Difference between revisions of "Part:BBa K1632011:Design"
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===Materials and Methods=== | ===Materials and Methods=== | ||
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<b>1. Construction</b><br> | <b>1. Construction</b><br> | ||
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/ | + | A. PBAD/''araC''_fimE(wild-type) (pSB6A1) + ''fim'' switch[default ON](wild-type)_rbs_gfp (pSB3K3) <br> |
| − | B. PBAD/ | + | B. PBAD/''araC''_fimE(wild-type) (pSB6A1) + ''fim'' switch[default OFF](wild-type)_rbs_gfp (pSB3K3) <br> |
C. pSB6A1 + ''fim'' switch[default ON](wild-type)_rbs_gfp (pSB3K3) …positive control 1<br> | C. pSB6A1 + ''fim'' switch[default ON](wild-type)_rbs_gfp (pSB3K3) …positive control 1<br> | ||
D. pSB6A1 + ''fim'' switch[default OFF](wild-type)_rbs_gfp (pSB3K3) …negative control 1<br> | D. pSB6A1 + ''fim'' switch[default OFF](wild-type)_rbs_gfp (pSB3K3) …negative control 1<br> | ||
| − | E. PBAD/ | + | E. PBAD/''araC''_fimE(wild-type) (pSB6A1) + J23119_rbs_gfp (pSB3K3) …positive control 2 <br> |
| − | F. PBAD/ | + | F. PBAD/''araC''_fimE(wild-type) (pSB6A1) + rbs_gfp (pSB3K3) …negative control 2 <br> |
| − | [[Image:Tokyo_Tech_FimE_assay.png|thumb|center| | + | [[Image:Tokyo_Tech_FimE_assay.png|thumb|center|800px|<b>Fig. 1. </b>Plasmids]]<br> |
<b>2. Assay protocol</b><br> | <b>2. Assay protocol</b><br> | ||
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15. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)<br> | 15. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)<br> | ||
16. Dispense all of each suspension into a disposable tube through a cell strainer.<br> | 16. Dispense all of each suspension into a disposable tube through a cell strainer.<br> | ||
| − | 17. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)<br> | + | 17. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)<br><b>2. Assay protocol</b><br> |
===Source=== | ===Source=== | ||
Revision as of 11:30, 17 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) (pSB6A1) + fim switch[default ON](wild-type)_rbs_gfp (pSB3K3)
B. PBAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default OFF](wild-type)_rbs_gfp (pSB3K3)
C. pSB6A1 + fim switch[default ON](wild-type)_rbs_gfp (pSB3K3) …positive control 1
D. pSB6A1 + fim switch[default OFF](wild-type)_rbs_gfp (pSB3K3) …negative control 1
E. PBAD/araC_fimE(wild-type) (pSB6A1) + J23119_rbs_gfp (pSB3K3) …positive control 2
F. PBAD/araC_fimE(wild-type) (pSB6A1) + rbs_gfp (pSB3K3) …negative control 2
2. Assay protocol
1. Prepare overnight cultures for the each sample in 3 mL of LB medium containing ampicillin (50 microg / mL), kanamycin (30 microg / mL) and glucose (final concentration of mass of glucose is 1.0 %) at 37 ℃ for 12h.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration of mass of glucose is 1.0 %).
3.Grow the cells at 37 ℃, shaking at 180 rpm until the observed OD590 reaches 0.4 (Fresh Culture).
4. After incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃
5. Remove the supernatant.
6. Add 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃
7. Remove the supernatant.
8. Take the samples, and centrifuge at 5000x g, 1 min, 25 ℃
9. Remove the supernatant.
10. Suspend the pellet in 1mL of LB containing Amp and Kan.
11. Add 30 microL of suspension in the following medium.
① 3 mL of LB containing Amp, Kan and 30 microL of sterile water
② 3 mL of LB containing Amp, Kan and 30 microL of 500 microM arabinose (final concentration of arabinose is 5 microM)
③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)
④ 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)
※ As for C and D, the suspension were added only in medium ① and ④.
12. Incubate the samples at 37 ℃ for 6 hours, shaking at 180 rpm. (Measure the OD590 of all the samples every hour.)
13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.
14. Remove the supernatant.
15. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)
16. Dispense all of each suspension into a disposable tube through a cell strainer.
17. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)
2. Assay protocol
Source
PCR from MG1655
===References===