User:Nkuldell:M13 redesign
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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'TAGAATTCTATGCA
- ChangePst2RI_btm
- 5'ACGTATCTTAAGAT
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°
