User:Nkuldell:M13 redesign: Difference between revisions
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*pUC118 [[http://seq.yeastgenome.org/vectordb/vector_descrip/PUC118.html]] or 119 [[http://seq.yeastgenome.org/vectordb/vector_descrip/PUC119.html]] | *pUC118 [[http://seq.yeastgenome.org/vectordb/vector_descrip/PUC118.html]] or 119 [[http://seq.yeastgenome.org/vectordb/vector_descrip/PUC119.html]] | ||
*M13K07 (helper phage)[[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/sequences/m13ko7.txt |sequence]][[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/sequences/m13ko7.txt]] | *M13K07 (helper phage)[[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/sequences/m13ko7.txt |sequence]][[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/sequences/m13ko7.txt]] | ||
===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° | |||
==Makeover ideas== | ==Makeover ideas== | ||
===phage or phage coat rewrite=== | ===phage or phage coat rewrite=== | ||
===separate 8, 9, 3=== | ===separate 8, 9, 3=== | ||
===two copies of 8=== | ===two copies of 8=== |
Revision as of 07:49, 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'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°