Team:University of Sheffield/Dimtry

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{| style="color:#888888;background-color:##888888;" cellpadding="5" cellspacing="2" border="2" bordercolor=#888888 width="85%" align="center"
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!align="center"|[[Team:University_of_Sheffield |Introduction]]
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!align="center"|[[Team:University_of_Sheffield /Project|Our project]]
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!align="center"|[[Team:University_of_Sheffield /Modelling|Modelling]]
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!align="center"|[[Team:University_of_Sheffield /Wet Lab|Wet Lab]]
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!align="center"|[[Team:University_of_Sheffield /Lab Books| Our team]]
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!align="center"|[[Team:University_of_Sheffield /Timetable| Timetable]]
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!align="center"|[[Team:University_of_Sheffield /Misc| Miscellaneous]]
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{| style="color:#888888;background-color:##888888;" cellpadding="2" cellspacing="1" border="1" bordercolor=#888888 width="85%" align="center"
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!align="center"|[[Team:University_of_Sheffield /Wet Lab|Introduction]]
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!align="center"|[[Team:University_of_Sheffield /Protocols|Protocols]]
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==Lab book: Production and extraction of CAI==  
==Lab book: Production and extraction of CAI==  

Revision as of 02:16, 29 October 2008

UniShefBanner.jpg


Introduction Our project Modelling Wet Lab Our team Timetable Miscellaneous
Introduction Protocols


Lab book: Production and extraction of CAI

The original protocol of CAI production, which is a slightly modified version of the protocol described in the paper,[1] was as follows:

1.) Transform plasmid pTrc99A in E.coli DH5a How? Heat shock

2.) Grow at room temp, with aeration, overnight Medium: 2.4 Litres of M9 medium with

  • 9.6 g glucose (4g/l)
  • 1.2g leucine(0.5g/l)
  • 0.24g ampicillin (0.1g/l)

3.) Induce cqsA expression with 0.5 mM IPTG for 16 hours

4.) Centrifuge at 10000 x g for 60s

5.) Pass through a 0.22μm filter

6.) Mix the now cell-free culture fluid with 0.6v (?) dichloromethane (DCM) in separatory funnel. Isolate organic phase, then evaporate.

7.) Dissolve in DCM, and purify through silica gel with 0.5L of 7:3 DCM:methanol. Concentrate with evaporation

8.) 50-100mg of concentrate dissolved in ~1ml DCM

9.) Injected in (2x25cm) ethyl-pyridine HPLC column and eluted using a gradient of increasing ethyl acetate in hexanes (0-80%) at 20mL/min.

After taking cost and time considerations into account, it was decided that the usage of the HPLC in our in experiment was not possible, but since the purity of the CAI was not essential as long as the CAI was in high concentration, it was not a problem (we expected our sensor to sense bacteria in water in not perfect conditions). Therefore steps 7-9 were dropped.


Next is the copy of the day-to-day lab book:


3-10 Sept 08

A series of unsuccessful attempts to grow the cells in M9 at room temperature overnight. All medium and human error minimised: Different M9 stocks Different method of adding cells to media


11-18 Sept The experiment was changed. A variety of temperatures was tested, the overnight growth was replaced by a 36h and LB was used a control. The results were as follows:




References

[1] The Major Vibrio cholerae Autoinducer CAI-1 Supplementary Information" Nature 450, 883-886 (6 December 2007) by Douglas A. Higgins, Megan E. Pomianek, Christina M. Kraml, Ronald K. Taylor, Martin F. Semmelhack & Bonnie L. Bassler

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