Difference between revisions of "Part:BBa K551000:Design"

(→‎Design Notes)
(→‎Design Notes)
 
(2 intermediate revisions by the same user not shown)
Line 6: Line 6:
  
 
===Design Notes===
 
===Design Notes===
−
The initial part (BBa_J61025) wasn't giving the resistance, so we remade it from the intermediate CmR-FRT (BBa_J61024). For that we made a PCR with a primer containing the FRT sequence and the RFC10 prefix; this means there is no scar between the first FRT and CmR. This is the reason we encoded it as a new biobrick to differentiate it from BBa_J61025.
+
The initial part (BBa_J61025) we wanted to use don't confer the chloramphenicol resistance; so we remade it from the CmR biobrick (BBa_P1004). This Cm resistance gene was amplified by PCR using primers containing a mutated FRT sequence (FRT').  As the wild-type FRT sequence contains a Xba1 restriction site, a mutation was introduced to mutate the Xba1 site in order to be able to use this biobrick in iGEM standard 10. This FRT' is the same as BBa_J61020. Since there is no biobrick scar between the FRT' and the CmR gene we encoded it as a new biobrick part compared to BBa_J61025.
  
 
===Source===
 
===Source===
  
−
It comes from PCR of a registry part
+
PCR from BBa_J61024.
  
 
===References===
 
===References===
 +
 +
"One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products" Datsenko KA, Wanner BL. PNAS 2000.

Latest revision as of 16:11, 21 September 2011

FRT-CM-FRT


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The initial part (BBa_J61025) we wanted to use don't confer the chloramphenicol resistance; so we remade it from the CmR biobrick (BBa_P1004). This Cm resistance gene was amplified by PCR using primers containing a mutated FRT sequence (FRT'). As the wild-type FRT sequence contains a Xba1 restriction site, a mutation was introduced to mutate the Xba1 site in order to be able to use this biobrick in iGEM standard 10. This FRT' is the same as BBa_J61020. Since there is no biobrick scar between the FRT' and the CmR gene we encoded it as a new biobrick part compared to BBa_J61025.

Source

PCR from BBa_J61024.

References

"One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products" Datsenko KA, Wanner BL. PNAS 2000.