagenesis for GCT to GCA (silent at Ala, first residue in mature protein) AddPst2gVIII_fwd

  • CGT TCCGATGCTG TCTTTCGCTG CaGAGGGTGA CGATCCCGCA AAAGCGGCC
  • Tm 74°

AddPst2gVIII_rev

  • GGC CGC TTT TGC GGG ATC GTC ACC CTC TGC AGC GAA AGA CAG CAT CGG AAC G
  • Tm 74°

M13K07 sequencing primers

M13KO7_seq1100_fwd

  • TAAGTAACATG GAGCAGGTCG CGGATTTCGA CAC
  • Tm 65°

Makeover ideas

phage or phage coat rewrite

separate 8, 9, 3

two copies of 8

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