Team:Newcastle/9 July 2010

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==Transformation==
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=Research=
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Today we initiated with our research in urease which would lead to calcium carbonate production inside the crack.
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=== Aims ===
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#Wray LV, Ferson E and Fisher SH. (1997). "Expression of the Bacillus subtilis ureABC operon is controlled by multiple regulatory factors including CodY, GlnR, TnrA, and Spo0H". ''Journal of bacteriology''. 179(17). 5494-501.
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The aim of this experiment is to transform competent ''E. coli'' DH5α with pSB1AT3 vectors containing the lacI insert (in the ratios of 1:3 and 1:5 of vector to insert).
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#Kim JK, Mulrooney SB and Hausinger RP. (2005). "Biosynthesis of Active Bacillus subtilis Urease in the Absence of Known Urease Accessory Proteins". ''Journal of Bacteriology''. 187(20). 7150-7154.  
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===Materials===
 
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Each of the following five tubes contain 200 µl of competent ''E. coli'' DH5α. To this the DNA to be transformed was added.
 
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# 1:3 (contains LacI insert and pSB1AT3 vector in the proportion of 1:3)
 
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# 1:5 (contains LacI insert and pSB1AT3 vector in the proportion of 1:5)
 
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# Negative control for ligation (contains vector with no insert)
 
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# Control for transformation (without plasmid)
 
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# Control for transformation (with plasmid, pSB1AT3)
 
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===Protocol===
 
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The [https://2010.igem.org/TeamNewcastleTransformation_of_E._coli#Protocol| transformation protocol] was followed using the tubes above:
 
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# Thaw a 200 µl aliquot of ''E. coli'' DH5α and add the transforming DNA. For tubes 1-3 5 µl of vector was added, for tube 4 no vector was added and for tube 5 only 1 µl of vector was added. This is because tubes 1 to 3 have been ligated and will contain cells in different forms of the ligated vector whereas tube 5 contains the ligated vector (our control).
 
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# Incubate for 45 mins on ice.
 
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# Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.
 
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# Add 1 ml of LB broth to each tube and incubate the cells at 37°C for 1.5 hr (static for initial 20 mins, shaking for remaining time).
 
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# Plate out 200 µl of solution from the 5 tubes above on LB plates (agar at 1.5%) using the glass balls - spread plating.
 
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#*10 LB plates were used (9 with ampicillin and 1 without ampicillin)
 
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#**3 plates for 1:3
 
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#**3 plates for 1:5
 
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#**Vector only plate (with no insert) i.e. 1:0
 
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#**2 plates for without plasmid (one on LB with ampicillin and one without ampicillin)
 
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#**1 plate with pSB1AT3 plasmid (only 1 µl of plasmid purified by SW and DY (198 ng/µl) was added)
 
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# The plates were incubated overnight at 37°C and left in the fridge till Monday.
 
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===Results===
 
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Please see next day (link)
 
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Latest revision as of 19:16, 25 October 2010

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Research

Today we initiated with our research in urease which would lead to calcium carbonate production inside the crack.

  1. Wray LV, Ferson E and Fisher SH. (1997). "Expression of the Bacillus subtilis ureABC operon is controlled by multiple regulatory factors including CodY, GlnR, TnrA, and Spo0H". Journal of bacteriology. 179(17). 5494-501.
  2. Kim JK, Mulrooney SB and Hausinger RP. (2005). "Biosynthesis of Active Bacillus subtilis Urease in the Absence of Known Urease Accessory Proteins". Journal of Bacteriology. 187(20). 7150-7154.


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