Latest revision as of 02:54, 22 October 2009

Results

cheZ knockout in YBE01 and YBS01

cheZ knockout is a crucial 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

Figure.1 Colony PCR for cheZ knockout in E.coli and S. typhimurium strains. 1, YBS01; 2, YBE01

By using recombineering technology, cheZ could be precisely replaced by selective resistance marker with homologous arms (about 50-bp). To test the result, colonies were selected on the agar plate with chloramphenicol resistance followed by colony PCR test. In the PCR reaction, 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 400 bp. The DNA agrose gel picture indicated the cheZ gene 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 was used for testing the swimming ability. After observing the spread of the bacteria 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.

Figure. 2 Swimming assays of E.coli and S. typhimurium strains. a, YBE01; b, cheZ knock-out YBE01 (YBE02); c, YBS01; d, cheZ knock-out YBS01 (YBS02).

Regulation of cheZ expression

To control the expression level of cheZ, a series of plasmids were constructed, such as plasmid plac/ara-his-cheZ-cm ( [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283014 BBa_K283014] ), which could be induced by IPTG or arabinose. This plasmid ( [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283014 BBa_K283014] ) was transformed to YBS02 and YBE02. Different concentrations 1, 2, 3.3 uM of IPTG were added into the cultivating solution LB to induce cheZ expression.

Western Blot

The samples were collected from different 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.

Figure. 3 Western blotting result of CheZ expression in YBE02 with different concentration of IPTG. 1, without IPTG; 2, with 1uM IPTG; 3, with 2uM IPTG; 4, with 3.3uM IPTG

Although [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283014 BBa_K283014] could control the expression of CheZ, the leak expression could cause the failure of swimming control. Therefore, more powerful system [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 BBa_K283002] must be constructed to prevent the leak.

Speed control

The speed was detected by the swimming assay. The swimming with TetR repress [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 BBa_K283002], the swimming radius was measured each hour continuously for 8 hours. The results indicated that without aTc inducer, the bacteria only tumbled; and when aTC was added into cultivation LB broth, the bacteria were started to swim. And the swimming speed of bacteria could be controlled 'Speed up' with increasing aTc concentration. This result indicated that the alternation of aTc concentrations could be an effective way to control speed.

Figure. 4 Swimming speed assay by different concentration inducer aTc.

Conclusion

We successfully knock-out cheZ gene in both YBE01 and YBS01 strains. Re-introduction of cheZ gene restored their swimming ability. Moreover, the swimming speed of these two strains was shown to be adjustable by different amounts of chemical inducers.

@@ Line 5: / Line 5: @@
 =Results=
-=='''CheZ Knockout detection in ''E. coli 2443'' and ''Salmonella SL7207'''''==
+=='''''cheZ'' knockout in YBE01 and YBS01'''==
-===Colony PCR test===
+''cheZ'' knockout is a crucial 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.
-[[Image:HKU-HKBU_speed_control_experiments_PCR_test.png | center]]
+===Colony PCR===
-The expected DNA size of this test was about 400bp, which showed the success recombination procedure.
+[[Image:HKU-HKBU delta cheZ2.png|center|thumb|'''Figure.1''' Colony PCR for ''cheZ'' knockout in ''E.coli'' and ''S. typhimurium'' strains. 1, YBS01; 2, YBE01|300px]]
+By using recombineering technology, ''cheZ'' could be precisely replaced by selective resistance marker with homologous arms (about 50-bp). To test the result, colonies were selected on the agar plate with chloramphenicol resistance followed by colony PCR test. In the PCR reaction, 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 400 bp. The DNA agrose gel picture indicated the ''cheZ'' gene was knocked out both in YBE01 and YBS01 strains.
 ===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 YBE01'' and ''S.typhimurium YBS01''.
+Based on the theory of speed control, the ''cheZ'' knockout strain could not swim at all. Therefore 3 percent half solid agar was used for testing the swimming ability. After observing the spread of the bacteria 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.
-[[Image:HKU-HKBU_speed_control_experiments_Fig2.png | center]]
-==Regulation of CheZ expression==
+[[Image:HKU-HKBU_speed_control_experiments_Fig3.png |center|thumb|'''Figure. 2''' Swimming assays of ''E.coli'' and ''S. typhimurium'' strains.''' a,''' YBE01; '''b,''' ''cheZ'' knock-out YBE01 (YBE02); '''c,''' YBS01; '''d,''' ''cheZ'' knock-out YBS01 (YBS02).
+|500px]]
-===Swimming test===
+==Regulation of ''cheZ'' expression==
-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====
+To control the expression level of ''cheZ'', a series of plasmids were constructed, such as plasmid plac/ara-his-cheZ-cm ( [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283014 '''BBa_K283014'''] ), which could be induced by IPTG or arabinose. This plasmid ( [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283014 BBa_K283014] ) was transformed to YBS02 and YBE02. Different concentrations 1, 2, 3.3 uM of IPTG were added into the cultivating solution LB to induce ''cheZ'' expression.
-[[Image:HKU-HKBU_speed_control_experiments_MG3_with_Chloramphenicol.png|center]]
-====Chloramphenicol added====
+===Western Blot===
-[[Image:HKU-HKBU_speed_control_experiments_MG3_without_Chloramphenicol.png|center]]
-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 samples were collected from different 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.
+[[Image:HKU-HKBU speed control western4.png |center|thumb|'''Figure. 3''' Western blotting result of CheZ expression in YBE02 with different concentration of IPTG. 1, without IPTG; 2, with 1uM IPTG; 3, with 2uM IPTG;  4, with 3.3uM IPTG
+|500px]]
+Although [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283014 '''BBa_K283014'''] could control the expression of CheZ, the leak expression could cause the failure of swimming control. Therefore, more powerful system [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 '''BBa_K283002'''] must be constructed to prevent the leak.
+===Speed control ===
+The speed was detected by the swimming assay. The swimming with TetR repress [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 '''BBa_K283002'''], the swimming radius was measured each hour continuously for 8 hours. The results indicated that without aTc inducer, the bacteria only tumbled; and when aTC was added into cultivation LB broth, the bacteria were started to swim. And the swimming speed of bacteria could be controlled 'Speed up' with increasing aTc concentration. This result indicated that the alternation of aTc concentrations could be an effective way to control speed.
-===Western Blotting===
-[[Image:HKU-HKBU_speed_control_experiments_QQ_western.png | center]]
+[[Image:Speed control aTc2.JPG|center|thumb|'''Figure. 4''' Swimming speed assay by different concentration  inducer aTc.
-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.
+|500px]]
+=='''Conclusion'''==
-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.
+We successfully knock-out ''cheZ'' gene in both YBE01 and YBS01 strains. Re-introduction of ''cheZ'' gene restored their swimming ability. Moreover, the swimming speed of these two strains was shown to be adjustable by different amounts of chemical inducers.
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Team:HKU-HKBU/Speed Control Results

From 2009.igem.org