Part:BBa_K1632011:Design

fimE (wild-type)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 22
  
• 1000
  COMPATIBLE WITH RFC[1000]

2008_Caltech FimE(BBa_K137007) didn’t match the nucleotide sequence of fimE of wild type. So, we designed FimE(wild-type) (BBa_K1632011) which completely match the nucleotide sequence of fimE of wild type. Compared with these two parts, two differences were confirmed. First, fimE of 2008_Caltech lacks the nucleotide sequence of N-terminal 15 residues. Second, fimE of 2008_Caltech is different from the nucleotide sequence of C-terminal 2 residues as shown as below.
2008_Caltech : 5’-...-TAA-TAA-Suffix-3’
2015_TokyoTech : 5’-...-GTT-TGA-Suffix-3’

Design Notes

sequence confirmed

Materials and Methods

1. Construction
All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.

A. PBAD/araC_FimE(wild-type) (6A1) +fim switch[default ON](wild-type)_rbs_gfp (3K3)
B. PBAD/araC_FimE(wild-type) (6A1) +fim switch[default OFF](wild-type)_rbs_gfp (3K3)
C. rbs_M256IcysE (6A1) + fimswitch[default ON](wild-type)_rbs_gfp (3K3) …positive control 1
D. rbs_M256IcysE (6A1) + fimswitch[default OFF](wild-type)_rbs_gfp (3K3) …negative control 1
E. PBAD/araC_FimE(wild-type) (6A1) + J23119_rbs_gfp (3K3) …positive control 2
F. PBAD/araC_FimE(wild-type) (6A1)+rbs_gfp (3K3) …negative control 2

Fig. 1. Plasmids

2. Assay protocol

Source

PCR from MG1655

===References===