Team:Newcastle/1 July 2010

From 2010.igem.org

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===Aim of this experiment===
===Aim of this experiment===
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The aim of this experiment was to determine if Bacillus subtilis 168 is able to produce urease and to degrade urea.
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The aim of this experiment was to determine if ''Bacillus subtilis'' 168 is able to produce urease and to degrade urea.
===Materials and methods===
===Materials and methods===
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Note: Ensure that the work was done using aseptic technique.
Note: Ensure that the work was done using aseptic technique.
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Transfer 10ml of the molten base to universal tube and allow the agar to set in a slanted position.
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Transfer 10ml of the molten base to universal tube and allow the agar to set in a slanted position.
 +
Store the harden agar in the fridge.
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*
 
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*''spac'' forward and reverse primers
 
*Pipettes
*Pipettes
*Universal tubes
*Universal tubes
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*Eppendorf tubes
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*Streaking loop
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*Centrifuge
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*PCR (see PCR protocol from Lab training)
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*Gel electrophoresis (see Gel electrophoresis from Lab training)
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''ara'' and ''spac'' are two genes that exist in the ATCC 6633 strain. ''ara'' and ''spac'' forward and reverse primers are two tests, which will be used in PCR. At the end, Gel Electrophersis can be used to distinguish th bands. If the bands from either of ''ara'' or ''spac'' show up at the expected bps against the DNA ladder, this proves that the genomic DNA is sucessfully extracted.
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===Gram-positive Bacteria Chromosomal DNA Extraction Protocol===
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===Urease test===
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====Puregene DNA isolation====
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# Perform the experiment using aseptic technique.
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# Grow overnight 7.5ml cultures in THYB containing 30μg/ml hyaluronidase.
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# Pick up single colony of ''B. subtilis'' 168 and streaking it onto the slanted agar tube.
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# Loosen up the cap to allow air exchange between the interior of the tube and the external environment.
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# Incubate the tubes overnight at 37°C.
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====Cell Lysis====
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Negative control - Without ''B. subtilis'' 168
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# Two sets of chromosomal DNA were being done.
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Test 1 (Duplicate) - Inoculated with ''B. subtilis'' 168
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# Cells go through 3600 x g centrifugation for 15 minutes to pellet.
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Test 2 (Duplicate) - Inoculated with ''B. subtilis'' 168
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# The supernatant in the tubes are poured away.
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# 0.5ml of cell suspension solution is added to the eppendorf tubes. The solution in the tubes are pipetted up and down to resuspend cells. The solution is then transferred to another 1.5ml tube.
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# 50μl of lysozyme and 7.5μl of lytic enzyme solution is added into each tube. The tubes are then inverted 25 times to mix the solution.
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# We then incubate them at 37°C for 30 minutes in order to digest the cell walls. The tubes are inverted occasionally during incubation.
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# To pellet the cells, we centrifuged it for 10 minutes and poured away the supernatant after that.
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# 0.5ml of cell lysis solution is added to the pelleted cells and the solution is pipetted up and down in order to lyse the cells. The samples are then heated for about 5 minutes at 80°C.
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====RNase Treatment====
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===Expected results===
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# 6μl of RNase A solution is added into the cell lysate.
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Christensen's Urea Agar was formulated to detect and differentiate urolytic and urea degrading microorganisms.
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# The tubes are inverted 25 times in order to allow the solutions to mix. They are then incubated at 60minutes at 37°C.
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The addition of gelatine peptone, dextrose and reduced content of buffer supports a luxuriant growth at an early stage. Urea is the substrate and can be degraded by certain organisms, which results in ammonia building. The ammonia makes the media alkaline and therefore the indicator phenol red will change from orange color to pink-red color.
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====Protein Precipitation====
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Therefo
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# The samples are cooled on ice.
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# 0.5ml of protein precipitation solution is added into each tube.
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# The solutions are vortexed at high speed for about 20 seconds for the protein precipitation solution to mix properly with the cell lysate. The samples are then placed on ice for 5 minutes.
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# They are then centrifuged at 13000rpm for 1 minute for the proteins to form a tight pellet.
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====DNA Precipitation====
 
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# The supernatant containing the DNA are poured into a clean eppendorf tube.
 
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# 0.5ml of isopropanol is then added into each tube.
 
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# The solutions are then mixed by inverting gently for 50 times.
 
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# The mixture is centrifuged at 13000rpm for 1 minute. The DNA can be seen as a small white pellet at the bottom of the tubes.
 
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# The supernatant is poured off and the tubes are drained on a clean absorbent paper. 0.5ml of 70% ethanol is added into the tubes and are inverted a few times to wash the DNA.
 
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# They are then centrifuged for 1 minute at 13000rpm. After that, ethanol is poured off leaving the DNA behind.
 
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# The tubes are drained on clean absorbent paper. They are allowed to air dry for 10-15 minutes.
 
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====DNA Hydration====
 
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# 100μl of DNA hydration solution is added to each tube.
 
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#DNA is rehydrated by incubating the sample for one hour at 65°C and overnight at room temperature.
 
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===Results===
 
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Results were as expected
 
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see: https://2010.igem.org/Team:Newcastle/7_July_2010
 
===Conclusion===  
===Conclusion===  
See: https://2010.igem.org/Team:Newcastle/7_July_2010
See: https://2010.igem.org/Team:Newcastle/7_July_2010

Revision as of 13:54, 21 July 2010

Contents

Aim of this experiment

The aim of this experiment was to determine if Bacillus subtilis 168 is able to produce urease and to degrade urea.

Materials and methods

  • Christensen's Urea Agar

Make up 1 liter of the agar mixture containing:

  1. Gelatine peptone 1.0g
  2. Potassium dihydrogen phosphate 2.0g
  3. Sodium chloride 5.0g
  4. Agar 20g

Top up to 1 liter and apply gentle heating to dissolve. Sterilize at 115°C for at least 20min.

Add the following to the molten base:

  1. D(+)-Glucose 1.0g
  2. Phenol red, 0.2% 6ml

Note: Ensure that the work was done using aseptic technique.

Transfer 10ml of the molten base to universal tube and allow the agar to set in a slanted position. Store the harden agar in the fridge.

  • Pipettes
  • Universal tubes
  • Streaking loop

Urease test

  1. Perform the experiment using aseptic technique.
  2. Pick up single colony of B. subtilis 168 and streaking it onto the slanted agar tube.
  3. Loosen up the cap to allow air exchange between the interior of the tube and the external environment.
  4. Incubate the tubes overnight at 37°C.

Negative control - Without B. subtilis 168 Test 1 (Duplicate) - Inoculated with B. subtilis 168 Test 2 (Duplicate) - Inoculated with B. subtilis 168

Expected results

Christensen's Urea Agar was formulated to detect and differentiate urolytic and urea degrading microorganisms. The addition of gelatine peptone, dextrose and reduced content of buffer supports a luxuriant growth at an early stage. Urea is the substrate and can be degraded by certain organisms, which results in ammonia building. The ammonia makes the media alkaline and therefore the indicator phenol red will change from orange color to pink-red color.

Therefo


Conclusion

See: https://2010.igem.org/Team:Newcastle/7_July_2010

Retrieved from "http://2010.igem.org/Team:Newcastle/1_July_2010"