Difference between revisions of "Part:BBa K1632012:Design"
(→‎Materials and Methods)
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===Materials and Methods===
 
===Materials and Methods===
  
−
<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/araC_fimB(pSB6A1) +''fim'' switch[default ON](wild-type)_rbs_gfp (pSB3K3) <br>
+
A. PBAD/araC_fimB (pSB6A1) + ''fim'' switch[default ON](wild-type)_rbs_gfp (pSB3K3) <br>
−
B. PBAD/araC_fimB(pSB6A1) +''fim'' switch[default OFF](wild-type)_rbs_gfp (pSB3K3) <br>
+
B. PBAD/araC_fimB (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_fimB(6A1) + J23119_rbs_gfp (pSB3K3) …positive control 2 <br>
+
E. PBAD/araC_fimB (pSB6A1) + J23119_rbs_gfp(pSB3K3) …positive control 2 <br>
−
F. PBAD/araC_fimB(6A1)+rbs_gfp (pSB3K3) …negative control 2 <br>
+
F. PBAD/araC_fimB (pSB6A1) + rbs_gfp(pSB3K3) …negative control 2 <br>
  
−
[[Image:Tokyo_Tech_FimB_assay.png |thumb|center|00px|<b>Fig. 1. </b>Plasmids]]<br>
+
[[Image:Tokyo_Tech_FimB_assay.png |thumb|center|900px|<b>Fig. 1. </b>Plasmids]]<br>
  
 
<b>2. Assay protocol</b><br>
 
<b>2. Assay protocol</b><br>
  
−
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 0.5 percent) at 37 ℃ for 12h.<br>
+
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 0.5 %) at 37 ℃ for 12h.<br>
−
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration of mass of glucose is 0.5 percent).<br>
+
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration of mass of glucose is 0.5 %).<br>
−
3.Grow the cells at 37 ℃ until the observed OD590 reaches 0.4 (Fresh Culture)<br>
+
3.Grow the cells at 37 ℃, shaking at 180 rpm until the observed OD590 reaches 0.4 (Fresh Culture).<br>
 
4. After incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 
4. After incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
−
5. Remove the supernatant by using P1000 pipette<br>
+
5. Remove the supernatant.<br>
−
6. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
+
6. Add 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
−
7. Remove the supernatant by using P1000 pipette<br>
+
7. Remove the supernatant.<br>
 
8. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 
8. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
−
9. Remove the supernatant by using P1000 pipette<br>
+
9. Remove the supernatant.<br>
 
10. Add 1 mL of LB containing Amp and Kan, and suspend.<br>
 
10. Add 1 mL of LB containing Amp and Kan, and suspend.<br>
 
11. Add 30 microL of suspension in the following medium.<br>
 
11. Add 30 microL of suspension in the following medium.<br>
−
<span style="margin-left: 20px;">① 3 mL of LB containing Amp, Kan, glucose (final concentration of mass of glucose is 0.5 percent) and 30 microL of sterile water<br>
+
<span style="margin-left: 20px;">① 3 mL of LB containing Amp, Kan, glucose (final concentration of mass of glucose is 0.5 %) and 30 microL of sterile water<br>
−
<span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)<br>
+
<span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 2 mM　arabinose (final concentration of arabinose is 20 microM)<br>
 
<span style="margin-left: 20px;">③ 3 mL of LB containing Amp, Kan and 30 microL of 20 mM arabinose (final concentration of arabinose is 200 microM)<br>
 
<span style="margin-left: 20px;">③ 3 mL of LB containing Amp, Kan and 30 microL of 20 mM arabinose (final concentration of arabinose is 200 microM)<br>
 
<span style="margin-left: 20px;">④ 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)<br>
 
<span style="margin-left: 20px;">④ 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)<br>
−
12. Grow the samples at 37 ℃ for 6.5 hours.<br>
+
<span style="margin-left: 20px;">※ As for C and D, the suspension were added only in medium ① and ④. <br>
−
13. Measure OD590 of all the samples every hour.<br>
+
12. Incubate the samples at 37 ℃, shaking at 180 rpm for 6.5 hours. (Measure OD590 of all the samples every hour.)<br>
−
14. Start preparing the flow cytometer 1 h before the end of incubation.<br>
+
13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.<br>
−
15. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.<br>
+
14. Remove the supernatant.<br>
−
16. Remove the supernatant by using P1000 pipette<br>
+
15. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)<br>
−
17. 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>
−
18. 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>
−
19. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)<br>
+
  
 
===Source===
 
===Source===

Revision as of 07:08, 17 September 2015

PBAD/araC_rbs_fimB(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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


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_fimB (pSB6A1) + fim switch[default ON](wild-type)_rbs_gfp (pSB3K3)
B. PBAD/araC_fimB (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_fimB (pSB6A1) + J23119_rbs_gfp(pSB3K3) …positive control 2
F. PBAD/araC_fimB (pSB6A1) + rbs_gfp(pSB3K3) …negative control 2

Fig. 1. Plasmids

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 0.5 %) 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 0.5 %).
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. 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. Add 1 mL of LB containing Amp and Kan, and suspend.
11. Add 30 microL of suspension in the following medium.
① 3 mL of LB containing Amp, Kan, glucose (final concentration of mass of glucose is 0.5 %) and 30 microL of sterile water
② 3 mL of LB containing Amp, Kan and 30 microL of 2 mM　arabinose (final concentration of arabinose is 20 microM)
③ 3 mL of LB containing Amp, Kan and 30 microL of 20 mM arabinose (final concentration of arabinose is 200 microM)
④ 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)
※ As for C and D, the suspension were added only in medium ① and ④.
12. Incubate the samples at 37 ℃, shaking at 180 rpm for 6.5 hours. (Measure 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

PCR from MG1655

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
Hung M. et al. (2014) Modulating the frequency and bias of stochastic switching to control phenotypic variation. Nat Commun 5:4574. doi:10.1038/ncomms5574