Difference between revisions of "Part:BBa K1632013:Design"

(→‎Assay protocol)
(→‎Materials and Methods)
Line 14: Line 14:
 
All the samples were DH5alpha strain with antibiotic resistance to ampicillin and kanamycin.<br>
 
All the samples were DH5alpha strain with antibiotic resistance to ampicillin and kanamycin.<br>
  
−
A. PBAD/''araC''_fimE(wild-type) (pSB6A1) + ''fim'' switch[default ON](wild-type)_rbs_gfp (pSB3K3) <br>
+
A. PBAD/''araC''_<i>fimE</i>(wild-type) (pSB6A1) + ''fim'' switch[default ON](wild-type)_rbs_<i>gfp</i> (pSB3K3) <br>
−
B. PBAD/''araC''_fimE(wild-type) (pSB6A1) + ''fim'' switch[default OFF](wild-type)_rbs_gfp (pSB3K3) <br>
+
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/''araC''_fimE(wild-type) (pSB6A1) + J23119_rbs_gfp (pSB3K3) …positive control 2 <br>
+
E. PBAD/''araC''_''fimE''(wild-type) (pSB6A1) + J23119_rbs_''gfp'' (pSB3K3) …positive control 2 <br>
−
F. PBAD/''araC''_fimE(wild-type) (pSB6A1) + rbs_gfp (pSB3K3) …negative control 2 <br>
+
F. PBAD/''araC''_''fimE''(wild-type) (pSB6A1) + rbs_''gfp'' (pSB3K3) …negative control 2 <br>
  
 
[[Image:Tokyo_Tech_FimE_assay.png|thumb|center|800px|<b>Fig. 1. </b>Plasmids]]<br>
 
[[Image:Tokyo_Tech_FimE_assay.png|thumb|center|800px|<b>Fig. 1. </b>Plasmids]]<br>

Revision as of 21:58, 18 September 2015

PBAD/araC_rbs_fimE(wild-type)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 1260
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961

Design Notes

sequence confirmed

Materials and Methods

Construction

All the samples were DH5alpha 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

Fig. 1. Plasmids

Assay protocol

1. Prepare overnight cultures for 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. Incubate the cells at 37 ℃, shaking at 180 rpm until the observed OD590 reaches 0.4 (Fresh Culture).
4. After the incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃
5. Remove the supernatant.
6. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃
7. Remove the supernatant.
8. Suspend the pellet in 1 mL of LB containing Amp and Kan, 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.)

Source

Composite of BBa_I0500, BBa_B0034, BBa_K1632011

References

Timothy S. Ham et al. (2006) A Tightly Regulated Inducible Expression System Utilizing the fim Inversion Recombination Switch. Biotechnol Bioeng 94(1):1-4