Aberdeen_Scotland 2009

The initial gel worked although the obvious confirmation was not possible due to the very small fragment size (49bp).But later it was confirmed following its usage by forming the part BBa_K182002 through the PCR gel analysis and the sequencing.The plasmid miniprep also worked.

Aberdeen_Scotland 2009

The transformation, miniprep and the gel (undigested and digested with the EcoRI and SpeI) worked as expected.

Aberdeen_Scotland 2009

The transformation, miniprep and the gel (undigested and digested with the EcoRI and SpeI) worked. Further testings were performed by the formation of the BBa_K182002, PCR analysis, sequencing and everything worked flawlessly according to our expectation.

Aberdeen_Scotland 2009

The plasmid miniprep and the gel worked as expected. However, since we did not use this part for our cloning step we cannot comment on the sequencing or the other features of this part.

Aberdeen_Scotland 2009

The initial gel worked although the obvious confirmation was not possible due to the very small fragment size (54bp).But later it was confirmed following its usage by forming the part BBa_K182001 through the PCR gel analysis and the sequencing.The plasmid miniprep also worked.

Aberdeen_Scotland 2009

The gel analysis and the plasmid miniprep worked quite nicely as expected. But later the construct was further tested and characterised by combining with the bio-brick BBa_K182002 which contained a tet repressor under the control of a CI operator. So, the CFP expression of BBa_I13600was repressed as expected when tested with the BBa_K182002. With the addition of Anhydrotetracycline (an analogue of tetracycline) the CFP expression was restored and this was confirmed performing the fluorescent microscopy assay.

Aberdeen_Scotland 2009

Experimental Design
The J37032 BioBrick expresses GFP under control of a LuxR responsive promoter. The J37032 part was provided by iGEM-HQ, was succesfully mini-preppred and transformed into E.coli. J37032 was used to test our constitutive LuxR/LuxI operon ([http://partsregistry.org/Part:BBa_K182200 BBa_K182200]). For that purpose SCS1 E.coli was transformed either with K182200 alone (referred to as pIR in figure below), J37032 alone, or a combination of both plasmids. The single transformants acted as negative controls.

Results
It would be expected that the K182200 transformant would not fluoresce, since it does not express GFP. This was confirmed (top panel, figure below, showing light microscope pictures on the left, and fluorescent images on the right). Similarly, the single J37032 transformant was not expected to exhibit much fluorescence, since there is no endogenous expression of LuxR or LuxI in E.coli. A combination of K182200 and J37032 would be expected to produce high amounts of fluorescence at high cell densities as quorum sensing triggers the Lux promoter of J37032. However, in overnight stationary phase cultures at high density, it was found that the J37032 construct expresses the same high amount of GFP fluorescence with or without the presence of LuxR (compare middle panel[J37032 single transformant] with bottom panel [J37032/pIR double transformant])

Conclusion
We conclude that J37032 is either not responsive to LuxR, or that the pLux promoter is extremely leaky, driving significant GFP expression in the absence of LuxR. It therefore cannot easily be used to test quorum sensing due to this high background level of GFP expression.

Aberdeen_Scotland 2009

The gel analysis and the plasmid miniprep worked quite nicely as expected. In addition, the construct was further tested by adding IPTG which took off the lacRepressor of the LacO and hence enabling the CFP expression.