Difference between revisions of "Part:BBa K574009:Experience"

(→‎Applications of BBa_K574009)
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'''Description:''' we assembled E0840 (''<partinfo>E0840</partinfo>'') under the pLas promoter (''<partinfo>R0079</partinfo>'') that was contained into K574009. We kept pSB1A2 as the scaffold vector.
 
'''Description:''' we assembled E0840 (''<partinfo>E0840</partinfo>'') under the pLas promoter (''<partinfo>R0079</partinfo>'') that was contained into K574009. We kept pSB1A2 as the scaffold vector.
 
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'''Methods:''' we ligated E0840 (''<partinfo>E0840</partinfo>'') downstream of K574009, and the positive colony was cultured at 37°C, 220 rpm for 12 hours, as well as the DH5alpha. Then we diluted 1:25 the former in 12 falcon tubes (4 ml cultures), each 3 tubes as a group. The later was also diluted in 3 tubes as the negative control. After Cultured for another 2 hours, the groups were induced with 3OC12HSL 0, 10^-6M, 10^-5M and 10^-4M respectively. We took samples after 1 hours, 4 hours, 8 hours and 24 hours, and then the samples were measured through the flow cytometer.
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'''Methods:''' <br>
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1. ligate E0840 (''<partinfo>E0840</partinfo>'') downstream of K574009, which acts as a reporter. <br>
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2. culture the positive colony at 37°C, 220 rpm for 12 hours, as well as the DH5alpha. <br>
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3. dilute 1:25 the former in 12 falcon tubes (4 ml cultures), each 3 tubes as a group. The later is also diluted in 3 tubes as the negative control. <br>
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4. after culturing for another 2 hours, as their OD600 reach 0.2, induce with 3OC12HSL 0, 10^-6M, 10^-5M and 10^-4M respectively. <br>
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5. take samples after 1 hour, 4 hours, 8 hours and 24 hours, that is, centrifugalizate 0.5 mL of each culture and suspend them by 1 mL of PBS (phosphate-buffered saline).  <br>
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6. measure the fluorescence of the samples with the flow cytometer.
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'''Results:''' From the chart, we can see that cells were hardly induced in the control group, and with the concentration of inducer growing, the intensity of GFP increased by groups. The most efficient concentration of inducer was around 10^-6M, and to most groups, the intensity of GFP reached its maxium after 4 hours.
 
'''Results:''' From the chart, we can see that cells were hardly induced in the control group, and with the concentration of inducer growing, the intensity of GFP increased by groups. The most efficient concentration of inducer was around 10^-6M, and to most groups, the intensity of GFP reached its maxium after 4 hours.
 
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Revision as of 02:53, 5 October 2011

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Please enter how you used this part and how it worked out.

Applications of BBa_K574009

Description: we assembled E0840 (BBa_E0840) under the pLas promoter (BBa_R0079) that was contained into K574009. We kept pSB1A2 as the scaffold vector.
Methods:
1. ligate E0840 (BBa_E0840) downstream of K574009, which acts as a reporter.
2. culture the positive colony at 37°C, 220 rpm for 12 hours, as well as the DH5alpha.
3. dilute 1:25 the former in 12 falcon tubes (4 ml cultures), each 3 tubes as a group. The later is also diluted in 3 tubes as the negative control.
4. after culturing for another 2 hours, as their OD600 reach 0.2, induce with 3OC12HSL 0, 10^-6M, 10^-5M and 10^-4M respectively.
5. take samples after 1 hour, 4 hours, 8 hours and 24 hours, that is, centrifugalizate 0.5 mL of each culture and suspend them by 1 mL of PBS (phosphate-buffered saline).
6. measure the fluorescence of the samples with the flow cytometer.

Results: From the chart, we can see that cells were hardly induced in the control group, and with the concentration of inducer growing, the intensity of GFP increased by groups. The most efficient concentration of inducer was around 10^-6M, and to most groups, the intensity of GFP reached its maxium after 4 hours.

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