PCANTAB5E Plasmid

2015-01-13

azim58 - PCANTAB5E Plasmid

Length: 4472 bp
Origins: f1, pBR322 (pMB1 ori)
The origin of replication or ori site in this plasmid (pBR322) is pMB1 (a
close relative of ColE1)
http://en.wikipedia.org/wiki/PBR322
These are both low copy origins of replication.
Antibiotic Resistance: ampicillin
Information about plasmid:
-The phagemid vector pCANTAB 5 E is widely used for the selection of
antibody fragments from corresponding libraries.
http://www.ncbi.nlm.nih.gov/pubmed/11849927
-PCANTAB5E is a phagemid plasmid
-
McCafferty et al. described in 1990 the fd-CATI original phage vector
forantibody display (27). These vectors contain all the genetic
information encoding the phage life cycle, in this context, an
alternative system has been used upto now. This system involves cloning
into phagemid vectors that contain acopy of the gene 3 and phage
packaging signal sequence. Thus, antibody fragments can be displayed as a
fusion with the gene 3 protein and the genetic infonnation is packaged
thanks to the packaging signal. In our laboratory we have used the
phagemid vector pCANTAB 5 E included in the Pharmacia RPAS kit. This
vector allows cloning of antibody genes into Sfi I and Not Isites. This
vector incorporates an amber codon between the C-terminus of the cloned
scFv and (he start of gene 3 sequence aIIowin the recombinant antibody to
be made as a soluble protein. This vector also includes a peptide
tag,allowing the detection of the single-chain antibody.
http://books.google.com/books?id=h0lx0ACckP8C&amp;pg=PA137&amp;lpg=PA137&am
p;dq=what+is+the+pcantab+plasmid+used+for&amp;source=bl&amp;ots=t2mBV2oY07&
amp;sig=TImrbx0PEgQe3P2JnwCBJidXhYg&amp;hl=en&amp;ei=HY9STq6wAszisQL2tojYBg
&amp;sa=X&amp;oi=book_result&amp;ct=result&amp;resnum=1&amp;sqi=2&amp;ved=0
CB4Q6AEwAA#v=onepage&amp;q&amp;f=false

* ORF1:
  o (2218,3594)
  o Length: 1376
  o Description: This ORF basically seems to code for the p3 protein
    of M13 phage. When an scFv fragment is inserted between the SfiI and
    NotI sites, a recombinant p3 scFv protein is produced.
  o Most Similar Blast result:
    - Type 3 trypsin release phage display vector f3TR1, complete
      sequence
    - http://www.ncbi.nlm.nih.gov/nucleotide/307776653?report=genbank
      &amp;log$=nucltop&amp;blast_rank=1&amp;RID=554H7MGA01N
    - The displayed peptide is connected to the bulk of pIII
      through a trypsin-sensitive tether.

o Sequence:
    - PCANTAB5E ORF1 Sequence (pIII protein)

o M13F at 3620
    - actggccgtcgttttac

o M13R at 2208
    - ggaaacagctatgaccatg

Miscellaneous Info:
M13 F sequence: 5'-GTCACGACGTTGTAAAACGACGGCCAGTCTGACTTTGGCAGCCCTT-3'
M13_R sequence:
5' CAGGAAACAGCTATGACCATG 3'
NotI_F sequence (goes over border of plasmid and insert):
5' CTGCGGCCGCCCGTTT 3'

If I want to check whether a plasmid has an insert, I should use the NotI
primer and the M13_R primer (located in Tien's scFv phage 3 box).
Now it would actually be better to check for an insert using the same
primers that were used to construct the scFv.

* To check a kappa insert, use these primers
  o MHV_Back_SfiI + MKV_For_NotI

* To checka lambda insert, use these primers
  o MHV_Back_SfiI + MLV_For_NotI

* see Primers to check for scFv

NotI: 2368-2375
gcggccgc
SfiI: 2316-2328
ggcccagccggcc
ggcc[nnnnn]ggcc

fragment between NotI and SfiI: (2368-2328)= 40 bp

Sequence:
PCANTAB5E Sequence
Source of Sequence:
https://www.lablife.org/ll?a=showvecinfo&amp;vectorid=5225

Questions:

ScFv inserted into the PCANTAB5E vector will eventually result in
recombinant p3 scFv proteins. However, won't 2/3 of the inserted scFv
cause a frameshift resulting in a faulty p3 protein? Perhaps the last
part of the p3 protein is not critical for forming a functional M13 phage.

azim58wiki: