Difference between revisions of "User:Etienne Robillard/Notebook/Agent Ecoli:Plasmid cloning vectors for gene therapy with phosphohistidine based oligos"

From OpenWetWare
Jump to: navigation, search
(Artificial DNA primers: phosphoramidate-based oligonucleotide synthesis)
(Artificial DNA primers: phosphoramidate-based oligonucleotide synthesis)
Line 1: Line 1:
 
= Plasmid cloning vectors for gene therapy with phosphohistidine-based oligos =
 
= Plasmid cloning vectors for gene therapy with phosphohistidine-based oligos =
 
__TOC__
 
__TOC__
== Artificial DNA primers: phosphoramidate-based oligonucleotide synthesis ==
+
== Artificial DNA promoters: phosphoramidate-based oligonucleotide synthesis ==
  
 
cDNA:
 
cDNA:
Line 22: Line 22:
 
[http://openwetware.org/wiki/20.109(S07):Start-up_genome_engineering#Part_3:_Digest_M13K07 M13K07] cloning in ''E. coli'':
 
[http://openwetware.org/wiki/20.109(S07):Start-up_genome_engineering#Part_3:_Digest_M13K07 M13K07] cloning in ''E. coli'':
 
* amino-acid sequence signature (Myc epitope tag) = EQKLISEEDL
 
* amino-acid sequence signature (Myc epitope tag) = EQKLISEEDL
 +
 +
References:
 +
* http://openwetware.org/images/1/1c/TheBacterialPromoter.pdf
  
 
== References ==
 
== References ==

Revision as of 16:25, 9 November 2012

Plasmid cloning vectors for gene therapy with phosphohistidine-based oligos

Artificial DNA promoters: phosphoramidate-based oligonucleotide synthesis

cDNA:

  • N-6-benzoyl-deoxyadenosine phosphoramidite - Notice the P-N bound indicating the linkage of a phosphonate diester to a benzoyl atom :)
  • N-3-oxo-hexanoyl-L-homoserine lactone - A light-inducible phosphohistidine-bound AHL ? A(denosine)TP/cAMP dependent.
  • Reminder:
    • NHC = (N heterocycle carbene) - Chemical compounds with a N chiral prodrug switch - highly enantioselective molecules
    • N = indicates a Nitrogen atom
    • P = a Phosphonate (inorganic state) - a compound for oligo synthesis using the nucleoside phosphoramidite method.
    • P-N = phosphoramidate (a Nitrogen atom bound to a Phosphonate ester in nucleophilic substitution).

deoxynucleotide synthesis:

  • cDNA synthesis building block = deoxynucleoside + 3'-phosphonate = synthetic nucleotide analog (dATP) 1

oligodeoxyribonucleotide synthesis:

  • short 50-200(?) bp restriction endonucleases enzymes for cDNA catalysis/plasmid vector cloning in E. coli
  • building blocks for oligonucleotides assembly with phosphoramidites method 1
  • see also Oligonucleotide Synthesis

M13K07 cloning in E. coli:

  • amino-acid sequence signature (Myc epitope tag) = EQKLISEEDL

References:

References