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

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.
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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-==Transformation==
+=Research=
+Today we initiated with our research in urease which would lead to calcium carbonate production inside the crack.
-=== Aims ===
+#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.
-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).
+#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.
-===Materials===
-Each of the following five tubes contain 200 µl of competent ''E. coli'' DH5α. To this the DNA to be transformed was added.
-# 1:3 (contains LacI insert and pSB1AT3 vector in the proportion of 1:3)
-# 1:5 (contains LacI insert and pSB1AT3 vector in the proportion of 1:5)
-# Negative control for ligation (contains vector with no insert)
-# Control for transformation (without plasmid)
-# Control for transformation (with plasmid, pSB1AT3)
-===Protocol===
-The [[https://2010.igem.org/TeamNewcastleTransformation_of_E._coli#Protocol|transformation protocol]] was followed using the tubes above:
-# 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).
-# Incubate for 45 mins on ice.
-# Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.
-# 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).
-# Plate out 200 µl of solutiion from the 5 tubes above on LB plates (agar at 1.5%) using the glass balls.
-#*10 LB plates were used (9 with ampicillin and 1 without ampicillin)
-#**3 plates for 1:3
-#**3 plates for 1:5
-#**Vector only plate (with no insert) i.e. 1:0
-#**2 plates for without plasmid (one on LB with ampicillin and one without ampicillin)
-#**1 plate with pSB1AT3 plasmid (only 1 µl of plasmid purified by SW and DY (198 ng/µl) was added)
-# Incubate plates overnight at 37°C.
-===Results===
-Please see next day (link)
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Team:Newcastle/9 July 2010

From 2010.igem.org

Latest revision as of 19:16, 25 October 2010

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