Team:HKU-HKBU/Speed Control Results

From 2009.igem.org

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(Regulation of cheZ expression)
(Regulation of cheZ expression)
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===Western Blotting===
===Western Blotting===
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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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The samples were collected from differnt cultivation time and various IPTG concentrations. Obvious concentration gradient was observed with the induce time of 24 hours with different IPTG concentrations, which indicated that the inducer IPTG could control the expression level of ''cheZ'', which in turn could speed up and slow down the swimming speed of the bacteria.  
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[[Image:HKU-HKBU_speed_control_experiments_QQ_western.png | center|thumb]]
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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.
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[[Image:HKU-HKBU_speed_control_experiments_QQ_western.png |center|thumb]]
===Speed control test===
===Speed control test===
The speed was detected by the swimming assay. The swimming radius was measured each hour. The results indicated  
The speed was detected by the swimming assay. The swimming radius was measured each hour. The results indicated  
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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.
From the results above, it can be seen that the CheZ concentration
From the results above, it can be seen that the CheZ concentration
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  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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[[Image:HKU-BU-IPTG-speed-control-yu.JPG|center|thumb]]
[[Image:HKU-BU-IPTG-speed-control-yu.JPG|center|thumb]]

Revision as of 16:27, 16 October 2009

Contents

Results

cheZ knockout detection in YBE01 and YBS01

cheZ knockout is a crutial step for speed control. We believed that by controlling the expression level of cheZ, we could manipulate the ratio of flagella rotating direction (clockwise & counterclockwise). Therefore, higher concentration of expressed CheZ could lead to faster swimming speed. This part played a key role as an "adapter" of bactomotor.

Colony PCR test

2HKU-HKBU colony delta CheZ.png

By using recombineering technology, cheZ could be precisely replaced by seletive resistance marker with homologous arms(about 50bp). To test the result, colonies were selected on the agar plate with chloramphenicol resistance followed by colony PCR test. In PCR, forward primers were designed in the upstream of cheZ locus in the chromosome and reverse primer was designed inside the chlromphenicol gene. The result of colony PCR test could examine whether the recombination procedure was successful or not. The expected DNA size of this test was about 400bp. The DNA agrose gel picture indicated the cheZ was knocked out both in YBE01 and YBS01 strains.

Swimming Test

Based on the theory of speed control, the cheZ knockout strain could not swim at all. Therefore 3 percent half solid agar were used for testing the swimming ability. After obseveing swimming assay constantly for 8 hours, the patterns of YBE01 and YBS01 strains were formed as follows and the cheZ knockout strains showed that the ineffectiveness of the swimming ability of the two bacteria strains, which was an indication of the successful knockout of cheZ gene in YBE01 and YBS01.

HKU-HKBU speed control experiments Fig2.png

Regulation of cheZ expression

For controlling the expression level of cheZ, a series of plasmids were constructed, such as plsmid plac/ara-his-cheZ-cm, which could be induced by IPTG or arabinose. This plasmid was transformed to YBS02 and YBE02. Different concentrations 0.001, 0.002, 0.004, 0.008, 0.012mmol/L of IPTG were added into the cultivating solution LB to induce cheZ expression.

Western Blotting

The samples were collected from differnt cultivation time and various IPTG concentrations. Obvious concentration gradient was observed with the induce time of 24 hours with different IPTG concentrations, which indicated that the inducer IPTG could control the expression level of cheZ, which in turn could speed up and slow down the swimming speed of the bacteria.

HKU-HKBU speed control experiments QQ western.png

Speed control test

The speed was detected by the swimming assay. The swimming radius was measured each hour. The results indicated

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.

From the results above, it can be seen that the CheZ concentration


HKU-BU-IPTG-speed-control-yu.JPG



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