Difference between revisions of "Part:BBa K1632011:Design"
Line 6: Line 6:
 
<br>
 
<br>
 
<br>
 
<br>
−
<span style="margin-left: 10px;">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.<br>
+
<span style="margin-left: 10px;">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.<br>
 
2008_Caltech : 5’-...-TAA-TAA-Suffix-3’<br>
 
2008_Caltech : 5’-...-TAA-TAA-Suffix-3’<br>
 
2015_TokyoTech : 5’-...-GTT-TGA-Suffix-3’<br>
 
2015_TokyoTech : 5’-...-GTT-TGA-Suffix-3’<br>
Line 18: Line 18:
 
All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.<br>
 
All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.<br>
  
−
A. PBAD/araC_FimE(wild-type) (6A1) +fim switch[default ON](wild-type)_rbs_gfp (3K3) <br>
+
A. PBAD/araC_fimE(wild-type) (6A1) +''fim'' switch[default ON](wild-type)_rbs_gfp (3K3) <br>
−
B. PBAD/araC_FimE(wild-type) (6A1) +fim switch[default OFF](wild-type)_rbs_gfp (3K3) <br>
+
B. PBAD/araC_fimE(wild-type) (6A1) +''fim'' switch[default OFF](wild-type)_rbs_gfp (3K3) <br>
−
C. rbs_M256IcysE (6A1) + fimswitch[default ON](wild-type)_rbs_gfp (3K3) …positive control 1<br>
+
C. rbs_M256IcysE (6A1) + ''fim'' switch[default ON](wild-type)_rbs_gfp (3K3) …positive control 1<br>
−
D. rbs_M256IcysE (6A1) + fimswitch[default OFF](wild-type)_rbs_gfp (3K3) …negative control 1<br>
+
D. rbs_M256IcysE (6A1) + ''fim'' switch[default OFF](wild-type)_rbs_gfp (3K3) …negative control 1<br>
−
E. PBAD/araC_FimE(wild-type) (6A1) + J23119_rbs_gfp (3K3) …positive control 2 <br>
+
E. PBAD/araC_fimE(wild-type) (6A1) + J23119_rbs_gfp (3K3) …positive control 2 <br>
−
F. PBAD/araC_FimE(wild-type) (6A1)+rbs_gfp (3K3) …negative control 2 <br>
+
F. PBAD/araC_fimE(wild-type) (6A1)+rbs_gfp (3K3) …negative control 2 <br>
 
[[Image:Tokyo_Tech_FimE_assay.png|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br>
 
[[Image:Tokyo_Tech_FimE_assay.png|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br>
  

Revision as of 13:41, 15 September 2015

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) + fim switch[default ON](wild-type)_rbs_gfp (3K3) …positive control 1
D. rbs_M256IcysE (6A1) + fim switch[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===