Difference between revisions of "Part:BBa K1632010:Experience"
(→‎Materials and Methods)
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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_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 (pSB6A1) + 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 (pSB6A1) + 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|800px|<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>
+
  
 
===Results===
 
===Results===

Revision as of 07:09, 17 September 2015

fimB (wild-type)

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.)

===Results===