Difference between revisions of "Part:BBa K1632011:Design"
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===Materials and Methods=== | ===Materials and Methods=== | ||
+ | <b>1. Construction</b><br> | ||
+ | All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.<br> | ||
+ | |||
+ | A. PBAD/araC_FimE(6A1) +Fimswitch[default ON]_gfp(3K3) <br> | ||
+ | B. PBAD/araC_FimE(6A1) +Fimswitch[default ON]_gfp(3K3) <br> | ||
+ | C. rbs_M256IcysE(6A1) + Fimswitch[ON]_gfp(3K3) …positive control 1<br> | ||
+ | D. rbs_M256IcysE(6A1) + Fimswitch[OFF]_gfp(3K3) …negative control 1<br> | ||
+ | E. PBAD/araC_FimE(6A1) + J23119_gfp(3K3) …positive control 2 <br> | ||
+ | F. PBAD/araC_FimE(6A1)+rbs_gfp(3K3) …negative control 2 <br> | ||
+ | [[Image:Tokyo_Tech_FimE_assay.png|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br> | ||
+ | |||
+ | <b>2. Assay protocol</b><br> | ||
===Source=== | ===Source=== |
Revision as of 23:12, 13 September 2015
fimE (wild-type)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 22
- 1000COMPATIBLE WITH RFC[1000]
2008_Caltech FimE(BBa_K137007) didn’t match the nucleotide sequence of fimE of wild type. So, we designed FimE (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(6A1) +Fimswitch[default ON]_gfp(3K3)
B. PBAD/araC_FimE(6A1) +Fimswitch[default ON]_gfp(3K3)
C. rbs_M256IcysE(6A1) + Fimswitch[ON]_gfp(3K3) …positive control 1
D. rbs_M256IcysE(6A1) + Fimswitch[OFF]_gfp(3K3) …negative control 1
E. PBAD/araC_FimE(6A1) + J23119_gfp(3K3) …positive control 2
F. PBAD/araC_FimE(6A1)+rbs_gfp(3K3) …negative control 2
2. Assay protocol
Source
PCR from MG1655