Team:University of Sheffield/Dimtry

From 2008.igem.org

Introduction	Our project	Modelling	Wet Lab	Our team	Timetable	Miscellaneous

Introduction	Protocols

Lab book: Production and extraction of CAI

Original Protocol

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.

Early adaptations

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.

Day-to-day lab book

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

In the end there was no growth in the medium.

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:

In temperatures above 30C the growth was very poor. There was no noticeable growth at 37C.
LB yeilded a slightly higher growth rate.
In the end several 36-48H cultures were were induced and the used in the CAI extraction.

During this time, extraction of CAI was experimented. There were no new changes to the method. In the end several samples were obtained. They are yet to be tested.

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