User:Nkuldell:M13 redesign: Difference between revisions
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====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 | |||
*5' | *5'ATAG AAT TCT ATG CA | ||
ChangePst2RI_btm | |||
*5'ACG TTAT CTT AAG AT | |||
*5' | |||
====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 | |||
*CGT TCCGATGCTG TCTTTCGCTG CaGAGGGTGA CGATCCCGCA AAAGCGGCC | *CGT TCCGATGCTG TCTTTCGCTG CaGAGGGTGA CGATCCCGCA AAAGCGGCC | ||
* Tm 74° | * Tm 74° | ||
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 | |||
*TAAGTAACATG GAGCAGGTCG CGGATTTCGA CAC | *TAAGTAACATG GAGCAGGTCG CGGATTTCGA CAC | ||
*Tm 65° | *Tm 65° | ||
Revision as of 08:10, 24 October 2006
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°
