Team:HKU-HKBU/speed control experiments

From 2009.igem.org

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{{Team:HKU-HKBU/header}}
{{Team:HKU-HKBU/header}}
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=Experiments=
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=Speed Control - Experiments=
          
          
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==Plasmid extraction==
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==CheZ knockout of ''E. coli 2443'' and ''Salmonella SL7207''==
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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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! 0uM !! 1uM !! 2uM !! 3.3uM
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| 1hr
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| 2hr
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| 3hr
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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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==Experiment using E.Coli 2443==
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===CheZ knockout of E.coli 2443 and Salmonella SL7207===
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Two methods of detection of whether the CheZ gene were combined to make sure the success of recombination for CheZ knock out.  
Two methods of detection of whether the CheZ gene were combined to make sure the success of recombination for CheZ knock out.  
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====Swimming Test====
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===Swimming Test===
After 8 hours’ bacteria swimming, the patterns three strains formed below showed that the ineffectiveness of the swimming ability of the bacteria, which was an indication of the successful knockout of CheZ gene in E.coli 2443 and Salmonella SL7207. The third panel of  E.coli 2443 acted as a positive control in this test.
After 8 hours’ bacteria swimming, the patterns three strains formed below showed that the ineffectiveness of the swimming ability of the bacteria, which was an indication of the successful knockout of CheZ gene in E.coli 2443 and Salmonella SL7207. The third panel of  E.coli 2443 acted as a positive control in this test.
<gallery align="center">
<gallery align="center">
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Image:HKU-HKBU_speed_control_experiments_Salmonella_SL7207_delta_cheZ.png‎ | frame | ''Salmonella SL7207'' (delta CheZ)
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Image:HKU-HKBU_speed_control_experiments_Salmonella_SL7207_delta_cheZ.png‎ | ''Salmonella SL7207''<br />(delta CheZ)
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Image:HKU-HKBU_speed_control_experiments_E_coli_2443_delta_cheZ.png‎ | frame | ''E. coli'' 2443 (delta CheZ)
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Image:HKU-HKBU_speed_control_experiments_E_coli_2443_delta_cheZ.png‎ | ''E. coli 2443''<br />(delta CheZ)
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Image:HKU-HKBU_speed_control_experiments_E_coli_2443_positive_control.png‎ | frame | ''E. coli'' 2443 (positive control)
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Image:HKU-HKBU_speed_control_experiments_E_coli_2443_positive_control.png |‎ ''E. coli 2443''<br />(positive control)
</gallery>
</gallery>
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====PCR test====
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===PCR test===
The expected DNA size of this test was about 400bp, which showed the success recombination procedure.
The expected DNA size of this test was about 400bp, which showed the success recombination procedure.
[[Image:HKU-HKBU_speed_control_experiments_PCR_test.png‎ | center]]
[[Image:HKU-HKBU_speed_control_experiments_PCR_test.png‎ | center]]
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===Regulation of CheZ expression===
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==Regulation of CheZ expression==
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====Swimming test====
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===Swimming test===
Test plac-his-cheZ-cm under 0.0005, 0.001, 0.002, 0.004, 0.008, 0.012mmol/L IPTG in MG3 stain.
Test plac-his-cheZ-cm under 0.0005, 0.001, 0.002, 0.004, 0.008, 0.012mmol/L IPTG in MG3 stain.
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=====No Chloramphenicol=====
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====No Chloramphenicol====
[[Image:HKU-HKBU_speed_control_experiments_MG3_with_Chloramphenicol.png|center]]
[[Image:HKU-HKBU_speed_control_experiments_MG3_with_Chloramphenicol.png|center]]
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=====Chloramphenicol added=====
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====Chloramphenicol added====
[[Image:HKU-HKBU_speed_control_experiments_MG3_without_Chloramphenicol.png|center]]
[[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.
 
The results above are ambiguous, and we tried to verify if we were successful in achieving differential levels of CheZ in MG3 by doing western blotting.
The results above are ambiguous, and we tried to verify if we were successful in achieving differential levels of CheZ in MG3 by doing western blotting.
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====Western Blotting====
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===Western Blotting===
   
   
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[[Image:HKU-HKBU_speed_control_experiments_QQ_western.png‎ | center]]
From the results above, it can be seen that the CheZ concentration in MG3 was proportional to the concentration of IPTG added to the cultures when the induced time was about 24 hours. The samples with induced time less than 24 hours showed no obvious bands in the Western Blotting.
From the results above, it can be seen that the CheZ concentration in MG3 was proportional to the concentration of IPTG added to the cultures when the induced time was about 24 hours. The samples with induced time less than 24 hours showed no obvious bands in the Western Blotting.
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According to the result of Western Blotting, we could explain the similar swimming abilities of bacteria with different IPTG concentrations. As the speeds of swimming were tested within 9 hours, which were too short for the induced reactions to undergo, the speeds were more or less the same. Another problem was that even negative control strain (no IPTG added) swam, which was probably caused by leaky expression.
According to the result of Western Blotting, we could explain the similar swimming abilities of bacteria with different IPTG concentrations. As the speeds of swimming were tested within 9 hours, which were too short for the induced reactions to undergo, the speeds were more or less the same. Another problem was that even negative control strain (no IPTG added) swam, which was probably caused by leaky expression.
{{Team:HKU-HKBU/footer}}
{{Team:HKU-HKBU/footer}}

Latest revision as of 03:43, 12 October 2009

Contents

Speed Control - Experiments

CheZ knockout of E. coli 2443 and Salmonella SL7207

Two methods of detection of whether the CheZ gene were combined to make sure the success of recombination for CheZ knock out.

Swimming Test

After 8 hours’ bacteria swimming, the patterns three strains formed below showed that the ineffectiveness of the swimming ability of the bacteria, which was an indication of the successful knockout of CheZ gene in E.coli 2443 and Salmonella SL7207. The third panel of E.coli 2443 acted as a positive control in this test.

Salmonella SL7207
(delta CheZ)

E. coli 2443
(delta CheZ)

‎ E. coli 2443
(positive control)

PCR test

The expected DNA size of this test was about 400bp, which showed the success recombination procedure.

HKU-HKBU speed control experiments PCR test.png

Regulation of CheZ expression

Swimming test

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

No Chloramphenicol

HKU-HKBU speed control experiments MG3 with Chloramphenicol.png

Chloramphenicol added

HKU-HKBU speed control experiments MG3 without Chloramphenicol.png

The results above are ambiguous, and we tried to verify if we were successful in achieving differential levels of CheZ in MG3 by doing western blotting.

Western Blotting

HKU-HKBU speed control experiments QQ western.png

From the results above, it can be seen that the CheZ concentration in MG3 was proportional to the concentration of IPTG added to the cultures when the induced time was about 24 hours. The samples with induced time less than 24 hours showed no obvious bands in the Western Blotting.

According to the result of Western Blotting, we could explain the similar swimming abilities of bacteria with different IPTG concentrations. As the speeds of swimming were tested within 9 hours, which were too short for the induced reactions to undergo, the speeds were more or less the same. Another problem was that even negative control strain (no IPTG added) swam, which was probably caused by leaky expression.

Sponsors

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