Difference between revisions of "Part:BBa K358019:Experience"

(→‎Experiment 2 Quantitative characterization of lytic activity of λ　lysis cassette)
(→‎Experiment 2 Quantitative characterization of lytic activity of λ　lysis cassette)
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A steady state of cell lysis caused by λ lysis cassette exists. So we evaluated the lytic activity by measuring OD550 of the cultures that were steady state of cell lysis.  
 
A steady state of cell lysis caused by λ lysis cassette exists. So we evaluated the lytic activity by measuring OD550 of the cultures that were steady state of cell lysis.  
  
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Revision as of 14:11, 31 October 2010

Experiment1 Characterization of lytic activity with time

To characterize the lytic activity of λ lysis cassette in more detail, we focused on when cell lysis occurs after induction by IPTG. We did the experiment as this protocol, and we got the following data. Protocol

  1. Pour 3mL of supplemented M9 medium to a Falcon tube.
  2. Pick out a colony on the plate of E.coli and put into the Falcon tube.
  3. Grow it at 30 degreees for 16h.
  4. Dilute it 50 folds with the same media and incubate until OD550 is about 0.15.
  5. Ditribute 3ml of the culture to falcon tubes and add certain amount of IPTG.
  6. Incubate the cultures at 30 degrees and measure OD550 of them every 30 min.
KyotoGrp101028-2.png


As this figure shows, when E.coli starts to lyse depends on the strength of induction of IPTG. When the concentration of IPTG is 1mM, E.coli starts to lyse after 1.5h, in contrast, when it is 0.03mM, E.coli starts to lyse after 6h. This result suggests the following two facts.,

  1. To evaluate the lytic activity quantitatively, we must evaluate it after cell lysis becomes a steady state.
  2. We can regulate freely when E.coli are lysed, by changing the strength of induction of λ Lysis cassette.

Experiment 2 Quantitative characterization of lytic activity of λ　lysis cassette

A steady state of cell lysis caused by λ lysis cassette exists. So we evaluated the lytic activity by measuring OD550 of the cultures that were steady state of cell lysis.

KyotoFigA007.png
Incubating 16h 18h 20h 16h 18h 20h 16h 18h 20h
Number of colony 1 1 1 2 2 2 3 3 3
0mM 2.13 2.17 2.35 2.05 2.04 2.14 2.22 2.21 2.33
0.01mM 2.02 2.01 2.15 1.96 2.23 2.23 2.21 2.07 2.29
0.02mM 2.21 2.12 2.16 1.86 1.86 1.95 1.76 1.93 1.71
0.03mM 1.44 1.41 1.39 1.70 1.62 1.59 0.82 1.02 0.83
0.05mM 0.33 0.25 0.17 0.25 0.25 0.24 0.26 0.33 0.24
0.07mM 0.15 0.17 0.23 0.21 0.20 0.23 0.18 0.18 0.19
0.1mM 0.13 0.20 0.36 0.20 0.18 0.23 0.17 0.15 0.23
0.3mM 0.10 0.21 0.31 0.08 0.11 0.14 0.11 0.07 0.20
0.5mM 0.07 0.08 0.10 0.08 0.08 0.09 0.11 0.08 0.25
0.8mM 0.25 0.10 0.08 0.29 0.22 0.11 0.18 0.22 0.10
1.0mM 0.04 0.15 0.21 0.18 0.27 0.27 0.18 0.06 0.24

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