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Introduction System Modeling Lab note Result Reference
=>Experiment-Assay- Terminator Leak/ Location Sequence/ Phage MS2

Introduction

The object of the assay is to test whether antisense RNA used in our construct works or not. In order to block the unnecessary information transformed by the virus from the other grid, we use antisense RNA to block ribosome to bind the region around the ribosome binding site (rbs) and prevent the expression of protein. In our construct, information is carried by virus and antisense RNA is transcribed constantly inside the cell. Once the unnecessary information was transformed, the antisense will come and shut out all the RNA chain excluded by virus. In this assay, we used pBAD as the promoter to start translating grid information which will be transformed by virus in our construct. The strength of the promoter depends on the concentration of arabinose. On the other hand, we used c-pro as the promoter to start translating antisense RNA. This c-pro is the strongest constitutive promoter submitted in igem parts. In assay I, we examined the relative strength of pBAD (with eight different concentration of arabinose) and c-pro. By using the proper concentration of arabinose which was determined by assay I, in assay II, we inspect observe whether our antisense RNA works or not. In these two assay, gfp was used as a reporter protein.

Assay I

The assay to observe how strong pBAD expresses depending on concentration of arabinose.

1st day
Transform the miniprep product of pBAD-LS-24, pBAD-4, cpro-24, cpro-4. Incubate those plates at 37 degrees C for about twelve hours (until the diameter of the colony grows to nearly 1 cm).

2nd day
Make full growth medium for eight samples (A to H).

• Sample A; pBAD-LS-24, add 1.0x10^-2 M arabinose solution.
  
• Sample B; pBAD-LS-24, add 1.0x10^-3 M arabinose solution.
  
• Sample C; pBAD-LS-24, add 1.0x10^-4 M arabinose solution.
  
• Sample D; pBAD-LS-24, add 1.0x10^-5 M arabinose solution.
  
• Sample E; pBAD-LS-24, add 1.0x10^-6 M arabinose solution.
  
• Sample F; pBAD-LS-4
  
• Sample G; cpro-24
  
• Sample H; cpro-4
  

Pour 15 mL of LB-10 medium (with 100 uL ampicillin) into Erlenmeyer flask, add tip which picked up one colony from the plate of each sample.

Incubate flask at 37 degree C with shaking at 180 rpm for more than 12 hours (until the medium become the state of full growth).

3rd day
Measure Optical Density (600).

- Prepare 8 Erlenmeyer flask, pour 100mL LB broth, add 100 uL ampicillin, and 1 mL full growth medium.
- Make arabinose diluted solutions.
1. Add “L-(+)-Arabinose, minimum 99%” 1.5 g into 10ml arabinose to make master mix solution of 1.0x10^(-2)M arabinose for sample A.
2. Add 150 ul master mix solution to 1350 ul LB broth to make 1.0x10^-3M arabinose solution for sample B.
3. Add 150 ul 1.0x10^-3M arabinose solution to 1350 ul LB broth to make 1.0x10^-4 arabinose solution for sample C.
4. Add 150 ul 1.0x10^-4M arabinose solution to 1350 ul LB broth to make 1.0x10^-5 arabinose solution for sample D.
5. Add 150 ul 1.0x10^-5M arabinose solution to 1350 ul LB broth to make 1.0x10^-6 arabinose solution for sample E.

-Put flasks into shaking incubator (25 degree C, 180 rpm) and begin incubation.
-Measure OD600 of each sample every one hour.

• Add arabinose solution of each concentration to the medium when the OD600 almost reached 2.0.
  

-Extract 1ml medium from the flask and put into 1.5ml Eppendorf tube every time measuring OD. Put the tube into icebox as soon as possible. Centrifuge the tube and throw away supernatant to make E.coli pellet. Preserve pellet in -20 degrees C freezer.

4th day
Measure Fluorescence
1. Add 100 ul 8M urea buffer into each pellet and make suspension solution.
2. Put the pellet in room temperature for 30 minutes.
3. Use sonicator to smash the cell. (10 seconds, 10% power.)
4. Put the tube into icebox and cool it down.
5. Do 3 and 4 again.
6. Centrifuge the tube. 150rpm, 2 minutes, 4 degrees C.
7. Measure the fluorescence.

Assay II

Test whether location sequence works or not.

1st day
Transform the miniprep product of 4 samples bellow.
-4-gfp-L2-pBAD(rev)-cpro-L2’-4
-4-gfp-L2-pBAD(rev)-cpro-L4’-4
-4-gfp-L4-pBAD(rev)-cpro-L2’-4
-4-gfg-L4-pBAD(rev)-cpro-L4’-4
Incubate those plates at 37 degrees C for about twelve hours (until the diameter of the colony grows to nearly 1 cm).

2nd day
Make full growth medium for eight samples (A to H).
Sample A; 4-gfp-L2-pBAD(rev)-cpro-L2’-4, add 1.0x10^-2 M arabinose solution.
Sample B; 4-gfp-L2-pBAD(rev)-cpro-L4’-4, add 1.0x10^-2 M arabinose solution.
Sample C; 4-gfp-L4-pBAD(rev)-cpro-L2’-4, add 1.0x10^-2 M arabinose solution.
Sample D; 4-gfg-L4-pBAD(rev)-cpro-L4’-4, add 1.0x10^-2 M arabinose solution.
Sample E; 4-gfp-L2-pBAD(rev)-cpro-L2’-4, add 1.0x10^-4 M arabinose solution.
Sample F; 4-gfp-L2-pBAD(rev)-cpro-L4’-4, add 1.0x10^-4 M arabinose solution.
Sample G; 4-gfp-L4-pBAD(rev)-cpro-L2’-4, add 1.0x10^-4 M arabinose solution.
Sample H; 4-gfg-L4-pBAD(rev)-cpro-L4’-4, add 1.0x10^-4 M arabinose solution.

Pour 15 mL of LB-10 medium (with 100 uL ampicillin) into Erlenmeyer flask, add tip which picked up one colony from the plate of each sample.

Incubate flask at 37 degree C with shaking at 180 rpm for more than 12 hours (until the medium become the state of full growth).

3rd day
Measure Optical Density (600).

-Prepare 8 Erlenmeyer flask, pour 100mL LB broth, add 100 uL ampicillin, and 1 mL full growth medium.
-Put flasks into shaking incubator (25 degree C, 180 rpm) and begin incubation.
-Measure OD600 of each sample every one hour.

• Add arabinose solution of each concentration to the medium when the OD600 almost reached 2.0.
  

-Extract 1ml medium from the flask and put into 1.5ml Eppendorf tube every time measuring OD. Put the tube into icebox as soon as possible. Centrifuge the tube and throw away supernatant to make E.coli pellet. Preserve pellet in -20 degrees C freezer.

4th day
Measure Fluorescence

1.　Add 100 ul 8M urea buffer into each pellet and make suspension solution.
2.　Put the pellet in room temperature for 30 minutes.
3.　Use sonicator to smash the cell. (10 seconds, 10% power.)
4.　Put the tube into icebox and cool it down.
5.　Do 3 and 4 again.
6.　Centrifuge the tube. 150rpm, 2 minutes, 4 degrees C.
7.　Measure the fluorescence.

Result

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Discussion

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Detail Protocol

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