Team:Newcastle/1 July 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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==Urease Test==
 
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===Aims===
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=Urease Test=
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==Aims==
The aim of this experiment was to determine if ''Bacillus subtilis'' 168 is able to produce urease and degrade urea.
The aim of this experiment was to determine if ''Bacillus subtilis'' 168 is able to produce urease and degrade urea.
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===Materials===
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==Procedures==
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*Pipettes
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* Please refer to [[Team:Newcastle/Urease test| urease test]]
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*Universal tubes
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*Streaking loop
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*Christensen's Urea Agar
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**Make up 1 liter of the agar mixture containing:
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*# Gelatine peptone 1.0g
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*# Potassium dihydrogen phosphate 2.0g
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*# Sodium chloride 5.0g
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*# Agar 20g
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**Top up to 1 liter and apply gentle heating to dissolve. Sterilize at 115°C for at least 20min.
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**Add the following to the molten base:
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*# D(+)-Glucose 1.0g
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*# Phenol red, 0.2% 6ml
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**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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**Store the harden agar in the fridge.
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===Protocol===
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==Inference==
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# Perform the experiment using aseptic technique.
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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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# Set up the tubes as indicated:
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##Negative control - Without ''B. subtilis'' 168
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##Test 1 (Duplicate) - Inoculated with ''B. subtilis'' 168
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##Test 2 (Duplicate) - Inoculated with ''B. subtilis'' 168
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===Inference===
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Christensen's Urea Agar was formulated to detect and differentiate urolytic and urea degrading microorganisms.
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.
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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*Test 2 (Duplicate) - Pink-red color
*Test 2 (Duplicate) - Pink-red color
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For actual results, please see 02.07.10 lab results.
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For actual results, please see [[Team:Newcastle/2 July 2010#Results|02.07.10 lab notebook.]]
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==LacI BioBrick Construction==
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===Aims===
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*To use PCR to extract ''lacI'' (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).
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===Materials===
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*''E. coli'' DH5alpha strain
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*pMutin4 plasmid
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*pSB1AT3 plasmid
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===Protocol===
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* [[TeamNewcastleTransformation of E. coli|Transformation of ''E. coli'']] DH5alpha with pMutin4 and pSB1AT3 separately.
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===Inference===
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*''Ecoli'' DH5alpha is used as a bioreactor to replicate our plasmids to create a large stock ready for extraction from cells.
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==Competence==
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=Competent ''E. coli'' Production=
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===Aims===
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==Aims==
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*
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*To make a stock of competent ''E. coli'' (DH5α strain) ready for transformation.
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===Materials===
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==Materials and Protocol==
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*''E. coli'' DH5alpha strain
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* Set up [[Team:Newcastle/Growing a liquid culture| liquid culture]] consisting of ''E. coli'' DH5α.
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*pMutin4 plasmid
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*pSB1AT3 plasmid
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===Protocol===
 
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*
 
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===Inference===
 
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*
 
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 14:11, 25 October 2010

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Contents

Urease Test

Aims

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

Procedures

Inference

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.

  • Negative control - No color change (Orange color)
  • Test 1 (Duplicate) - Pink-red color
  • Test 2 (Duplicate) - Pink-red color

For actual results, please see 02.07.10 lab notebook.

Competent E. coli Production

Aims

  • To make a stock of competent E. coli (DH5α strain) ready for transformation.

Materials and Protocol


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