Difference between revisions of "Part:BBa K1632007:Experience"

(→‎Invertion assay with FimB)
Line 5: Line 5:
 
====Invertion assay with FimB====
 
====Invertion assay with FimB====
  
−
<b>1. Construction</b><br>
+
=====1. Construction=====
  
 
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>
  
−
(1) PBAD/araC_fimB (pSB6A1) + ''fim'' switch[default ON](wild-type)_rbs_gfp (pSB3K3) <br>
+
(1) PBAD/araC_fimB (pSB6A1) + ''fim'' switch[default ON](wild-type)_gfp (pSB3K3) <br>
−
(2) PBAD/araC_fimB (pSB6A1) + ''fim'' switch[default OFF](wild-type)_rbs_gfp (pSB3K3) <br>
+
(2) PBAD/araC_fimB (pSB6A1) + ''fim'' switch[default OFF](wild-type)_gfp (pSB3K3) <br>
−
(3) pSB6A1 + ''fim'' switch[default ON](wild-type)_rbs_gfp (pSB3K3) …positive control 1<br>
+
(3) pSB6A1 + ''fim'' switch[default ON](wild-type)_gfp (pSB3K3) …positive control 1<br>
−
(4) pSB6A1 + ''fim'' switch[default OFF](wild-type)_rbs_gfp (pSB3K3) …negative control 1<br>
+
(4) pSB6A1 + ''fim'' switch[default OFF](wild-type)_gfp (pSB3K3) …negative control 1<br>
−
(5) PBAD/araC_fimB (pSB6A1) + J23119_rbs_gfp(pSB3K3) …positive control 2 <br>
+
(5) PBAD/araC_fimB (pSB6A1) + J23119_gfp(pSB3K3) …positive control 2 <br>
 
(6) PBAD/araC_fimB (pSB6A1) + rbs_gfp(pSB3K3) …negative control 2 <br>
 
(6) PBAD/araC_fimB (pSB6A1) + rbs_gfp(pSB3K3) …negative control 2 <br>
  
 
[[Image:Tokyo_Tech_FimB_assay.png |thumb|center|900px|<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>
+
=====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.<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>
Line 42: Line 42:
 
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>
 +
 +
=====3. Results=====
 +
 +
=====4. Discussion=====
 +
 +
====Invertion assay with FimE====
 +
 +
=====1. Construction=====
 +
 +
All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.<br>
 +
 +
(1) PBAD/''araC''_fimE(wild-type) (pSB6A1) + ''fim'' switch[default ON](wild-type)_gfp (pSB3K3) <br>
 +
(2) PBAD/''araC''_fimE(wild-type) (pSB6A1) + ''fim'' switch[default OFF](wild-type)_gfp (pSB3K3) <br>
 +
(3) pSB6A1 + ''fim'' switch[default ON](wild-type)_gfp (pSB3K3) …positive control 1<br>
 +
(4) pSB6A1 + ''fim'' switch[default OFF](wild-type)_gfp (pSB3K3) …negative control 1<br>
 +
(5) PBAD/''araC''_fimE(wild-type) (pSB6A1) + J23119_gfp (pSB3K3) …positive control 2 <br>
 +
(6) PBAD/''araC''_fimE(wild-type) (pSB6A1) + rbs_gfp (pSB3K3) …negative control 2 <br>
 +
 +
[[Image:Tokyo_Tech_FimE_assay.png|thumb|center|900px|<b>Fig. 2. </b>Plasmids]]<br>
 +
 +
=====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.<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 1.0 %).<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>
 +
5. Remove the supernatant.<br>
 +
6. Add 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 +
7. Remove the supernatant.<br>
 +
8. Take the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 +
9. Remove the supernatant.<br>
 +
10. Suspend the pellet in 1mL of LB containing Amp and Kan.<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 and 30 microL of sterile water<br>
 +
<span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 500 microM　arabinose (final concentration of arabinose is 5 microM)<br>
 +
<span style="margin-left: 20px;">③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)<br>
 +
<span style="margin-left: 20px;">④ 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;">※ As for C and D, the suspension were added only in medium ① and ④. <br>
 +
12. Incubate the samples at 37 ℃ for 6 hours, shaking at 180 rpm. (Measure the OD590 of all the samples every hour.)<br>
 +
13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.<br>
 +
14. Remove the supernatant.<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>
 +
17. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)<br>
 +
 +
=====3. Results=====
 +
 +
=====4. Discussion=====
  
 
===Applications of BBa_K1632007===
 
===Applications of BBa_K1632007===

Revision as of 11:44, 17 September 2015

Materials and Methods

Invertion assay with FimB

1. Construction

All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.

(1) PBAD/araC_fimB (pSB6A1) + fim switch[default ON](wild-type)_gfp (pSB3K3)
(2) PBAD/araC_fimB (pSB6A1) + fim switch[default OFF](wild-type)_gfp (pSB3K3)
(3) pSB6A1 + fim switch[default ON](wild-type)_gfp (pSB3K3) …positive control 1
(4) pSB6A1 + fim switch[default OFF](wild-type)_gfp (pSB3K3) …negative control 1
(5) PBAD/araC_fimB (pSB6A1) + J23119_gfp(pSB3K3) …positive control 2
(6) 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.)

3. Results
4. Discussion

Invertion assay with FimE

1. Construction

All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.

(1) PBAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default ON](wild-type)_gfp (pSB3K3)
(2) PBAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default OFF](wild-type)_gfp (pSB3K3)
(3) pSB6A1 + fim switch[default ON](wild-type)_gfp (pSB3K3) …positive control 1
(4) pSB6A1 + fim switch[default OFF](wild-type)_gfp (pSB3K3) …negative control 1
(5) PBAD/araC_fimE(wild-type) (pSB6A1) + J23119_gfp (pSB3K3) …positive control 2
(6) PBAD/araC_fimE(wild-type) (pSB6A1) + rbs_gfp (pSB3K3) …negative control 2

Fig. 2. 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 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.)

3. Results
4. Discussion

Applications of BBa_K1632007

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