Team:Newcastle/2 July 2010

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{{Team:Newcastle/mainbanner}}
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==Urease Test==
 
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===Aims===
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'''2 July 2010'''
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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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===Protocol===
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==Procedures==
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The experiment was performed on 01.07.10. For the experimental protocol, please see 01.07.10 lab notebook.
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The experiment was performed on 01.07.10. Please refer to [[Team:Newcastle/Urease test| Urease test]].
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===Results===
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==Results==
|[[Image:Newcastle_urease_test_020710.png|400px ]]
|[[Image:Newcastle_urease_test_020710.png|400px ]]
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# Test 2 (Duplicate) - Pink-red color
# Test 2 (Duplicate) - Pink-red color
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===Discussion===  
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==Discussion==
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''B. subtilis'' 168 is able to produce urease, therefore, urea which is the substrate and can be found in the agar was degraded, 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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''B. subtilis'' 168 is able to produce urease, therefore, urea which is the substrate and can be found in the agar was degraded, 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 negative control did not turn pink-red color, therefore indicating that no contamination had occurred.
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===Conclusion===
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The negative control did not turn pink-red color, therefore indicating that no contamination had occurred.
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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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*Competent ''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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==Competent ''E. coli'' Production==
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===Aims===
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*To make a stock of competent ''E. coli'' (DH5alpha strain) ready for transformation.
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===Materials===
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*''E. coli'' DH5alpha strain
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===Protocol===
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==Conclusion==  
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* Set up [[Team:Newcastle/Growing a liquid culture| liquid culture]] of ''E. coli'' DH5alpha
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From the experiment it can be concluded that ''Bacillus subtilis'' 168 produces urease enzyme in order to break down urea and produces ammonium and carbonate ions.
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===Inference===
 
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*Grow up a stock of ''E.coli'' DH5alpha strain ready to be made competent
 
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=Competent ''E. coli'' DH5α Production=
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==Aims==
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*To make a stock of competent ''E. coli'' (DH5α strain) ready for transformation.
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* Produce [[Team:Newcastle/E. coli Competence|competent ''E. coli'']] (DH5alpha strain)
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==Materials and Protocol==
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Please refer to:[[Team:Newcastle/E. coli Competence|competent]] for materials and protocol.
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==Inference==
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*We created a stock of competent ''E. coli'' (DH5α strain) that an be used for future transformations to grow up/replicate plasmids.
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 14:19, 25 October 2010

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2 July 2010

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

The experiment was performed on 01.07.10. Please refer to Urease test.

Results

|Newcastle urease test 020710.png

Figure 1: Urease test results

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

Discussion

B. subtilis 168 is able to produce urease, therefore, urea which is the substrate and can be found in the agar was degraded, 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 negative control did not turn pink-red color, therefore indicating that no contamination had occurred.

Conclusion

From the experiment it can be concluded that Bacillus subtilis 168 produces urease enzyme in order to break down urea and produces ammonium and carbonate ions.


Competent E. coli DH5α Production

Aims

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

Materials and Protocol

Please refer to:competent for materials and protocol.

Inference

  • We created a stock of competent E. coli (DH5α strain) that an be used for future transformations to grow up/replicate plasmids.
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