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 ''YBE01'' and ''YBS01'''''==
+=='''''cheZ'' knockout 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.
+''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===
-[[Image:HKU-HKBU_delta_cheZ.png|center|300px]]
+[[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 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.
+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 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''.
+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|500px]]
+[[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]]
 ==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.
+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 Blotting===
+===Western Blot===
-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''.
+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 western2.png |center|500px]]
+[[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 radius was measured each hour continously for 8 hours. The results indicated that without IPTG inducer, the bacteria only tumbled; and when IPTG was added into cultivation LB broth, the bacteria were started and swam. However, similar speed bacteria with differnt IPTG concentration indicated that the alternation of IPTG concentrations was not an effective speed control method. Although it was hard to manipulate the speed via this method, the turn-on and turn-off of swimming could be achieved.
+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.
-[[Image:HKU-BU-IPTG-speed-control-yu2.png|center|500px]]
+[[Image:Speed control aTc2.JPG|center|thumb|'''Figure. 4''' Swimming speed assay by different concentration  inducer aTc.
+|500px]]
+=='''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.
 {{Team:HKU-HKBU/footer}}

Team:HKU-HKBU/Speed Control Results

From 2009.igem.org