Team:HKU-HKBU/speed control experiments

From 2009.igem.org

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=Experiments=
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==Plasmid extraction==
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5µl DH10B, containing the plasmid added into 5ml (anti-chloramphenicol) LB-Broth, 32 , overnight for plasmid extraction.
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==Experiment using MG3==
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===Prepare competent cell: MG3 (delta CheZ strain)===
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===Transformation: electroporation===
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===Recover for 30 minutes===
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===Culture with IPTG===
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{| align="center" border="2px" cellpadding="4px"
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! rowspan="2" | Culture Time !! colspan="4" | IPTG concentration
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|-
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! 0uM !! 1uM !! 2uM !! 3.3uM
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|-
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| 1hr
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|-
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| 2hr
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|-
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| 3hr
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|-
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| 24hr
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|}
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===Western Blotting===
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====Equal loading====
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2ul sample -> 96 well plate -> add 100ul BCA solution (50:1). After measurement at  wavelength of 562nm, total amount of protein = sample concentration x sample volume. Adjust the sample volume to the same loading volume by adding the mixture buffer.
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====Result====
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[[Image:HKU-HKBU_speed_control_experiments_MG3_western.png|center]]
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According to the results from western Blotting, there is no obvious positive relation between the cheZ expressing and the concentration gradient of IPTG, in the range from 0 to 3.3uM in the first three hours . Above is the expressing of cheZ overnight, 0, 1,2 and 3.3 uM from left to right respectively. The positive relationship is much more obvious after 24 hours.
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===Swimming test===
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Test MG3-plD-his-cheZ-cm under 0.0005, 0.001, 0.002, 0.004, 0.008, 0.012mmol/L IPTG.
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=====No Chloramphenicol=====
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[[Image:HKU-HKBU_speed_control_experiments_MG3_with_Chloramphenicol.png|center]]
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=====2nd time: Chloramphenicol added=====
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[[Image:HKU-HKBU_speed_control_experiments_MG3_without_Chloramphenicol.png|center]]
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====Analysis====
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# Difference between various IPTG conc. gradients not significant. The expressing level of cheZ under various concentration gradients of IPTG is not significantly different within 8 hours, leading to the similarity of swim behaviors.
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# Even negative control (no IPTG added) swims, probably due to leak-expressing.
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# The speed enters plateau after 4 hours, about 2.25-2.5mm/hr.
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==Experiment using E.Coli 2443==
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===cheZ knock-out: Refer to QQ===
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===Co-transformation: Failure===
{{Team:HKU-HKBU/footer}}
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Revision as of 02:36, 12 October 2009

Contents

Experiments

Plasmid extraction

5µl DH10B, containing the plasmid added into 5ml (anti-chloramphenicol) LB-Broth, 32 , overnight for plasmid extraction.

Experiment using MG3

Prepare competent cell: MG3 (delta CheZ strain)

Transformation: electroporation

Recover for 30 minutes

Culture with IPTG

Culture Time IPTG concentration
0uM 1uM 2uM 3.3uM
1hr
2hr
3hr
24hr

Western Blotting

Equal loading

2ul sample -> 96 well plate -> add 100ul BCA solution (50:1). After measurement at wavelength of 562nm, total amount of protein = sample concentration x sample volume. Adjust the sample volume to the same loading volume by adding the mixture buffer.

Result

HKU-HKBU speed control experiments MG3 western.png

According to the results from western Blotting, there is no obvious positive relation between the cheZ expressing and the concentration gradient of IPTG, in the range from 0 to 3.3uM in the first three hours . Above is the expressing of cheZ overnight, 0, 1,2 and 3.3 uM from left to right respectively. The positive relationship is much more obvious after 24 hours.

Swimming test

Test MG3-plD-his-cheZ-cm under 0.0005, 0.001, 0.002, 0.004, 0.008, 0.012mmol/L IPTG.

No Chloramphenicol
HKU-HKBU speed control experiments MG3 with Chloramphenicol.png
2nd time: Chloramphenicol added
HKU-HKBU speed control experiments MG3 without Chloramphenicol.png

Analysis

  1. Difference between various IPTG conc. gradients not significant. The expressing level of cheZ under various concentration gradients of IPTG is not significantly different within 8 hours, leading to the similarity of swim behaviors.
  2. Even negative control (no IPTG added) swims, probably due to leak-expressing.
  3. The speed enters plateau after 4 hours, about 2.25-2.5mm/hr.

Experiment using E.Coli 2443

cheZ knock-out: Refer to QQ

Co-transformation: Failure

Sponsors

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