From 2008.igem.org

Introduction	Our project	Modelling	Wet Lab	Our team	Timetable	Miscellaneous

Introduction	Protocols

Eva's Notebook

Management

Along with two other team members,we were actually running the first iGEM project within the University of Sheffield, even though this University has nearly the best Molecular Biology and Biotechnology Department in the UK.

Sponsorship

What is more we managed to collect material sponsorship as well as financial contribution worth of about £10 000. This includes free lab space and chemical materials in the [http://www.shef.ac.uk/bioincubator/ Sheffield Bioincubator], a newly built commercial business and research facility, also free primers and CqsS gene synthesis worth more than £1000 from the IDTDNA comercial company. Finally, financial support towards traveling and living expenses from MBB department, Prof. R. Poole, [http://www.fusionip.co.uk/ Biofusion], [http://cms.icheme.org/MainWebSite/Homepage3bf36337.aspx?Map=1B65ED76D4B00E9E78817E89DC003264 iCHEME ].

Biobrick Characterisation

The initial idea was to insert the GFP under the pgaABCD operon controlled by small regulatory RNA - CsrA. Once the CsrA inhibition of this operon is releaved under the sensing event of the fusion kinase, the GFP would be expressed. What is more, the project was to have the GFP with an LVA tag (AANDENYALAA). This means the GFP would have a tag targeted by proteases and would be degraded within 45 minutes once there is no more stimulation. An idea to include degradable GFP was taken from the Andersen, J.B et al. (1998) New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria. Applied and Environmental Microbiology.

A Biobrick present in the Registry (GFP with the LVA tag) would be the best choice for our idea, as it would make the sensing bacteria re-usable within about 45 minutes after the triggering of the fusion kinase stops. Also, the Biobrick present in the booklet is under lac promoter, which means that we could vary the IPTG concentration to have the optimal GFP expression rate.

Introduction

However, this plan was based on achieving a successful gene knock-out, which we failed to achieve. After extensive transformation attempts using various protocols only one possible transformant was present. As we suspected a possible contamination, the characterization of it was carried out so as to prove it.

Schedule

16th August 2008 - the transformation was carried out with CaCl2 chemically - competent DH5-alpha strains using the transformation method described in Sambrock.
The transformation was very "weak", meaning that there was only one colony growing after transformation. It might have been due to increased kanamycin concentration used on the plates (50ugrams/ml instead of 30 ugrams/ml) as well as short recovery time (the cells were incubated at 37 degrees Celcius only for 30 minutes) . Nonetheless, possible contamination was also suspected.
The transformant was replated and the fluorescence measurement was carried out using Tecan high-flow cytometer.
- Protocol used for characterization :

1.Grow 5ml of overnight cultures of DH5-alpha cells containing the plasmid with GFP-LVA.
2.Resuspend the overnight cultures in 50 ml of LB medium until the OD600 reaches about 0.6.
3.Quickly add 0.2 mM of IPTG to the medium and plate that into 96 well plate.
4.Use the high flow cytometry machine TecanR to measure the fluorescence every 15 minutes for 8 hours (excitation wv – 485 nm, emission wv- 535 nm )(including shaking = aeration of the cultures)

Results

Even repeating the protocol for 5 times the results were consistent.

1.A control sample with LB + IPTG had more fluorescence than LB media with cultures (this is due to turbidity caused by the growth of cultures, which reduces the shining of LB) (Fluorescence varied : 47108 to 41136 units) 2.LB + DH5-alpha had a range of fluorescence from about 42 000 units decreasing to 32 770 units. 3.LB + DH5-alpha + IPTG (expected high levels of fluorescence) the fluorescence measurement showed from about 46 000 units decreasing to 41 700 units over 8 hours of measurement

The measurement was taken 7 times, varying the amount of IPTG added to the medium from (0.3 mM to 0.05 mM) this did not seem to affect the measurements in any noticeable way. This proved that most likely the transformant colonies present were a contamination.

Further actions

Together with this GFP – LVA characterization we decided to transform the cells using RFP (tested - working) and RFP-LVA (not characterised previously) and plating them directly on Amp + IPTG plates to straight-forwardly check for red transformant cells (Figure 1).

Cells expressing RFP under UV light

Cells expressing RFP visible under naked eye

Cells expressing RFP in separate colonies

As it can be seen in the Figure 1 the RFP with no LVA tag was used by other iGEM teams. The transformed cells can be easily visualized under UV light in UV box as well as are visible under naked eye.

The transformation with RFP and RFP-LVA was carried out on the 15th October
The DNA content was checked before the actual procedure. The spectrophotometer indicated 91 ng/ul and 121 ng/ ul for RFP-LVA and RFP respectively
The transformation was unsuccessful even the pre-checked chemically competent DH5-alpha cells were used, the positive controls worked and as a last attempt, the transformation was made by a Ph.D. student.

Proof of faulty Biobrick booklet

As the Tecan measurement for GFP - LVA Biobrick proved no fluorescence present, and the transformations with RFP and RFP-LVA were unsuccessful, the suspicions of faulty Biobrick booklet increased. So as to completely prove it various parts from the booklet were selected and eluted to TE, then loaded on the agarose gel. None of the bands were present even after two successive checks. Hypothesis of a faulty Biobrick booklet was proven.

Conclusion

Unfortunately, the step that seemed to be the easiest one - transformation failed even after excessive attempts. A lot of troubleshooting was done, however nothing seemed to let us figure out what was the problem. Only later on the suspicions of the faulty Biobrick booklet arose. After thorough investigation of the DNA present in the booklet, by randomly eluting it in TE and running the agarose gel, we came to conclusion that the Biobrick booklet was faulty.

Leaflet Production

This was my second responsibility.

Software

[http://www.scribus.net Scribus], an open source desktop publishing program was selected as being most suitable for this task.

Initial Design Work

This is still a work in progress.

Team:University of Sheffield/Eva