Team:Newcastle/12 July 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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'''12 July 2010'''
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=Research=
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=LacI BioBrick Construction=
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We continued with our research on urease pathway in ''Bacillus subtilis'' 168.
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[[Image:P7120450.JPG|300px|thumb|right]]
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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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Summary:
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#Garnett J, Baumberg S, Stockley PG and Phillips SEV. (2007)."Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor L-arginine". ''Acta crystallographica. Section F, Structural biology and crystallization communications''. 63(Pt 11). 918-21. International Union of Crystallography.
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# Colony PCR
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#Garnett J, Marincs F, Baumberg S, Stockley PG and Phillips SEV. (2008). "Structure and function of the arginine repressor-operator complex from Bacillus subtilis". ''Journal of molecular biology''. 379(2. 284-98.
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# Streak plate
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# Miniprep PCR - of colonies that worked
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==Materials==
 
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* PCR reagents
 
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* Agar plates
 
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* Gloves
 
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* Wire loop
 
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==Protocols==
 
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====Colony PCR====
 
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Of the 11 plates with colonies grown 7 were selected for screeneing by colony PCR and Streak plating.
 
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Colony PCR is a rapid way to check if the desired sequence is now present in the vector. Unfortunately none of the colonies had the desired sequence integrated into the vector.
 
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It seems likely that the colonies selected were present due to the degradation of ampicillin in the plates.
 
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[[Image:P7120427.JPG|200px|thumb|right]]
 
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7 Tubes were labelled 1-7 (+ a control)
 
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The quantities for the PCR are as follows:
 
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Please refer to [[Team:Newcastle/PCR|PCR]]. The melting temperature, Tm, of this experiment is 54°C.
 
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'''Note''' We used 1 minute of extension time because the size of lacI is 1.4kb.
 
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'''Note''' There are 2 possible reasons for red colonies formed:
 
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# partial digest - rejoins without insert
 
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# with insert 
 
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We need to tell the difference between the two.
 
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====Spread Plates====
 
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{|
 
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|-
 
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|[[Image:P7120408.JPG|thumb]]
 
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|[[Image:P7120409.JPG|thumb]]
 
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|[[Image:P7120418.JPG|thumb]]
 
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|}
 
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===Overnight culture===
 
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The seven colonies that grew on the plates were cultured overnight. The protocol for [[Team:Newcastle/Growing_an_overnight_cultures| growing an overnight culture]] was followed.
 
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==Tommorrow==
 
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We are going to take one of the colonies that have worked and possibly a whole plasmid prep.
 
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 19:32, 25 October 2010

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Research

We continued with our research on urease pathway in Bacillus subtilis 168.

  1. Garnett J, Baumberg S, Stockley PG and Phillips SEV. (2007)."Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor L-arginine". Acta crystallographica. Section F, Structural biology and crystallization communications. 63(Pt 11). 918-21. International Union of Crystallography.
  2. Garnett J, Marincs F, Baumberg S, Stockley PG and Phillips SEV. (2008). "Structure and function of the arginine repressor-operator complex from Bacillus subtilis". Journal of molecular biology. 379(2. 284-98.


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