Difference between revisions of "Part:BBa T9002:Experience"

Revision as of 14:32, 1 October 2011

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Applications of BBa_T9002

User Reviews

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2009 DNA Distribution quality control

The UNIPV-Pavia iGEM team sequenced T9002 part and found that it was completely confirmed, while iGEM QC results classified it as "inconsistent". DNA was resuspended from well 9A, kit plate 2, transformed in TOP10 E. coli and amplified inoculating a single colony from the grown LB agar plate in LB medium. Finally DNA has been miniprepped from the grown culture and sent to a BMR Genomics (Padova, Italy) for sequencing.

Experimental measurements

The UNIPV-Pavia iGEM team tested T9002 BioBrick in several working conditions. Results are reported in BBa_F2620 Experience page.

The Brown iGEM team conducted tests on this part in the summer of 2007. The results are depicted in the graphs below.

The first graph indicates that until a critical point is reached, increasing AHL concentration does increase GFP output. This is most easily noticeable between the concentrations of 20 nM, after which point the amount of GFP produced by cells begins to decrease. This may be due to one of two things: AHL quenches the signal from GFP, or too much AHL disrupts the cell's functions in a way that either kills it or prevents it from making as much GFP. This second hypothesis is partially confirmed by the second graph, which shows that adding more than 20 nM AHL causes a decline in cell density. On each graph, the different colored lines represent different time points after AHL was added to the cells. They are 4 hours, 5 hours, etc.

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••••• UNIPV-Pavia iGEM 2011

BioBrick BBa_T9002 is an HSL biosensor, which provides a non linear relationship between HSL input and Scell output. More precisely, the characteristic sigmoidal curve requires synthetic parameters for its accurate identification. These are the minimum and maximum values, the swtich point (i.e., the curve inflection point), and the upper and lower boundaries of linearity. This biosensor revealed greatly reliable, providing measurement repeatability and minimal experimental noise. Referring to its activation formula, the calibration curve is shown below. Minimum [Scell] Maximum [Scell] Switch point [nM] Lower boundary of linearity [nM] Upper boundary of linearity [nM] 17.31 739.4 1.39 0.38 5.07 In order to determine the threshold sensitivity of T9002 biosensor, experiments were performed with several HSL inductions minimally interspaced in the region of low detectability. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests), the minimum detectable HSL concentration is 3 nM. This biosensor was used to measure HSL concentration for parts producing or degrading this signalling molecule. For each of these experiments, a calibration curve of the biosensor was built, inducing it with known HSL concentrations, evaluating for each of them the Scell signal and finally estimating the Hill curve parameters. Once identified the parameters, the unknown concentration of a sample can be evaluated from its Scell (provided that it has a value included in the linear zone of the biosensor), as shown below: It is necessary, then, to multiply the measurement for the dilution factor used (in our experiments it was 20).

Sevilla 2011

We used this construction as a model for a GFP-based characterization method. We tried different concentrations, that turned to be too high, for it seems that the media was quite saturated.

Fluorescence O.D.(600nm)

Fluorescence/Absorbance ratio and average of these data

Variation of the fluo/Abs ratio in time

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