Difference between revisions of "Part:BBa K1632010:Experience"

 
Line 1: Line 1:
 +
<partinfo>BBa_K1529301 short</partinfo>
 +
__TOC__
 +
===Materials and Methods===
 +
<b>1. Construction</b><br>
 +
All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.<br>
  
−
__NOTOC__
+
A. Ptet_RhlR (6A1)　Prhl-GFP (3K3) <br>
−
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
+
B. Ptet_RhlR (6A1)　Prhl(RR)-GFP (3K3) <br>
−
how you used this part and how it worked out.
+
C. Ptet_RhlR (6A1)　Prhl(LR)-GFP (3K3) <br>
 +
D. Ptet_RhlR (6A1)　Prhl(RL)-GFP (3K3) <br>
 +
E. Ptet_RhlR (6A1)　 Placuv5-GFP (3K3) …positive control<br>
 +
F. Ptet_RhlR (6A1)　promoter less-GFP(3K3) …negative control<br>
  
−
===Applications of BBa_K1632010===
+
[[Image:Improved_Prhl_Promoter_Assay_Construction.png|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br>
  
−
===User Reviews===
+
<b>2. Assay protocol</b><br>
−
<!-- DON'T DELETE --><partinfo>BBa_K1632010 StartReviews</partinfo>
+
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>
−
<!-- Template for a user review
+
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>
−
{|width='80%' style='border:1px solid gray'
+
3.Grow the cells at 37 ℃ 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>
−
|width='10%'|
+
5. Remove the supernatant by using P1000 pipette<br>
−
<partinfo>BBa_K1632010 AddReview number</partinfo>
+
6. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
−
<I>Username</I>
+
7. Remove the supernatant by using P1000 pipette<br>
−
|width='60%' valign='top'|
+
8. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
−
Enter the review inofrmation here.
+
9. Remove the supernatant by using P1000 pipette<br>
−
|};
+
10. Add 1 mL of LB containing Amp and Kan, and suspend.<br>
−
<!-- End of the user review template -->
+
11. Add 30 microL of suspension in the following medium.<br>
−
<!-- DON'T DELETE --><partinfo>BBa_K1632010 EndReviews</partinfo>
+
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>
 +
② 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)<br>
 +
③ 3 mL of LB containing Amp, Kan and 30 microL of 20 mM arabinose (final concentration of arabinose is 200 microM)<br>
 +
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>
 +
13. Measure OD590 of all the samples every hour.<br>
 +
14. Start preparing the flow cytometer 1 h before the end of incubation.<br>
 +
15. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.<br>
 +
16. Remove the supernatant by using P1000 pipette<br>
 +
17. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)<br>
 +
18. Dispense all of each suspension into a disposable tube through a cell strainer.<br>
 +
19. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)<br>

Revision as of 09:35, 13 September 2015

Prhl(RL)-GFP

Materials and Methods

1. Construction
All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.

A. Ptet_RhlR (6A1)　Prhl-GFP (3K3)
B. Ptet_RhlR (6A1)　Prhl(RR)-GFP (3K3)
C. Ptet_RhlR (6A1)　Prhl(LR)-GFP (3K3)
D. Ptet_RhlR (6A1)　Prhl(RL)-GFP (3K3)
E. Ptet_RhlR (6A1)　 Placuv5-GFP (3K3) …positive control
F. Ptet_RhlR (6A1)　promoter less-GFP(3K3) …negative control

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 percent) 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 percent).
3.Grow the cells at 37 ℃ 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 by using P1000 pipette
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 by using P1000 pipette
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 by using P1000 pipette
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 percent) 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)
12. Grow the samples at 37 ℃ for 6.5 hours.
13. Measure OD590 of all the samples every hour.
14. Start preparing the flow cytometer 1 h before the end of incubation.
15. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.
16. Remove the supernatant by using P1000 pipette
17. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)
18. Dispense all of each suspension into a disposable tube through a cell strainer.
19. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)