Difference between revisions of "Part:BBa K1632011:Design"
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− | 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.<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> | ||
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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 (6A1) +fim switch(wild-type) | + | A. PBAD/araC_FimE(wild-type) (6A1) +fim switch[default ON](wild-type)_rbs_gfp (3K3) <br> |
− | B. PBAD/araC_FimE (6A1) +fim switch(wild-type) | + | B. PBAD/araC_FimE(wild-type) (6A1) +fim switch[default OFF](wild-type)_rbs_gfp (3K3) <br> |
− | C. rbs_M256IcysE (6A1) + fimswitch(wild-type) | + | C. rbs_M256IcysE (6A1) + fimswitch[default ON](wild-type)_rbs_gfp (3K3) …positive control 1<br> |
− | D. rbs_M256IcysE (6A1) + fimswitch(wild-type) | + | D. rbs_M256IcysE (6A1) + fimswitch[default OFF](wild-type)_rbs_gfp (3K3) …negative control 1<br> |
− | E. PBAD/araC_FimE (6A1) + | + | E. PBAD/araC_FimE(wild-type) (6A1) + J23119_rbs_gfp (3K3) …positive control 2 <br> |
− | F. PBAD/araC_FimE (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 13:34, 15 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(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
2. Assay protocol
Source
PCR from MG1655
===References===