Part:BBa_K365004:Design

ClpX from E.coli (aa 61-425)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 699
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

We deleted 2 EcoRI sites and 2 Age1 sites by directed mutagenis at positions 522 and 867, and 183 and 921 respectively. This biobrick has standard RFC-25.

Problem: 2 AgeI and 2 EcoRI sides

Primers for cloning ClpX out of the E.Coli genome.
These primes were used to amplificate ClpX from the E.coli genome.

Forward primer (5’->3’) : 31bp
CGCAGTGCGCTACCGACGCCGCATGAAATTC

Reverse primer (5’->3’) : 32bp
TTCACCAGATGCCTGTTGCGCTTCCGGCTTGC

Primers for Pfu-mutagenese

1. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)       AgeI site

Forward primer (5’->3’) (31 bp)
CTGATCGGTCCGACTGGTTCCGGTAAAACGC

Reverse primer (5’->3’) (31 bp)
GCGTTTTACCGGAACCAGTCGGACCGATCAG

2. GAA=Glu GA(A,G), TTC=Phe TT(C,T)       EcoRI site

Forward primer (5’->3’) (28 bp)
CATCCGCAGCAGGAGTTCTTGCAGGTTG

Reverse primer (5’->3’) (28 bp)
CAACCTGCAAGAACTCCTGCTGCGGATG

3. GAA=Glu GA(A,G), TTC=Phe TT(C,T)       EcoRI site

Forward primer (5’->3’) (25 bp)
CGTGGATCTGGAGTTCCGTGACGAG

Reverse primer (5’->3’) (25 bp)
CTCGTCACGGAACTCCAGATCCACG

4. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)       AgeI site

Forward primer (5’->3’) (24 bp)
GGCGCGTAAAACTGGTGCCCGTGG

Reverse primer (5’->3’) (24 bp)
CCACGGGCACCAGTTTTACGCGCC

Primers for amplification of ClpX with fusion pre- and suffix
After mutagenesis of internal restriction sides, the fusion pre- and suffixes were added to the ClpX gene.

Forward primer (5’->3’):
GGATCCgaattcgcggccgcttctagatggccggcCGCAGTGCGCTACCGACGCCGC

Reverse primer (5’->3’):
CAGCTGctgcagcggccgctactagtattaaccggtTTCACCAGATGCCTGTTGCGC

Source

Extracted from the genomic sequence of E.coli.

References